U.S. patent application number 15/852762 was filed with the patent office on 2018-10-04 for bacteria engineered to treat a disease or disorder.
The applicant listed for this patent is Synlogic, Inc.. Invention is credited to Dean Falb, Adam B. Fisher, Vincent M. Isabella, Jonathan W. Kotula, Suman Machinani, Paul F. Miller, Yves Millet.
Application Number | 20180280451 15/852762 |
Document ID | / |
Family ID | 56117975 |
Filed Date | 2018-10-04 |
United States Patent
Application |
20180280451 |
Kind Code |
A9 |
Falb; Dean ; et al. |
October 4, 2018 |
BACTERIA ENGINEERED TO TREAT A DISEASE OR DISORDER
Abstract
Genetically programmed microorganisms, such as bacteria,
pharmaceutical compositions thereof, and methods of modulating and
treating a disease and/or disorder are disclosed.
Inventors: |
Falb; Dean; (Sherborn,
MA) ; Miller; Paul F.; (Salem, CT) ; Kotula;
Jonathan W.; (Somerville, MA) ; Isabella; Vincent
M.; (Cambridge, MA) ; Machinani; Suman;
(Cambridge, MA) ; Fisher; Adam B.; (Cambridge,
MA) ; Millet; Yves; (Newton, MA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Synlogic, Inc. |
Cambridge |
MA |
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20180169154 A1 |
June 21, 2018 |
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Family ID: |
56117975 |
Appl. No.: |
15/852762 |
Filed: |
December 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15319564 |
Dec 16, 2016 |
9889164 |
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PCT/US2016/032565 |
May 13, 2016 |
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15852762 |
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15154934 |
May 13, 2016 |
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15319564 |
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14960333 |
Dec 4, 2015 |
9487764 |
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15154934 |
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PCT/US2015/064140 |
Dec 4, 2015 |
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14960333 |
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PCT/US2016/020530 |
Mar 2, 2016 |
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PCT/US2015/064140 |
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PCT/US2016/032562 |
May 13, 2016 |
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PCT/US2016/020530 |
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62336338 |
May 13, 2016 |
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62335780 |
May 13, 2016 |
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62335940 |
May 13, 2016 |
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62336012 |
May 13, 2016 |
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62314322 |
Mar 28, 2016 |
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62313691 |
Mar 25, 2016 |
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62293749 |
Feb 10, 2016 |
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62277654 |
Jan 12, 2016 |
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62277413 |
Jan 11, 2016 |
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62277346 |
Jan 11, 2016 |
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62263329 |
Dec 4, 2015 |
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62256041 |
Nov 16, 2015 |
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62256039 |
Nov 16, 2015 |
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62256052 |
Nov 16, 2015 |
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62212223 |
Aug 31, 2015 |
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62199445 |
Jul 31, 2015 |
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62183935 |
Jun 24, 2015 |
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62173761 |
Jun 10, 2015 |
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62173706 |
Jun 10, 2015 |
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62173710 |
Jun 10, 2015 |
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62161137 |
May 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/741 20130101;
A61K 2035/11 20130101; C07K 14/195 20130101; A61K 35/74 20130101;
C12Y 104/03002 20130101; C12Y 403/01024 20130101; C12N 9/0022
20130101; C12N 15/70 20130101; Y02A 50/30 20180101; C12N 9/88
20130101; C12N 9/0014 20130101; C07K 14/245 20130101; A61K 39/02
20130101; A61P 3/00 20180101; A61K 38/00 20130101 |
International
Class: |
A61K 35/74 20150101
A61K035/74; C07K 14/195 20060101 C07K014/195 |
Claims
1. A genetically-engineered non-pathogenic bacterium for use in
metabolizing a substrate of interest, the bacterium comprising at
least one heterologous gene encoding a transporter for importing
said substrate, wherein the gene encoding the substrate transporter
is operably linked to a directly or indirectly inducible promoter
that is not associated with the substrate transporter in
nature.
2. The genetically engineered bacterium of claim 1, wherein the
bacterium further comprises at least one heterologous gene encoding
a polypeptide for metabolizing the substrate of interest, wherein
the gene encoding the polypeptide is operably linked to a directly
or indirectly inducible promoter that is not associated with the
polypeptide in nature.
3. The genetically engineered bacterium of claim 1 or claim 2,
wherein the promoter operably linked to the gene encoding the
transporter and the promoter operably linked to the gene encoding a
polypeptide are separate copies of the same promoter.
4. The genetically engineered bacterium of claim 1 or claim 2,
wherein the gene encoding the transporter and the gene encoding the
polypeptide are operably linked to the same copy of the same
promoter.
5. The genetically engineered bacterium of any one of claims 1-4,
wherein the promoter operably linked to the gene encoding the
transporter and the promoter operably linked to the gene encoding
the polypeptide are directly or indirectly induced by exogenous
environmental conditions.
6. The genetically engineered bacterium of any one of claims 1-5,
wherein the promoter operably linked to the gene encoding the
transporter and the promoter operably linked to the polypeptide are
directly or indirectly induced by exogenous environmental
conditions found in the gut of a mammal.
7. The genetically engineered bacterium of any one of claims 1-5,
wherein the promoter operably linked to the gene encoding the
transporter and the promoter operably linked to the polypeptide are
directly or indirectly induced by exogenous environmental
conditions found in the microenvironment of a tumor.
8. The genetically engineered bacterium of claim any one of claims
1-7, wherein the promoter operably linked to the gene encoding a
transporter and the promoter operably linked to the gene encoding
the polypeptide are directly or indirectly induced under low-oxygen
or anaerobic conditions.
9. The genetically engineered bacterium of any one of claims 1-8,
wherein the promoter operably linked to the gene encoding the
transporter and the promoter operably linked to the gene encoding
the polypeptide are selected from the group consisting of an
FNR-responsive promoter, an ANR-responsive promoter, and a
DNR-responsive promoter.
10. The genetically engineered bacterium of any one of claims 1-9
wherein the gene encoding the transporter is located on a
chromosome in the bacterium.
11. The genetically engineered bacterium of any one of claims 1-9,
wherein the gene encoding the transporter is located on a plasmid
in the bacterium.
12. The genetically engineered bacterium of any one of claims 1-11,
wherein the gene encoding the polypeptide is located on a plasmid
in the bacterium.
13. The genetically engineered bacterium of any one of claims 1-11,
wherein the gene encoding the polypeptide is located on a
chromosome in the bacterium.
14. The genetically engineered bacterium of any one of claims 1-13,
wherein the bacterium is a probiotic bacterium.
15. The genetically engineered bacterium of claim 14, wherein the
bacterium is selected from the group consisting of Bacteroides,
Bifidobacterium, Clostridium, Escherichia, Lactobacillus, and
Lactococcus.
16. The genetically engineered bacterium of claim 15, wherein the
bacterium is Escherichia coli strain Nissle.
17. The genetically engineered bacterium of any one of claims 1-16,
wherein the bacterium is an auxotroph in a gene that is
complemented when the bacterium is present in a mammalian gut.
18. The genetically engineered bacterium of claim 17, wherein
mammalian gut is a human gut.
19. The genetically engineered bacterium of claim 17 or 18, wherein
the bacterium is an auxotroph in diaminopimelic acid or an enzyme
in the thymidine biosynthetic pathway.
20. The genetically engineered bacterium of any one of claims 1-19,
wherein the bacterium is further engineered to harbor a gene
encoding a substance toxic to the bacterium, wherein the gene is
under the control of a promoter that is directly or indirectly
induced by an environmental factor not naturally present in a
mammalian gut.
21. A pharmaceutically acceptable composition comprising the
bacterium of any one of claims 1-20; and a pharmaceutically
acceptable carrier.
22. The composition of claim 21 formulated for oral
administration.
23. A method for treating a disease associated with the
accumulation of a toxic substrate comprising the step of
administering to a subject in need thereof, the composition of
claim 21 or 22.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/319,564, filed on Dec. 16, 2016, which is a 35 U.S.C. .sctn.
371 national stage filing of International Application No.
PCT/US2016/032565, filed on May 13, 2016, which in turn claims
priority to U.S. Provisional Patent Application No. 62/335,780,
filed on May 13, 2016; U.S. Provisional Patent Application No.
62/336,338, filed on May 13, 2016; U.S. Provisional Patent
Application No. 62/336,012, filed on May 13, 2016; U.S. Provisional
Patent Application No. 62/335,940, filed on May 13, 2016; U.S.
patent application Ser. No. 15/154,934, filed on May 13, 2016;
International Application No. PCT/US2016/032562, filed on May 13,
2016; U.S. Provisional Patent Application No. 62/277,654, filed on
Jan. 12, 2016; U.S. Provisional Patent Application No. 62/277,413,
filed on Jan. 11, 2016; U.S. Provisional Patent Application No.
62/293,749, filed on Feb. 10, 2016; International Application No.
PCT/US2016/020530, filed on Mar. 2, 2016; U.S. Provisional Patent
Application No. 62/173,761, filed on Jun. 10, 2015; U.S.
Provisional Patent Application No. 62/173,706, filed on Jun. 10,
2015; U.S. Provisional Patent Application No. 62/173,710, filed on
Jun. 10, 2015; U.S. Provisional Patent Application No. 62/277,346,
filed on Jan. 11, 2016; U.S. Provisional Patent Application No.
62/199,445, filed on Jul. 31, 2015; U.S. Provisional Patent
Application No. 62/314,322, filed on Mar. 28, 2016; U.S.
Provisional Patent Application No. 62/313,691, filed on Mar. 25,
2016; U.S. patent application Ser. No. 14/960,333, filed on Dec. 4,
2015; International Application No. PCT/US2015/064140, filed on
Dec. 4, 2015; U.S. Provisional Patent Application No. 62/263,329,
filed on Dec. 4, 2015; U.S. Provisional Patent Application No.
62/256,041, filed on Nov. 16, 2015; U.S. Provisional Patent
Application No. 62/256,039, filed on Nov. 16, 2015; U.S.
Provisional Patent Application No. 62/212,223, filed on Aug. 31,
2015; U.S. Provisional Patent Application No. 62/183,935, filed on
Jun. 24, 2015; U.S. Provisional Patent Application No. 62/256,052,
filed on Nov. 16, 2015; U.S. Provisional Patent Application No.
62/161,137, filed on May 13, 2015, the entire contents of each of
which are expressly incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 22, 2017, is named 126046-01403_Sequence_Listing.txt and is
355,163 bytes in size.
BACKGROUND OF THE INVENTION
[0003] It has recently been discovered that the microbiome in
mammals plays a large role in health and disease (see Cho and
Blaser, Nature Rev. Genet., 13:260-270, 2012 and Owyang and Wu,
Gastroenterol., 146(6):1433-1436, 2014). Indeed, bacteria-free
animals have abnormal gut epithelial and immune function,
suggesting that the microbiome in the gut plays a critical role in
the mammalian immune system. Specifically, the gut microbiome has
been shown to be involved in diseases, including, for example,
immune diseases (such as Inflammatory Bowel Disease), autism, liver
disease, cancer, food allergy, metabolic diseases (such as urea
cycle disorder, phenylketonuria, and maple syrup urine disease),
obesity, and infection, among many others.
[0004] Fecal transplantation of native microbial strains has
recently garnered much attention for its potential to treat certain
microbial infections and immune diseases in the gut (Owyang and Wu,
2014). There have also been recent efforts to engineer microbes to
produce, e.g., secrete, therapeutic molecules and administer them
to a subject in order to deliver the therapeutic molecule(s)
directly to the site where therapy is needed, such as various sites
in the gut. However, such efforts have been frustrated for several
reasons, mostly relating to the constitutive production of the
bacteria and its gene product(s). For example, the viability and
stability of the engineered microbes have been compromised due, in
part, to the constitutive production of large amounts of foreign
protein(s). Unfortunately, genetically engineered microbes which
have been engineered to express intracellular therapeutic enzymes
which degrade target molecules associated with disease states or
disorders, e.g., diseases or disorders associated with the
overexpression of a molecule which is harmful to a subject, have
also been shown to have low efficacy and enzyme activity levels in
vitro and in vivo. Accordingly, a need exists for improved
genetically engineered microbes which are useful for therapeutic
purposes.
SUMMARY
[0005] The instant invention surprisingly provides genetically
engineered microbes which express a heterologous transporter in
order to regulate, e.g., increase, the transport of target
molecules associated with disease into the genetically engineered
microbes in order to increase the therapeutic efficacy of the
microbe.
[0006] In one aspect, the invention provides a
genetically-engineered non-pathogenic microorganism comprising at
least one heterologous gene encoding a substrate transporter,
wherein the gene encoding the substrate transporter is operably
linked to an inducible promoter.
[0007] In one embodiment, the bacterium is a Gram-positive
bacterium. In one embodiment, the bacterium is a Gram-negative
bacterium. In one embodiment, the bacterium is an obligate
anaerobic bacterium. In one embodiment, the bacterium is a
facultative anaerobic bacterium.
[0008] In one embodiment, the bacterium is an aerobic bacterium. In
one embodiment, the bacterium is selected from Clostridium novyi
NT, Clostridium butyricum, E. coli Nissle, and E. Coli K-12.
[0009] In one embodiment, the inducible promoter is induced by
low-oxygen or anaerobic conditions. In one embodiment, the
inducible promoter is selected from a FNR-inducible promoter, an
ANR-inducible promoter, and a DNR-inducible promoter. In one
embodiment, the inducible promoter is P-fnrs promoter.
[0010] In one embodiment, the substrate transporter is capable of
importing into the bacterium a substrate selected from the group
consisting of an amino acid, a nucleoside, kynurenine,
prostaglandin E2, lactic acid, propionate, bile salt,
.gamma.-aminobutyric acid (GABA), manganese, a toxin, and a
peptide.
[0011] In one embodiment, the substrate transporter is an amino
acid transporter capable of importing into the bacterium an amino
acid selected from the group consisting of leucine, isoleucine,
valine, arginine, lysine, asparagine, serine, glycine, glutamine,
tryptophan, methionine, threonine, cysteine, tyrosine,
phenylalanine, glutamic acid, aspartic acid, alanine, histidine,
and proline.
[0012] In one embodiment, the heterologous gene encoding the amino
acid transporter is from Agrobacterium tumefaciens, Anabaena
cylindrical, Anabaena variabilis, Bacillus amyiquefaciens, Bacillus
atrophaeus, Bacillus halodurans, Bacillus methanolicus, Bacillus
subtilis, Caenorhabditis elegans, Clostridium botulinum,
Corynebacterium glutamicum, Escherichia coli, Flavobacterium
limosediminis, Helicobacter pylori, Klebsiella pneumonia,
Lactococcus lactis, Lactobacillus saniviri, Legionella pneumophila
Methylobacterium aquaticum, Mycobacterium bovis, Photorhabdus
luminescens, Pseudomonas aeruginosa, Pseudomonas fluorescens,
Saccharomyces cerevisiae, Salmonella enterica, Sinorhizobium
meliloti, or Ustilago maydis. In one embodiment, the heterologous
gene encoding the amino acid transporter has a sequence with at
least 90% identity to any one of SEQ ID NOs:9, 10, 13-18, 25, 26,
29, 35, 41-44, 46-48, 59-62, 69, 87, 91, 94-96, 98, or 103. In one
embodiment, the heterologous gene encoding the amino acid
transporter has a sequence comprising any one of SEQ ID NOs:9, 10,
13-18, 25, 26, 29, 35, 41-44, 46-48, 59-62, 69, 87, 91, 94-96, 98,
or 103. In one embodiment, the heterologous gene encoding the amino
acid transporter has a sequence consisting of any one of SEQ ID
NOs:9, 10, 13-18, 25, 26, 29, 35, 41-44, 46-48, 59-62, 69, 87, 91,
94-96, 98, or 103.
[0013] In one embodiment,the substrate transporter is a nucleoside
transporter capable of importing into the bacterium a nucleoside
selected from the group consisting of adenosine, guanosine,
uridine, inosine, xanthosine, thymidine and cytidine. In one
embodiment, the heterologous gene encoding the nucleoside
transporter is from Bacillus halodurans, Bacillus subtilis,
Caulobacter crescentus, Escherichia coli, Haemophilus influenzae,
Helicobacter pylori, Pseudomonas, Bacillus subtilis, Escherichia
coli, Prevotella intermedia, Porphytomonas gingivalis, Salmonella
typhimurium, Salmonella enterica, or Vibrio cholera.
[0014] In one embodiment, the heterologous gene encoding the
nucleoside transporter has a sequence with at least 90% identity to
any one of SEQ ID NOs:108-128. In one embodiment, the heterologous
gene encoding the amino acid transporter has a sequence comprising
any one of SEQ ID NOs:108-128. In one embodiment, the heterologous
gene encoding the amino acid transporter has a sequence consisting
of any one of SEQ ID NOs:108-128.
[0015] In one embodiment, the substrate transporter is a kynurenine
transporter capable of importing kynurenine into the bacterium. In
one embodiment, the heterologous gene encoding the kynurenine
transporter is from Escherichia coli, Saccharomyces cerevisiae or
Corynebacterium glutamicum. In one embodiment, the heterologous
gene encoding the kynurenine transporter has a sequence with at
least 90% identity to any one of SEQ ID NOs:46-48. In one
embodiment, the heterologous gene encoding the kynurenine
transporter has a sequence comprising any one of SEQ ID NOs:46-48.
In one embodiment, the heterologous gene encoding the amino acid
transporter has a sequence consisting of any one of SEQ ID
NOs:46-48.
[0016] In one embodiment, the substrate transporter is a
prostaglandin E2 transporter capable of importing prostaglandin E2
into the bacterium. In one embodiment, the heterologous gene
encoding the prostaglandin E2 (PGE2) transporter is from
Escherichia coli, Saccharomyces cerevisiae or Corynebacterium
glutamicum.
[0017] In one embodiment, the substrate transporter is a lactic
acid transporter capable of importing lactic acid into the
bacterium. In one embodiment,the heterologous gene encoding the
lactic acid transporter is from Escherichia coli, Saccharomyces
cerevisiae and Corynebacterium glutamicum.
[0018] In one embodiment, the substrate transporter is a propionate
transporter capable of importing propionate into the bacterium. In
one embodiment, the heterologous gene encoding the propionate
transporter is from Bacillus subtilis, Campylobacter jejuni,
Clostridium perfringens, Escherichia coli, Lactobacillus
delbrueckii, Mycobacterium smegmatis, Nocardia farcinica,
Pseudomonas aeruginosa, Salmonella typhimurium, Virgibacillus, or
Staphylococcus aureus. In one embodiment, the heterologous gene
encoding the propionate transporter has a sequence with at least
90% identity to any one of SEQ ID NOs:129-130. In one embodiment,
the heterologous gene encoding the propionate transporter has a
sequence comprising any one of SEQ ID NOs:129-130. In one
embodiment, the heterologous gene encoding the propionate
transporter has a sequence consisting of any one of SEQ ID
NOs:129-130.
[0019] In one embodiment, the substrate transporter is a bile salt
transporter capable of importing bile salt into the bacterium. In
one embodiment, the heterologous gene encoding the bile salt
transporter is from Lactobacillus johnsonni. In one embodiment, the
heterologous gene encoding the bile salt acid transporter has a
sequence with at least 90% identity to any one of SEQ ID
NOs:131-132. In one embodiment, the heterologous gene encoding the
bile salt transporter has a sequence comprising any one of SEQ ID
NOs:131-132. In one embodiment, the heterologous gene encoding the
bile salt transporter has a sequence consisting of any one of SEQ
ID NOs:131-132.
[0020] In one embodiment, the substrate transporter is a bile salt
transporter capable of importing ammonia into the bacterium. In one
embodiment, the heterologous gene encoding the ammonia transporter
is from Corynebacterium glutamicum, Escherichia coli, Streptomyces
coelicolor or Ruminococcus albus. In one embodiment, the
heterologous gene encoding the ammonia transporter has a sequence
with at least 90% identity to SEQ ID NO:133. In one embodiment, the
heterologous gene encoding the ammonia transporter has a sequence
comprising SEQ ID NO:133. In one embodiment, the heterologous gene
encoding the ammonia transporter has a sequence consisting of SEQ
ID NO:133.
[0021] In one embodiment, the substrate transporter is a
.gamma.-aminobutyric acid (GABA) transporter capable of importing
GABA into the bacterium. In one embodiment, the heterologous gene
encoding the GABA transporter is from Escherichia coli or Bacillus
subtilis. In one embodiment, the heterologous gene encoding the
GABA transporter has a sequence with at least 90% identity to SEQ
ID NO:134. In one embodiment, the heterologous gene encoding the
GABA transporter has a sequence comprising SEQ ID NO:134. In one
embodiment, the heterologous gene encoding the GABA transporter has
a sequence consisting of SEQ ID NO:134.
[0022] In one embodiment, the substrate transporter is a manganese
transporter capable of importing manganese into the bacterium. In
one embodiment, the heterologous gene encoding the manganese
transporter is from Bacillus subtilis, Staphylococcus aureus,
Salmonella typhimurium, Shigella flexneri, Yersinia pestis, or
Escherichia coli. In one embodiment, the heterologous gene encoding
the manganese transporter has a sequence with at least 90% identity
to SEQ ID NO:135. In one embodiment, the heterologous gene encoding
the manganese transporter has a sequence comprising SEQ ID NO:135.
In one embodiment, the heterologous gene encoding the manganese
transporter has a sequence consisting of SEQ ID NO:135.
[0023] In one embodiment, the substrate transporter is a toxin
transporter capable of importing a toxin into the bacterium. In one
embodiment, the substrate transporter is a peptide transporter
capable of importing a peptide into the bacterium.
[0024] In one embodiment, the heterologous gene encoding the
substrate transporter and operatively linked promoter are present
on a chromosome in the bacterium. In one embodiment, the
heterologous gene encoding the substrate transporter and
operatively linked promoter are present on a plasmid in the
bacterium.
[0025] In one embodiment, the bacterium is an auxotroph comprising
a deletion or mutation in a gene required for cell survival and/or
growth. In one embodiment, the gene required for cell survival
and/or growth is selected from thyA, dapD, and dapA.
[0026] In one embodiment, the bacterium comprises a kill
switch.
[0027] In one aspect, the present disclosure provides a
pharmaceutical composition comprising a recombinant bacterium
disclosed herein and a pharmaceutically acceptable carrier.
[0028] In another aspect, the present invention provides a method
of treating a disease in a subject in need thereof comprising the
step of administering to the subject a pharmaceutical composition
comprising a recombinant bacterium disclosed herein and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 depicts a synthetic biotic for treating
phenylketonuria (PKU) and disorders characterized by
hyperphenylalaninemia.
[0030] FIG. 2 depicts a synthetic biotic for treating
phenylketonuria (PKU) and disorders characterized by
hyperphenylalaninemia.
[0031] FIG. 3 is a graph showing that PheP and AroP Transport Phe
at the same rate.
[0032] FIG. 4 depicts a schematic representation of the
construction of a pheP knock-in strain, wherein recombineering is
used to insert a 2.sup.nd copy of pheP into the Nissle lacZ
gene.
[0033] FIG. 5 depicts the gene organization of an exemplary
construct comprising a gene encoding PheP, a gene coding TetR, and
a Tet promoter sequence for chromosomal insertion e.g., as for
example comprised in SYN-PKU203, SYN-PKU401, SYN-PKU402,
SYN-PKU302, and SYN-PKU303.
[0034] FIGS. 6A and 6B depict the gene organization of an exemplary
construct, comprising a cloned PAL3 gene under the control of an
FNR promoter sequence, on a low-copy, kanamycin-resistant plasmid
(pSC101 origin of replication, (FIG. 6A). Under anaerobic
conditions, PAL3 degrades phenylalanine to non-toxic
trans-cinnamate. FIG. 6B depicts an additional copy of the
endogenous E. coli high affinity phenylalanine transporter, pheP,
driven by the PfnrS promoter and inserted into the lacZ locus on
the Nissle chromosome.
[0035] FIGS. 7A, 7B, and 7C depict schematic diagrams of
non-limiting embodiments of the disclosure. FIG. 7A depicts
phenylalanine degradation components integrated into the E. coli
Nissle chromosome. In some embodiments, engineered plasmid-free
bacterial strains are used to prevent plasmid conjugation in vivo.
In some embodiments, multiple insertions of the PAL gene result in
increased copy number and/or increased phenylalanine degradation
activity. In some embodiments, a copy of the endogenous E. coli
high affinity phenylalanine transporter, pheP, is driven by the
PfnrS promoter and is inserted into the lacZ locus. FIG. 7B depicts
a schematic diagram of one non-limiting embodiment of the
disclosure, wherein the E. coli Nissle chromosome is engineered to
contain four copies of PfnrS-PAL inserted at four different
insertion sites across the genome (malE/K, yicS/nepI, agaI/rsmI,
and cea), and one copy of a phenylalanine transporter gene inserted
at a different insertion site (lacZ). In this embodiment, the PAL
gene is PAL3 derived from P. luminescens, and the phenylalanine
transporter gene is pheP derived from E. coli. In one embodiment,
the strain is SYN-PKU511. FIG. 7C depicts a schematic diagram of
one preferred embodiment of the disclosure, wherein the E. coli
Nissle chromosome is engineered to contain five copies of PAL under
the control of an oxygen level-dependent promoter (e.g.,
PfnrS-PAL3) inserted at different integration sites on the
chromosome (malE/K, yicS/nepI, malP/T, agaI/rsmI, and cea), and one
copy of a phenylalanine transporter gene under the control of an
oxygen level-dependent promoter (e.g., PfnrS-pheP) inserted at a
different integration site on the chromosome (lacZ). The genome is
further engineered to include a thyA auxotrophy, in which the thyA
gene is deleted and/or replaced with an unrelated gene, as well as
a kanamycin resistance gene.
[0036] FIG. 8A depicts phenylalanine concentrations in samples
comprising bacteria expressing PAL1 or PAL3 on low-copy (LC) or
high-copy (HC) plasmids, or further comprising a copy of pheP
driven by the Tet promoter integrated into the chromosome. Bacteria
were induced with ATC, and then grown in culture medium
supplemented with 4 mM (660,000 ng/mL) of phenylalanine to an
OD.sub.600 of 2.0. Samples were removed at 0 hrs, 2 hrs, and 4 hrs
post-induction and phenylalanine concentrations were determined by
mass spectrometry. Notably, the additional copy of pheP permitted
the degradation of phenylalanine (4 mM) in 4 hrs. FIG. 8B depicts
cinnamate levels in samples at 2 hrs and 4 hrs post-induction. In
some embodiments, cinnamate may be used as an alternative biomarker
for strain activity. PheP overexpression improves phenylalanine
metabolism in engineered bacteria. Strains analyzed in this data
set are SYN-PKU101, SYN-PKU102, SYN-PKU202, SYN-PKU201, SYN-PKU401,
SYN-PKU402, SYN-PKU203, SYN-PKU302, SYN-PKU303.
[0037] FIGS. 9A and 9B depict the state of one non-limiting
embodiment of the PAL construct under non-inducing (FIG. 9A) and
inducing (FIG. 9B) conditions. FIG. 9A depicts relatively low PAL
and PheP production under aerobic conditions due to oxygen
(O.sub.2) preventing FNR from dimerizing and activating PAL and/or
pheP gene expression. FIG. 9B depicts up-regulated PAL and PheP
production under anaerobic conditions due to FNR dimerizing and
inducing FNR promoter-mediated expression of PAL and pheP (squiggle
above "PAL" and "pheP"). Arrows adjacent to a single rectangle, or
a cluster of rectangles, depict the promoter responsible for
driving transcription (in the direction of the arrow) of such
gene(s). Arrows above each rectangle depict the expression product
of each gene.
[0038] FIG. 10 depicts phenylalanine concentrations in cultures of
synthetic probiotic strains, with and without an additional copy of
pheP inserted on the chromosome. After 1.5 hrs of growth, cultures
were placed in Coy anaerobic chamber supplying 90% N.sub.2, 5%
CO.sub.2, and 5% H.sub.2. After 4 hrs of induction, bacteria were
resuspended in assay buffer containing 4 mM phenylalanine. Aliquots
were removed from cell assays every 30 min for 3 hrs for
phenylalanine quantification by mass spectrometry. Phenylalanine
degradation rates in strains comprising an additional copy of pheP
(SYN-PKU304 and SYN-PKU305; left) were higher than strains lacking
an additional copy of pheP (SYN-PKU308 and SYN-PKU307; right).
[0039] FIG. 11. shows that pheP Overexpression Improves Phe
Degradation. Strains containing different PAL genes and an
additional copy of the gene encoding the pheP transporter were
compared with strains lacking the additional pheP gene. Notably,
the additional pheP copy permitted the complete degradation of 4 mM
Phe in 4 hours in this experiment.
[0040] FIG. 12. Shows that cinnamate production is enhanced in
pheP+ Strains. Cinnamate production is directly correlated with Phe
degradation. pheP+ refers to the Nissle parent strain containing an
additional integrated copy of the pheP gene but lacking a PAL
circuit.
[0041] FIG. 13 depicts diseases associated with branched chain
amino acid degradative pathways.
[0042] FIG. 14 depicts aspects of the branched chain amino acid
degradative pathway
[0043] FIG. 15 depicts aspects of the branched chain amino acid
degradative pathway
[0044] FIG. 16 depicts aspects of the branched chain amino acid
degradative pathway
[0045] FIG. 17 depicts possible components of a branched chain
amino acid synthetic biotic disclosed herein
[0046] FIG. 18 depicts possible components of a branched chain
amino acid synthetic biotic disclosed herein.
[0047] FIG. 19 depicts possible components of a branched chain
amino acid synthetic biotic disclosed herein
[0048] FIG. 20 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, aldehyde dehydrogenase 2 (Adh2) from
Saccharomyces cerevisiae, and leucine dehydrogenase (Ldh) from
Pseudomonas aeruginosa. The genes for the leucine exporter (LeuE)
and ilvC have been deleted. The gene for ilvJ is added which can be
under the control of the native, FNR, or constitutive promoter
Ptac
[0049] FIG. 21 depicts exemplary components of a branched chain
amino acid synthetic biotic disclosed herein for leucine
degradation.
[0050] FIG. 22 depicts exemplary components of a branched chain
amino acid synthetic biotic disclosed herein for leucine
import.
[0051] FIG. 23 depicts one exemplary branched chain amino acid
circuit. Genes shown are low affinity BCAA transporter (BrnQ), the
branched chain a-ketoacid decarboxylase (KivD) from Lactococcus
lactis, aldehyde dehydrogenase from E. Coli K-12 (PadA), and leuDH
derived from Pseudomonas aeruginosa PAO1 or Bacillus cereus. The
genes for the leucine exporter (LeuE) and ilvC have been deleted.
The gene for ilvJ is added.
[0052] FIG. 24 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, aldehyde dehydrogenase from E. Coli K-12
(PadA), and leuDH derived from Pseudomonas aeruginosa PAO1 or
Bacillus cereus. The genes for the leucine exporter (LeuE) and ilvC
have been deleted. The gene for BrnQ is added.
[0053] FIG. 25 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, either aldehyde dehydrogenase from E. Coli K-12
(PadA), alcohol dehydrogenase YqhD from E. Coli, or alcohol
dehydrogenase Adh2 from S. cerevisiae, and L-AAD derived from
Proteus vulgaris or Proteus mirabilis. The genes for the leucine
exporter (LeuE) and ilvC have been deleted. The gene for BrnQ is
added.
[0054] FIG. 26 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, either aldehyde dehydrogenase from E. Coli K-12
(PadA), alcohol dehydrogenase YqhD from E. Coli, or alcohol
dehydrogenase Adh2 from S. cerevisiae, and L-AAD derived from
Proteus vulgaris or Proteus mirabilis. The genes for the leucine
exporter (LeuE) and ilvC have been deleted. The gene for BrnQ is
added. The genes are under the control of the FNR promoter.
[0055] FIG. 27 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, either aldehyde dehydrogenase from E. Coli K-12
(PadA), alcohol dehydrogenase YqhD from E. Coli, or alcohol
dehydrogenase Adh2 from S. cerevisiae, and LeuDh derived from
Pseudomonas aeruginosa PAO1 or Bacillus cereus. The genes for the
leucine exporter (LeuE) and ilvC have been deleted. The gene for
BrnQ is added. The genes are under the control of the FNR
promoter.
[0056] FIG. 28 depicts one exemplary branched chain amino acid
circuit. Genes shown are high affinity leucine transporter complex
(LivKHMGF), the branched chain a-ketoacid decarboxylase (KivD) from
Lactococcus lactis, either aldehyde dehydrogenase from E. Coli K-12
(PadA), alcohol dehydrogenase YqhD from E. Coli, or alcohol
dehydrogenase Adh2 from S. cerevisiae, and BCAA aminotransferase
ilvE. The genes for the leucine exporter (LeuE) and ilvC have been
deleted. The gene for BrnQ is added. The genes are under the
control of the FNR promoter.
[0057] FIG. 29 depicts examples of circuit components for ldh, kivD
and livKHMGF inducible expression in E. coli.
[0058] FIG. 30 depicts leucine levels in the Nissle .DELTA.leuE
deletion strain harboring a high-copy plasmid expressing kivD from
the Tet promoter or further with a copy of the livKHMGF operon
driven by the Tet promoter integrated into the chromosome at the
lacZ locus, which were induced with ATC and incubated in culture
medium supplemented with 2 mM leucine. Samples were removed at 0,
1.5, 6 and 18 h, and leucine concentration was determined by liquid
chromatography tandem mass spectrometry.
[0059] FIG. 31 depicts leucine degradation in the Nissle
.DELTA.leuE deletion strain harboring a high-copy plasmid
expressing the branch-chain keto-acid dehydrogenase (bkd) complex
with or without expression of a leucine dehydrogenase (ldh) from
the Tet promoter or further with a copy of the leucine importer
livKHMGF driven by the Tet promoter integrated into the chromosome
at the lacZ locus, which were induced with ATC and incubated in
culture medium supplemented with 2 mM leucine. Samples were removed
at 0, 1.5, 6 and 18 h, and leucine concentration was determined by
liquid chromatography tandem mass spectrometry.
[0060] FIGS. 32A, 32B, and 32C depict the simultaneous degradation
of leucine (FIG. 32A), isoleucine (FIG. 32B), and valine (FIG. 32C)
by E. coli Nissle and its .DELTA.leuE deletion strain harboring a
high-copy plasmid expressing the keto-acid decarboxylase kivD from
the Tet promoter or further with a copy of the livKHMGF operon
driven by the Tet promoter integrated into the chromosome at the
lacZ locus, which were induced with ATC and incubated in culture
medium supplemented with 2 mM leucine, 2 mM isoleucine and 2 mM
valine. Samples were removed at 0, 1.5, 6 and 18 h, and leucine
concentration was determined by liquid chromatography tandem mass
spectrometry.
[0061] FIG. 33 shows that overexpression of the low-affinity BCAA
transporter BrnQ greatly improves the rate of leucine degradation
in a LeuE and ilvC knockout bacterial strain having either LeuDH
derived from P. aeruginosa or LeuDH derived from Bacillus cereus,
kivD, and padA with and without the BCAA transporter brnQ under the
control of tet promoter as measured by leucine degradation, KIC
production, and isovalerate production.
[0062] FIG. 34 is schematic depicting an exemplary Adenosine
Degradation Circuit. Adenosine is imported into the cell through
expression of the E. coli Nucleoside Permease nupG transporter.
Adenosine is converted to Inosine through expression of Adenine
Deaminase add. Inosine is converted to hypoxyxanthine through
expression of Inosine Phosphorylase, xapA, and deoD. Hypoxanthine
is converted to Xanthine and Urate through expression of
Hypoxanthine Hydroxylase, xdhA, xdhB, xdhC. All of these genes are
optionally expressed from an inducible promoter, e.g., a
FNR-inducible promoter. The bacteria may also include an
auxotrophy, e.g., deletion of thyA (.DELTA.thyA; thymindine
dependence). Non-limiting example of a bacterial strain is
listed.
[0063] FIG. 35. is a schematic depicting an exemplary circuit for
depleting kynurenine.
[0064] FIG. 36. shows the results of an adaptive laboratory
evolution to select a bacterial mutant with enhanced kynurenine
import into the cell. The results of the initial checkerboard assay
are displayed as a function of optical density at 600 nm. The
X-axis shows decreasing KYNU concentration from left-to-right,
while the Z-axis shows decreasing ToxTrp concentration from
front-to-back with the very back row representing media with no
ToxTrp.
[0065] FIG. 37. shows the results of an adaptive laboratory
evolution to select a bacterial mutant with enhanced kynurenine
import into the cell.
[0066] FIG. 38. shows the results of an adaptive laboratory
evolution to select a bacterial mutant with enhanced kynurenine
import into the cell. The control strains SYN094 and trpE are shown
in M9+KYNU without any ToxTrp, as there was no growth detected from
either strain at any concentration of ToxTrp. The results of the
assay show that expression of the pseudoKYNase provides protection
against toxicity of ToxTrp and shows that growth is permitted
between 250-62.5 ug/mL of KYNU and 6.3-1.55 ug/mL of ToxTrp.
[0067] FIG. 39. is a schematic depicting an exemplary circuit for
treating hepatic encephalopathy.
[0068] FIG. 40. is a schematic depicting an exemplary circuit for
depleting bile salts.
DETAILED DESCRIPTION
[0069] The invention includes genetically engineered
microorganisms, e.g., genetically engineered bacteria,
pharmaceutical compositions thereof, and methods of modulating or
treating a disease.
[0070] In order that the disclosure may be more readily understood,
certain terms are first defined. These definitions should be read
in light of the remainder of the disclosure and as understood by a
person of ordinary skill in the art. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by a person of ordinary skill in the art.
Additional definitions are set forth throughout the detailed
description.
[0071] The articles "a" and "an," as used herein, should be
understood to mean "at least one," unless clearly indicated to the
contrary.
[0072] The phrase "and/or," when used between elements in a list,
is intended to mean either (1) that only a single listed element is
present, or (2) that more than one element of the list is present.
For example, "A, B, and/or C" indicates that the selection may be A
alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C.
The phrase "and/or" may be used interchangeably with "at least one
of" or "one or more of" the elements in a list.
[0073] As used herein, the term "amino acid" refers to a class of
organic compounds that contain at least one amino group and one
carboxyl group Amino acids include leucine, isoleucine, valine,
arginine, lysine, asparagine, serine, glycine, glutamine,
tryptophan, methionine, threonine, cysteine, tyrosine,
phenylalanine, glutamic acid, aspartic acid, alanine, histidine,
and proline.
[0074] As used herein, the term "auxotroph" or "auxotrophic" refers
to an organism that requires a specific factor, e.g., an amino
acid, a sugar, or other nutrient, to support its growth. An
"auxotrophic modification" is a genetic modification that causes
the organism to die in the absence of an exogenously added nutrient
essential for survival or growth because it is unable to produce
said nutrient. As used herein, the term "essential gene" refers to
a gene which is necessary to for cell growth and/or survival.
Essential genes are described in more detail infra and include, but
are not limited to, DNA synthesis genes (such as thyA), cell wall
synthesis genes (such as dapA), and amino acid genes (such as serA
and metA).
[0075] "Cancer" or "cancerous" is used to refer to a physiological
condition that is characterized by unregulated cell growth. In some
embodiments, cancer refers to a tumor. "Tumor" is used to refer to
any neoplastic cell growth or proliferation or any pre-cancerous or
cancerous cell or tissue. A tumor may be malignant or benign. Types
of cancer include, but are not limited to, adrenal cancer,
adrenocortical carcinoma, anal cancer, appendix cancer, bile duct
cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma tumors,
osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g.,
astrocytomas, brain stem glioma, craniopharyngioma, ependymoma),
bronchial tumors, central nervous system tumors, breast cancer,
Castleman disease, cervical cancer, colon cancer, rectal cancer,
colorectal cancer, endometrial cancer, esophageal cancer, eye
cancer, gallbladder cancer, gastrointestinal cancer,
gastrointestinal carcinoid tumors, gastrointestinal stromal tumors,
gestational trophoblastic disease, heart cancer, Kaposi sarcoma,
kidney cancer, largyngeal cancer, hypopharyngeal cancer, leukemia
(e.g., acute lymphoblastic leukemia, acute myeloid leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia), liver
cancer, lung cancer, lymphoma (e.g., AIDS-related lymphoma, Burkitt
lymphoma, cutaneous T cell lymphoma, Hodgkin lymphoma, Non-Hodgkin
lymphoma, primary central nervous system lymphoma), malignant
mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal
cavity cancer, paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oral cavity cancer, oropharyngeal cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer,
pituitary tumors, prostate cancer, retinoblastoma,
rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma,
skin cancer (e.g., basal cell carcinoma, melanoma), small intestine
cancer, stomach cancer, teratoid tumor, testicular cancer, throat
cancer, thymus cancer, thyroid cancer, unusual childhood cancers,
urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer,
vulvar cancer, Waldenstrom macrogloblulinemia, and Wilms tumor.
Side effects of cancer treatment may include, but are not limited
to, opportunistic autoimmune disorder(s), systemic toxicity,
anemia, loss of appetite, irritation of bladder lining, bleeding
and bruising (thrombocytopenia), changes in taste or smell,
constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair
loss (alopecia), infection, infertility, lymphedema, mouth sores,
nausea, pain, peripheral neuropathy, tooth decay, urinary tract
infections, and/or problems with memory and concentration (National
Cancer Institute).
[0076] As used herein, the term "coding region" refers to a
nucleotide sequence that codes for a specific amino acid sequence.
The term "regulatory sequence" refers to a nucleotide sequence
located upstream (5' non-coding sequences), within, or downstream
(3' non-coding sequences) of a coding sequence, and which
influences the transcription, RNA processing, RNA stability, or
translation of the associated coding sequence. Examples of
regulatory sequences include, but are not limited to, promoters,
translation leader sequences, effector binding sites, and stem-loop
structures. In one embodiment, the regulatory sequence comprises a
promoter, e.g., an FNR responsive promoter.
[0077] As used herein the term "codon-optimized" refers to the
modification of codons in a gene or a coding region of a nucleic
acid molecule to improve translation in a host cell or organism of
a transcript RNA molecule transcribed from the coding sequence, or
to improve transcription of a coding sequence. Codon optimization
includes, but is not limited to, processes including selecting
codons for the coding sequence to suit the codon preference of the
expression host organism. Such optimization includes replacing at
least one, or more than one, or a significant number, of codons
with one or more codons that are more frequently used in the genes
of the host organism.
[0078] Many organisms display a bias or preference for use of
particular codons to code for insertion of a particular amino acid
in a growing polypeptide chain. Codon preference or codon bias,
differences in codon usage between organisms, is allowed by the
degeneracy of the genetic code, and is well documented among many
organisms. Codon bias often correlates with the efficiency of
translation of messenger RNA (mRNA), which is in turn believed to
be dependent on, inter alia, the properties of the codons being
translated and the availability of particular transfer RNA (tRNA)
molecules. The predominance of selected tRNAs in a cell is
generally a reflection of the codons used most frequently in
peptide synthesis. Accordingly, genes can be tailored for optimal
gene expression in a given organism based on codon
optimization.
[0079] "Constitutive promoter" refers to a promoter that is capable
of facilitating continuous transcription of a coding sequence or
gene under its control and/or to which it is operably linked.
Constitutive promoters and variants are well known in the art and
include, but are not limited to, BBa_J23100, a constitutive
Escherichia coli .sigma..sup.s promoter (e.g., an osmY promoter
(International Genetically Engineered Machine (iGEM) Registry of
Standard Biological Parts Name BBa_J45992; BBa_J45993)), a
constitutive Escherichia coli .sigma..sup.32 promoter (e.g., htpG
heat shock promoter (BBa_J45504)), a constitutive Escherichia coli
.sigma..sup.70 promoter (e.g., lacq promoter (BBa_J54200;
BBa_J56015), E. coli CreABCD phosphate sensing operon promoter
(BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter
(BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101);
M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter
(BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V
promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07
gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a
constitutive Bacillus subtilis .sigma..sup.A promoter (e.g.,
promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P.sub.liaG
(BBa_K823000), P.sub.iepA (BBa_K823002), P.sub.veg (BBa_K823003)),
a constitutive Bacillus subtilis .sigma..sup.B promoter (e.g.,
promoter ctc (BBa_K143010), promoter gsiB (BBa_K143011)), a
Salmonella promoter (e.g., Pspv2 from Salmonella (BBa_K112706),
Pspv from Salmonella (BBa_K112707)), a bacteriophage T7 promoter
(e.g., T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814;
BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085;
BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252;
BBa_Z0253)), and a bacteriophage SP6 promoter (e.g., SP6 promoter
(BBa_J64998)).
[0080] The term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of
an active ingredient. Examples include, but are not limited to,
calcium bicarbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils,
polyethylene glycols, and surfactants, including, for example,
polysorbate 20.
[0081] "Exogenous environmental condition(s)" refer to setting(s)
or circumstance(s) under which the promoter described herein is
induced. The phrase "exogenous environmental conditions" is meant
to refer to the environmental conditions external to the intact
(unlysed) recombinant micororganism, but endogenous or native to
the host subject environment. Thus, "exogenous" and "endogenous"
may be used interchangeably to refer to environmental conditions in
which the environmental conditions are endogenous to a mammalian
body, but external or exogenous to an intact microorganism cell. In
some embodiments, the exogenous environmental conditions are
low-oxygen, microaerobic, or anaerobic conditions, such as hypoxic
and/or necrotic tissues. In some embodiments, the exogenous
environmental condition is a low-pH environment. In some
embodiments, the recombinant bacterial cell of the disclosure
comprise a pH-dependent promoter. In some embodiments, the
recombinant bacterial cell of the diclosure comprise an oxygen
level-dependent promoter. In some aspects, bacteria have evolved
transcription factors that are capable of sensing oxygen levels
(i.e., oxygen-level dependent transcription factors). Different
signaling pathways may be triggered by different oxygen levels and
occur with different kinetics. An "oxygen level-dependent promoter"
or "oxygen level-dependent regulatory region" refers to a nucleic
acid sequence to which one or more oxygen level-sensing
transcription factors is capable of binding, wherein the binding
and/or activation of the corresponding transcription factor
activates downstream gene expression.
[0082] Examples of oxygen level-dependent transcription factors
include, but are not limited to, FNR (fumarate and nitrate
reductase)-responsive promoters, ANR (anaerobic nitrate
respiration)-responsive promoters, and DNR (dissimilatory nitrate
respiration regulator)-responsive promoters. Multiple
FNR-responsive promoters, ANR-responsive promoters, and
DNR-responsive promoters which can be used in the present invention
are known in the art (see, e.g., Castiglione et al. (2009)
Microbiology 155(Pt. 9): 2838-44; Eiglmeier et al. (1989) Mol.
Microbiol. 3(7): 869-78; Galimand et al. (1991) J. Bacteriol.
173(5): 1598-1606; Hasegawa et al. (1998) FEMS Microbiol. Lett.
166(2): 213-217; Hoeren et al. (1993) Eur. J. Biochem. 218(1):
49-57; Salmon et al. (2003) J. Biol. Chem. 278(32): 29837-55), and
non-limiting examples are shown in Table 1.
TABLE-US-00001 TABLE 1 Examples of transcription factors and
responsive genes and regulatory regions Transcription Examples of
responsive genes, Factor promoters, and/or regulatory regions: FNR
nirB, ydfZ, pdhR, focA, ndH, hlyE, narK, narX, narG, yfiD, tdcD ANR
arcDABC DNR norb, norC
[0083] In a non-limiting example, a promoter (PfnrS) from the E.
coli Nissle fumarate and nitrate reductase gene S (fnrS) that is
known to be highly expressed under conditions of low or no
environmental oxygen can be used in the present invention (Durand
and Storz, 2010; Boysen et al, 2010). The PfnrS promoter is
activated under anaerobic conditions by the global transcriptional
regulator FNR that is naturally found in E. coli Nissle. Under
anaerobic conditions, FNR forms a dimer and binds to specific
sequences in the promoters of genes under its control, thereby
activating their expression. However, under aerobic conditions,
oxygen reacts with iron-sulfur clusters in FNR dimers and converts
them to an inactive form. In this way, the PfnrS inducible promoter
is adopted to modulate the expression of proteins or RNA. PfnrS is
used interchangeably in this application as "FNRS", "fnrs", "FNR",
"P-FNRS" promoter and other such related designations to indicate
the promoter PfnrS.
[0084] As used herein, the term "expression" refers to the
transcription and stable accumulation of sense (mRNA) or anti-sense
RNA derived from a nucleic acid, and/or to translation of an mRNA
into a polypeptide.
[0085] As used herein, the term "gene" refers to a nucleic acid
fragment that encodes a protein or a fragment thereof, optionally
including regulatory sequences preceding (5' non-coding sequences)
and following (3' non-coding sequences) the coding sequence. In one
embodiment, a "gene" does not include regulatory sequences
preceding and following the coding sequence. A "native gene" refers
to a gene as found in nature, optionally with its own regulatory
sequences preceding and following the coding sequence. A "chimeric
gene" refers to any gene that is not a native gene, optionally
comprising regulatory sequences preceding and following the coding
sequence, wherein the coding sequence and/or the regulatory
sequence, in whole or in part, are not found together in nature.
Thus, a chimeric gene may comprise a regulatory sequence and a
coding sequence, each derived from different sources, or a
regulatory and a coding sequence each derived from the same source,
but arranged differently than they are found in nature.
[0086] The term "genetic modification," as used herein, refers to
any genetic change. Exemplary genetic modifications include those
that increase, decrease, or abolish the expression of a gene,
including, for example, modifications of native chromosomal or
extrachromosomal genetic material. Exemplary genetic modifications
also include the introduction of at least one plasmid,
modification, mutation, base deletion, base addition, and/or codon
modification of chromosomal or extrachromosomal genetic
sequence(s), gene over-expression, gene amplification, gene
suppression, promoter modification or substitution, gene addition
(either single or multi-copy), antisense expression or suppression,
or any other change to the genetic elements of a host cell, whether
the change produces a change in phenotype or not. Genetic
modification can include the introduction of a plasmid, e.g., a
plasmid comprising at least one substrate transporter operably
linked to a promoter, into a bacterial cell. Genetic modification
can also involve a targeted replacement in the chromosome, e.g., to
replace a native gene promoter with an inducible promoter,
regulated promoter, strong promoter, weak promoter, or constitutive
promoter. Genetic modification can also involve gene amplification,
e.g., introduction of at least one additional copy of a native gene
into the chromosome of the cell. Alternatively, chromosomal genetic
modification can involve a genetic mutation.
[0087] As used herein, the term "genetic mutation" refers to a
change or multiple changes in a nucleotide sequence of a gene or
related regulatory region that alters the nucleotide sequence as
compared to its native or wild-type sequence. Mutations include,
for example, substitutions, insertions, and deletions, in whole or
in part, within the wild-type sequence. Such substitutions,
insertions, or deletions can be single nucleotide changes (e.g.,
one or more point mutations), or can be two or more nucleotide
changes, which may result in substantial changes to the sequence.
Mutations can occur within the coding region of the gene as well as
within the non-coding and regulatory sequence of the gene. The term
"genetic mutation" is intended to include silent and conservative
mutations within a coding region as well as changes which alter the
amino acid sequence of the polypeptide encoded by the gene. A
genetic mutation in a gene coding sequence may, for example,
increase, decrease, or otherwise alter the activity (e.g., import
activity) of the polypeptide product encoded by the gene. A genetic
mutation in a regulatory sequence may increase, decrease, or
otherwise alter the expression of sequences operably linked to the
altered regulatory sequence.
[0088] It is routine for one of ordinary skill in the art to make
mutations in a gene of interest. Mutations include substitutions,
insertions, deletions, and/or truncations of one or more specific
amino acid residues or of one or more specific nucleotides or
codons in the polypeptide or polynucleotide of interest.
Mutagenesis and directed evolution methods are well known in the
art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; U.S.
Pat. No. 6,586,182; U.S. Pat. No. 6,117,679; and Ling, et al.,
1999, "Approaches to DNA mutagenesis: an overview," Anal. Biochem.,
254(2):157-78; Smith, 1985, "In vitro mutagenesis," Ann. Rev.
Genet., 19:423-462; Carter, 1986, "Site-directed mutagenesis,"
Biochem. J., 237:1-7; and Minshull, et al., 1999, "Protein
evolution by molecular breeding," Current Opinion in Chemical
Biology, 3:284-290. For example, the lambda red system can be used
to knock-out genes in E. coli (see, e.g., Datta et al., Gene,
379:109-115 (2006)).
[0089] "Gut" refers to the organs, glands, tracts, and systems that
are responsible for the transfer and digestion of food, absorption
of nutrients, and excretion of waste. In humans, the gut comprises
the gastrointestinal (GI) tract, which starts at the mouth and ends
at the anus, and additionally comprises the esophagus, stomach,
small intestine, and large intestine. The gut also comprises
accessory organs and glands, such as the spleen, liver,
gallbladder, and pancreas. The upper gastrointestinal tract
comprises the esophagus, stomach, and duodenum of the small
intestine. The lower gastrointestinal tract comprises the remainder
of the small intestine, i.e., the jejunum and ileum, and all of the
large intestine, i.e., the cecum, colon, rectum, and anal canal.
Bacteria can be found throughout the gut, e.g., in the
gastrointestinal tract, and particularly in the intestines.
[0090] As used herein, "heterologous" as used in the context of a
nucleic acid or polypeptide sequence, "heterologous gene", or
"heterologous sequence", refers to a nucleotide or polypeptide
sequence that is not normally found in a given cell in nature. As
used herein, a heterologous sequence encompasses a nucleic acid
sequence that is exogenously introduced into a given cell.
"Heterologous gene" includes a native gene, or fragment thereof,
that has been introduced into the host cell in a form that is
different from the corresponding native gene. For example, a
heterologous gene may include a native coding sequence that is a
portion of a chimeric gene to include a native coding sequence that
is a portion of a chimeric gene to include non-native regulatory
regions that is reintroduced into the host cell. A heterologous
gene may also include a native gene, or fragment thereof,
introduced into a non-native host cell. Thus, a heterologous gene
may be foreign or native to the recipient cell; a nucleic acid
sequence that is naturally found in a given cell but expresses an
unnatural amount of the nucleic acid and/or the polypeptide which
it encodes; and/or two or more nucleic acid sequences that are not
found in the same relationship to each other in nature. As used
herein, the term "endogenous gene" refers to a native gene in its
natural location in the genome of an organism. As used herein, the
term "transgene" refers to a gene that has been introduced into the
host organism, e.g., host bacterial cell, genome.
[0091] The term "inactivated" as applied to a gene refers to any
genetic modification that decreases or eliminates the expression of
the gene and/or the functional activity of the corresponding gene
product (mRNA and/or protein). The term "inactivated" encompasses
complete or partial inactivation, suppression, deletion,
interruption, blockage, promoter alterations, antisense RNA, dsRNA,
or down-regulation of a gene. This can be accomplished, for
example, by gene "knockout," inactivation, mutation (e.g.,
insertion, deletion, point, or frameshift mutations that disrupt
the expression or activity of the gene product), or by use of
inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A
deletion may encompass all or part of a gene's coding sequence. The
term "knockout" refers to the deletion of most (at least about 95%,
at least about 96%, at least about 97%, at least about 98%, or at
least about 99%) or all (100%) of the coding sequence of a gene. In
some embodiments, any number of nucleotides can be deleted, from a
single base to an entire piece of a chromosome.
[0092] An "inducible promoter" refers to a regulatory nucleic acid
region that is operably linked to one or more genes, wherein
transcription of the gene(s) is increased in response to a stimulus
(e.g., an inducer) or an exogenous environmental condition. A
"directly inducible promoter" refers to a regulatory region,
wherein the regulatory region is operably linked to a gene encoding
a protein or polypeptide, where, in the presence of an inducer of
said regulatory region, the protein or polypeptide is expressed. An
"indirectly inducible promoter" refers to a regulatory system
comprising two or more regulatory regions, for example, a first
regulatory region that is operably linked to a first gene encoding
a first protein, polypeptide, or factor, e.g., a transcriptional
regulator, which is capable of regulating a second regulatory
region that is operably linked to a second gene, the second
regulatory region may be activated or repressed, thereby activating
or repressing expression of the second gene. Both a directly
inducible promoter and an indirectly inducible promoter are
encompassed by "inducible promoter." Examples of inducible
promoters include, but are not limited to, an FNR promoter, a
P.sub.araC promoter, a P.sub.araBAD promoter, a propionate
promoter, and a P.sub.TctR promoter, each of which are described in
more detail herein. Examples of other inducible promoters are
provided herein below.
[0093] An "isolated" polypeptide, or a fragment, variant, or
derivative thereof, refers to a polypeptide that is not in its
natural milieu. No particular level of purification is required.
Recombinantly-produced polypeptides and proteins expressed in host
cells, including but not limited to bacterial or mammalian cells,
are considered isolated for purposed of the invention, as are
native or recombinant polypeptides which have been separated,
fractionated, or partially or substantially purified by any
suitable technique. Recombinant peptides, polypeptides or proteins
refer to peptides, polypeptides or proteins produced by recombinant
DNA techniques, i.e. produced from cells, microbial or mammalian,
transformed by an exogenous recombinant DNA expression construct
encoding the polypeptide. Proteins or peptides expressed in most
bacterial cultures will typically be free of glycan. Fragments,
derivatives, analogs or variants of the foregoing polypeptides, and
any combination thereof are also included as polypeptides. The
terms "fragment," "variant," "derivative" and "analog" include
polypeptides having an amino acid sequence sufficiently similar to
the amino acid sequence of the original peptide and include any
polypeptides, which retain at least one or more properties of the
corresponding original polypeptide. Fragments of polypeptides of
the present invention include proteolytic fragments, as well as
deletion fragments. Fragments also include specific antibody or
bioactive framents or immunologically active fragments derived from
any polypeptides described herein. Variants may occur naturally or
be non-naturally occurring. Non-naturally occurring variants may be
produced using mutagenesis methods known in the art. Variant
polypeptides may comprise conservative or non-conservative amino
acid substitutions, deletions or additions.
[0094] As used herein the term "linker", "linker peptide" or
"peptide linkers" or "linker" refers to synthetic or non-native or
non-naturally-occurring amino acid sequences that connect or link
two polypeptide sequences, e.g., that link two polypeptide domains.
As used herein the term "synthetic" refers to amino acid sequences
that are not naturally occurring. Exemplary linkers are described
herein. Additional exemplary linkers are provided in U.S. Pat. No.
2014/0079701, the contents of which are herein incorporated by
reference in its entirety.
[0095] As used herein, the terms "modulate" and "treat" and their
cognates refer to an amelioration of a disease or condition, or at
least one discernible symptom thereof. In another embodiment,
"modulate" and "treat" refer to an amelioration of at least one
measurable physical parameter, not necessarily discernible by the
patient. In another embodiment, "modulate" and "treat" refer to
inhibiting the progression of a disease or condition, either
physically (e.g., stabilization of a discernible symptom),
physiologically (e.g., stabilization of a physical parameter), or
both. In another embodiment, "modulate" and "treat" refer to
slowing the progression or reversing the progression of a disease
or condition. As used herein, "prevent" and its cognates refer to
delaying the onset or reducing the risk of acquiring a given
disease or condition.
[0096] As used herein, a "non-native" nucleic acid sequence refers
to a nucleic acid sequence not normally present in a microorganism,
e.g., an extra copy of an endogenous sequence, or a heterologous
sequence such as a sequence from a different organism (e.g., an
organism from a different species, strain, or substrain of a
prokaryote or eukaryote), or a sequence that is modified and/or
mutated as compared to the unmodified native or wild-type sequence.
In some embodiments, the non-native nucleic acid sequence is a
synthetic, non-naturally occurring sequence (see, e.g., Purcell et
al., 2013). The non-native nucleic acid sequence may be a
regulatory region, a promoter, a gene, and/or one or more genes
(e.g., genes in a gene cassette or operon). In some embodiments,
"non-native" refers to two or more nucleic acid sequences that are
not found in the same relationship to each other in nature. The
non-native nucleic acid sequence may be present on a plasmid or
chromosome. In some embodiments, the genetically engineered
bacteria of the disclosure comprise a gene that is operably linked
to a directly or indirectly inducible promoter that is not
associated with said gene in nature, e.g., an FNR-responsive
promoter (or other promoter described herein) operably linked to a
gene encoding a substrate transporter.
[0097] "Microorganism" refers to an organism or microbe of
microscopic, submicroscopic, or ultramicroscopic size that
typically consists of a single cell. Examples of microrganisms
include bacteria, viruses, parasites, fungi, certain algae, and
protozoa. In some aspects, the microorganism is engineered
("engineered microorganism") to produce one or more anti-cancer
molecules. In certain embodiments, the engineered microorganism is
an engineered bacteria. In certain embodiments, the engineered
microorganism is an engineered oncolytic virus.
[0098] "Non-pathogenic bacteria" refer to bacteria that are not
capable of causing disease or harmful responses in a host. In some
embodiments, non-pathogenic bacteria are commensal bacteria.
Examples of non-pathogenic bacteria include, but are not limited to
Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium,
Enterococcus, Escherichia coli, Lactobacillus, Lactococcus,
Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans,
Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis,
Bacteroides thetaiotaomicron, Bifidobacterium bifidum,
Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium
longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus
acidophilus, Lactobacillus bulgaricus, Lactobacillus casei,
Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, and
Lactococcus lactis (Sonnenborn et al., 2009; Dinleyici et al.,
2014; U.S. Pat. No. 6,835,376; U.S. Pat. No. 6,203,797; U.S. Pat.
No. 5,589,168; U.S. Pat. No. 7,731,976). Naturally pathogenic
bacteria may be genetically engineered to provide reduce or
eliminate pathogenicity.
[0099] "Operably linked" refers to the association of nucleic acid
sequences on a single nucleic acid fragment so that the function of
one is affected by the other. A regulatory element is operably
linked with a coding sequence when it is capable of affecting the
expression of the gene coding sequence, regardless of the distance
between the regulatory element and the coding sequence. More
specifically, operably linked refers to a nucleic acid sequence
that is joined to a regulatory sequence in a manner which allows
expression of the nucleic acid sequence. In other words, the
regulatory sequence acts in cis. In one embodiment, a gene may be
"directly linked" to a regulatory sequence in a manner which allows
expression of the gene. In another embodiment, a gene may be
"indirectly linked" to a regulatory sequence in a manner which
allows expression of the gene. In one embodiment, two or more genes
may be directly or indirectly linked to a regulatory sequence in a
manner which allows expression of the two or more genes.
[0100] As used herein, "payload" refers to one or more molecules of
interest to be produced by a genetically engineered microorganism,
such as a bacteria. In some embodiments, the payload is a
therapeutic payload. In some embodiments, the payload is a
regulatory molecule, e.g., a transcriptional regulator such as FNR.
In some embodiments, the payload comprises a regulatory element,
such as a promoter or a repressor. In some embodiments, the payload
comprises an inducible promoter, such as from FNRS. In some
embodiments the payload comprises a repressor element, such as a
kill switch. In some embodiments, the payload is encoded by a gene
or multiple genes or an operon. In alternate embodiments, the
payload is produced by a biosynthetic or biochemical pathway,
wherein the biosynthetic or biochemical pathway may optionally be
endogenous to the microorganism. In some embodiments, the
genetically engineered microorganism comprises two or more
payloads.
[0101] As used herein a "pharmaceutical composition" refers to a
preparation of bacterial cells disclosed herein with other
components such as a physiologically suitable carrier and/or
excipient.
[0102] The phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be used
interchangeably refer to a carrier or a diluent that does not cause
significant irritation to an organism and does not abrogate the
biological activity and properties of the administered bacterial
compound. An adjuvant is included under these phrases.
[0103] As used herein, the term "plasmid" or "vector" refers to an
extrachromosomal nucleic acid, e.g., DNA, construct that is not
integrated into a bacterial cell's genome. Plasmids are usually
circular and capable of autonomous replication. Plasmids may be
low-copy, medium-copy, or high-copy, as is well known in the art.
Plasmids may optionally comprise a selectable marker, such as an
antibiotic resistance gene, which helps select for bacterial cells
containing the plasmid and which ensures that the plasmid is
retained in the bacterial cell. A plasmid disclosed herein may
comprise a nucleic acid sequence encoding a heterologous gene,
e.g., a gene encoding at least one substrate transporter.
[0104] As used herein, the term "polypeptide" includes
"polypeptide" as well as "polypeptides," and refers to a molecule
composed of amino acid monomers linearly linked by amide bonds
(i.e., peptide bonds). The term "polypeptide" refers to any chain
or chains of two or more amino acids, and does not refer to a
specific length. Thus, "peptides," "dipeptides," "tripeptides,
"oligopeptides," "protein," "amino acid chain," or any other term
used to refer to a chain or chains of two or more amino acids, are
included within the definition of "polypeptide," and the term
"polypeptide" may be used instead of, or interchangeably with, any
of these terms. The term "polypeptide" is also intended to refer to
the products of post-expression modifications of the polypeptide,
including but not limited to glycosylation, acetylation,
phosphorylation, amidation, derivatization, proteolytic cleavage,
or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology. In other embodiments, the
polypeptide is produced by the genetically engineered bacteria of
the current invention. In some embodiments, a polypeptide of the
invention may be of a size of about 3 or more, 5 or more, 10 or
more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more,
200 or more, 500 or more, 1,000 or more, or 2,000 or more amino
acids. Polypeptides may have a defined three-dimensional structure,
although they must not necessarily have such structure.
Polypeptides with a defined three-dimensional structure are
referred to as folded, and polypeptides, which do not possess a
defined three-dimensional structure, but rather can adopt a large
number of different conformations, are referred to herein as
unfolded.
[0105] Polypeptides also include fusion proteins. As used herein,
the term "variant" includes a fusion protein, which comprises a
sequence of the original peptide or sufficiently similar to the
original peptide. As used herein, the term "fusion protein" refers
to a chimeric protein comprising amino acid sequences of two or
more different proteins. Typically, fusion proteins result from
well known in vitro recombination techniques. Fusion proteins may
have a similar structural function (but not necessarily to the same
extent), and/or similar regulatory function (but not necessarily to
the same extent), and/or similar biochemical function (but not
necessarily to the same extent) and/or immunological activity (but
not necessarily to the same extent) as the individual original
proteins which are the components of the fusion proteins.
"Derivatives" include but are not limited to peptides, which
contain one or more naturally occurring amino acid derivatives of
the twenty standard amino acids. "Similarity" between two peptides
is determined by comparing the amino acid sequence of one peptide
to the sequence of a second peptide. An amino acid of one peptide
is similar to the corresponding amino acid of a second peptide if
it is identical or a conservative amino acid substitution.
Conservative substitutions include those described in Dayhoff, M.
O., ed., The Atlas of Protein Sequence and Structure 5, National
Biomedical Research Foundation, Washington, D.C. (1978), and in
Argos, EMBO J. 8 (1989), 779-785. For example, amino acids
belonging to one of the following groups represent conservative
changes or substitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys,
Ser, Tyr, Thr; -Val, lie, Leu, Met, Ala, Phe; -Lys, Arg, His; -Phe,
Tyr, Trp, His; and -Asp, Glu.
[0106] "Probiotic" is used to refer to live, non-pathogenic
microorganisms, e.g., bacteria, which can confer health benefits to
a host organism that contains an appropriate amount of the
microorganism. In some embodiments, the host organism is a mammal.
In some embodiments, the host organism is a human. Some species,
strains, and/or subtypes of non-pathogenic bacteria are currently
recognized as probiotic bacteria. Examples of probiotic bacteria
include, but are not limited to, Bifidobacteria, Escherichia coli,
and Lactobacillus, e.g., Bifidobacterium bifidum, Enterococcus
faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus,
Lactobacillus bulgaricus, Lactobacillus paracasei, and
Lactobacillus plantarum (Dinleyici et al., 2014; U.S. Pat. No.
5,589,168; U.S. Pat. No. 6,203,797; U.S. Pat. 6,835,376). The
probiotic may be a variant or a mutant strain of bacterium (Arthur
et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012;
Nougayrede et al., 2006). Non-pathogenic bacteria may be
genetically engineered to enhance or improve desired biological
properties, e.g., survivability. Non-pathogenic bacteria may be
genetically engineered to provide probiotic properties. Probiotic
bacteria may be genetically engineered to enhance or improve
probiotic properties.
[0107] A "promoter" as used herein, refers to a nucleotide sequence
that is capable of controlling the expression of a coding sequence
or gene. Promoters are generally located 5' of the sequence that
they regulate. Promoters may be derived in their entirety from a
native gene, or be composed of different elements derived from
promoters found in nature, and/or comprise synthetic nucleotide
segments. Those skilled in the art will readily ascertain that
different promoters may regulate expression of a coding sequence or
gene in response to a particular stimulus, e.g., in a cell- or
tissue-specific manner, in response to different environmental or
physiological conditions, or in response to specific compounds.
Prokaryotic promoters are typically classified into two classes:
inducible and constitutive.
[0108] As used herein, the term "recombinant bacterial cell",
"recombinant bacteria" or "genetically modified bacteria" refers to
a bacterial cell or bacteria that have been genetically modified
from their native state. For instance, a recombinant bacterial cell
may have nucleotide insertions, nucleotide deletions, nucleotide
rearrangements, and nucleotide modifications introduced into their
DNA. These genetic modifications may be present in the chromosome
of the bacteria or bacterial cell, or on a plasmid in the bacteria
or bacterial cell. Recombinant bacterial cells of the disclosure
may comprise exogenous nucleotide sequences on plasmids.
Alternatively, recombinant bacterial cells may comprise exogenous
nucleotide sequences stably incorporated into their chromosome.
[0109] As used herein, "stably maintained" or "stable" bacterium is
used to refer to a bacterial host cell carrying non-native genetic
material, e.g., an amino acid catabolism enzyme, that is
incorporated into the host genome or propagated on a
self-replicating extra-chromosomal plasmid, such that the
non-native genetic material is retained, expressed, and propagated.
The stable bacterium is capable of survival and/or growth in vitro,
e.g., in medium, and/or in vivo, e.g., in the gut. For example, the
stable bacterium may be a genetically engineered bacterium
comprising an substrate transporter gene, in which the plasmid or
chromosome carrying the substrate transporter gene is stably
maintained in the bacterium, such that the substrate transporter
can be expressed in the bacterium, and the bacterium is capable of
survival and/or growth in vitro and/or in vivo. In some
embodiments, copy number affects the stability of expression of the
non-native genetic material. In some embodiments, copy number
affects the level of expression of the non-native genetic
material.
[0110] As used herein, the term "sufficiently similar" means a
first amino acid sequence that contains a sufficient or minimum
number of identical or equivalent amino acid residues relative to a
second amino acid sequence such that the first and second amino
acid sequences have a common structural domain and/or common
functional activity. For example, amino acid sequences that
comprise a common structural domain that is at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 91%, at
least about 9:2%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99%, or at least about 100%, identical are
defined herein as sufficiently similar. Preferably, variants will
be sufficiently similar to the amino acid sequence of the peptides
of the invention. Such variants generally retain the functional
activity of the peptides of the present invention. Variants include
peptides that differ in amino acid sequence from the native and wt
peptide, respectively, by way of one or more amino acid
deletion(s), addition(s), and/or substitution(s). These may be
naturally occurring variants as well as artificially designed
ones.
[0111] The terms "therapeutically effective dose" and
"therapeutically effective amount" are used to refer to an amount
of a compound that results in prevention, delay of onset of
symptoms, or amelioration of symptoms of a condition or disease. A
therapeutically effective amount, as well as a therapeutically
effective frequency of administration, can be determined by methods
known in the art and discussed below.
[0112] As used herein, the term "transform" or "transformation"
refers to the transfer of a nucleic acid fragment into a host
bacterial cell, resulting in genetically-stable inheritance. Host
bacterial cells comprising the transformed nucleic acid fragment
are referred to as "recombinant" or "transgenic" or "transformed"
organisms.
[0113] As used herein, the term "toxin" refers to a protein,
enzyme, or polypeptide fragment thereof, or other molecule which is
capable of arresting, retarding, or inhibiting the growth,
division, multiplication or replication of the recombinant
bacterial cell of the disclosure, or which is capable of killing
the recombinant bacterial cell of the disclosure. The term "toxin"
is intended to include bacteriostatic proteins and bactericidal
proteins. The term "toxin" is intended to include, but not limited
to, lytic proteins, bacteriocins (e.g., microcins and colicins),
gyrase inhibitors, polymerase inhibitors, transcription inhibitors,
translation inhibitors, DNases, and RNases. The term "anti-toxin"
or "antitoxin," as used herein, refers to a protein or enzyme which
is capable of inhibiting the activity of a toxin. The term
anti-toxin is intended to include, but not limited to, immunity
modulators, and inhibitors of toxin expression. Examples of toxins
and antitoxins are known in the art and described in more detail
infra.
[0114] As used herein, the term "treat" and its cognates refer to
an amelioration of a disease, or at least one discernible symptom
thereof. In another embodiment, "treat" refers to an amelioration
of at least one measurable physical parameter, not necessarily
discernible by the patient. In another embodiment, "treat" refers
to inhibiting the progression of a disease, either physically
(e.g., stabilization of a discernible symptom), physiologically
(e.g., stabilization of a physical parameter), or both. In another
embodiment, "treat" refers to slowing the progression or reversing
the progression of a disease. As used herein, "prevent" and its
cognates refer to delaying the onset or reducing the risk of
acquiring a given disease.
[0115] Those in need of treatment may include individuals already
having a particular medical disease, as well as those at risk of
having, or who may ultimately acquire the disease. The need for
treatment is assessed, for example, by the presence of one or more
risk factors associated with the development of a disease, the
presence or progression of a disease, or likely receptiveness to
treatment of a subject having the disease. Disorders associated
with or involved with amino acid metabolism, e.g., cancer, may be
caused by inborn genetic mutations for which there are no known
cures. Diseases can also be secondary to other conditions, e.g., an
intestinal disorder or a bacterial infection. Treating diseases
associated with amino acid metabolism may encompass reducing normal
levels of one or more substrates, e.g., an amino acid, reducing
excess levels of one or more substrates, e.g., an amino acid, or
eliminating one or more substrates, e.g., an amino acid, and does
not necessarily encompass the elimination of the underlying
disease.
[0116] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50.
[0117] Bacterial Strains
[0118] The disclosure provides a bacterial cell that comprises a
heterologous gene encoding a substrate transporter. In some
embodiments, the bacterial cell is a non-pathogenic bacterial cell.
In some embodiments, the bacterial cell is a commensal bacterial
cell. In some embodiments, the bacterial cell is a probiotic
bacterial cell.
[0119] In certain embodiments, the bacterial cell is selected from
the group consisting of a Bacteroides fragilis, Bacteroides
thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis,
Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium
lactis, Clostridium butyricum, Clostridium scindens, Escherichia
coli, Lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter
formigenes bacterial cell. In one embodiment, the bacterial cell is
a Bacteroides fragilis bacterial cell. In one embodiment, the
bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In
one embodiment, the bacterial cell is a Bacteroides subtilis
bacterial cell. In one embodiment, the bacterial cell is a
Bifidobacterium animalis bacterial cell. In one embodiment, the
bacterial cell is a Bifidobacterium bifidum bacterial cell. In one
embodiment, the bacterial cell is a Bifidobacterium infantis
bacterial cell. In one embodiment, the bacterial cell is a
Bifidobacterium lactis bacterial cell. In one embodiment, the
bacterial cell is a Clostridium butyricum bacterial cell. In one
embodiment, the bacterial cell is a Clostridium scindens bacterial
cell. In one embodiment, the bacterial cell is an Escherichia coli
bacterial cell. In one embodiment, the bacterial cell is a
Lactobacillus acidophilus bacterial cell. In one embodiment, the
bacterial cell is a Lactobacillus plantarum bacterial cell. In one
embodiment, the bacterial cell is a Lactobacillus reuteri bacterial
cell. In one embodiment, the bacterial cell is a Lactococcus lactis
bacterial cell. In one embodiment, the bacterial cell is a
Oxalobacter formigenes bacterial cell. In another embodiment, the
bacterial cell does not include Oxalobacter formigenes.
[0120] In one embodiment, the bacterial cell is a Gram positive
bacterial cell. In another embodiment, the bacterial cell is a Gram
negative bacterial cell.
[0121] In some embodiments, the bacterial cell is Escherichia coli
strain Nissle 1917 (E. coli Nissle), a Gram-negative bacterium of
the Enterobacteriaceae family that "has evolved into one of the
best characterized probiotics" (Ukena et al., 2007). The strain is
characterized by its "complete harmlessness" (Schultz, 2008), and
"has GRAS (generally recognized as safe) status" (Reister et al.,
2014, emphasis added). Genomic sequencing confirmed that E. coli
Nissle "lacks prominent virulence factors (e.g., E. coli
a-hemolysin, P-fimbrial adhesins)" (Schultz, 2008), and E. coli
Nissle "does not carry pathogenic adhesion factors and does not
produce any enterotoxins or cytotoxins, it is not invasive, not
uropathogenic" (Sonnenborn et al., 2009). As early as in 1917, E.
coli Nissle was packaged into medicinal capsules, called Mutaflor,
for therapeutic use. E. coli Nissle has since been used to treat
ulcerative colitis in humans in vivo (Rembacken et al., 1999), to
treat inflammatory bowel disease, Crohn's disease, and pouchitis in
humans in vivo (Schultz, 2008), and to inhibit enteroinvasive
Salmonella, Legionella, Yersinia, and Shigella in vitro
(Altenhoefer et al., 2004). It is commonly accepted that E. coli
Nissle's "therapeutic efficacy and safety have convincingly been
proven" (Ukena et al., 2007).
[0122] In one embodiment, the recombinant bacterial cell of the
disclosure does not colonize the subject to whom the cell is
administered.
[0123] One of ordinary skill in the art would appreciate that the
genetic modifications disclosed herein may be adapted for other
species, strains, and subtypes of bacteria. Furthermore, genes from
one or more different species can be introduced into one
another.
[0124] In some embodiments, the bacterial cell is a genetically
engineered bacterial cell. In another embodiment, the bacterial
cell is a recombinant bacterial cell. In some embodiments, the
disclosure comprises a colony of recombinant bacterial cells.
[0125] In another aspect, the disclosure provides a recombinant
bacterial culture which comprises bacterial cells disclosed herein.
In one aspect, the disclosure provides a recombinant bacterial
culture which reduces levels of a substrate, e.g., an amino acid or
a peptide, in the media of the culture. In one embodiment, the
levels of substrate is reduced by about 50%, by about 60%, by about
70%, by about 75%, by about 80%, by about 90%, by about 95%, or
about 100% in the media of the cell culture. In another embodiment,
the levels of a substrate is reduced by about two-fold, three-fold,
four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or
fifty-fold, in the media of the cell culture. In one embodiment,
the levels of a substrate are reduced below the limit of detection
in the media of the cell culture.
[0126] In some embodiments of the above described recombinant
bacterial cells, the gene encoding a substrate transporter is
present on a plasmid in the bacterium and operatively linked on the
plasmid to the promoter that is induced under low-oxygen or
anaerobic conditions. In other embodiments, the gene encoding a
substrate transporter is present in the bacterial chromosome and is
operatively linked in the chromosome to the promoter that is
induced under low-oxygen or anaerobic conditions.
[0127] In some embodiments, the recombinant bacterial cell
comprising a heterologous substrate transporter is an auxotroph. In
one embodiment, the recombinant bacterial cell is an auxotroph
selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA,
hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB,
dapD, dapE, dapF, flhD, metB, metC, proAB, and thiI auxotroph. In
some embodiments, the recombinant bacterial cell has more than one
auxotrophy, for example, they may be a .DELTA.thyA and .DELTA.dapA
auxotroph.
[0128] In some embodiments, the recombinant bacterial cell
comprising a heterologous substrate transporter further comprises a
kill-switch circuit, such as any of the kill-switch circuits
provided herein. For example, in some embodiments, the recombinant
bacterial cells may further comprise one or more genes encoding one
or more recombinase(s) under the control of an inducible promoter,
and an inverted toxin sequence. In some embodiments, the
recombinant bacterial cell further comprises one or more genes
encoding an antitoxin. In some embodiments, the recombinant
bacterial cell further comprise one or more genes encoding one or
more recombinase(s) under the control of an inducible promoter and
one or more inverted excision genes, wherein the excision gene(s)
encode an enzyme that deletes an essential gene. In some
embodiments, the recombinant bacterial cell further comprise one or
more genes encoding an antitoxin. In some embodiments, the
recombinant bacterial cell further comprises one or more genes
encoding a toxin under the control of an promoter having a TetR
repressor binding site and a gene encoding the TetR under the
control of an inducible promoter that is induced by arabinose, such
as P.sub.araBAD. In some embodiments, the recombinant bacterial
cell further comprises one or more genes encoding an antitoxin.
[0129] In some embodiments, the recombinant bacterial cell is an
auxotroph comprising a heterologous substrate transporter gene and
further comprises a kill-switch circuit, such as any of the
kill-switch circuits described herein.
[0130] In some embodiments of the above described recombinant
bacterial cell, the heterologous gene encoding a substrate
transporter is present on a plasmid in the bacterium and
operatively linked on the plasmid to the promoter that is induced
under low-oxygen or anaerobic conditions. In other embodiments, the
gene encoding a substrate transporter is present in the bacterial
chromosome and is operatively linked in the chromosome to the
promoter that is induced under low-oxygen or anaerobic
conditions.
A. Amino Acid Transporters
[0131] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is an amino acid
transporter. In one embodiment, the amino acid transporter
transports at least one amino acid selected from the group
consisting of leucine, isoleucine, valine, arginine, lysine,
asparagine, serine, glycine, glutamine, tryptophan, methionine,
threonine, cysteine, tyrosine, phenylalanine, glutamic acid,
aspartic acid, alanine, histidine, and proline, into the cell.
[0132] The uptake of amino acids into bacterial cells is mediated
by proteins well known to those of skill in the art Amino acid
transporters may be expressed or modified in the bacteria in order
to enhance amino acid transport into the cell. Specifically, when
the amino acid transporter is expressed in the recombinant
bacterial cells, the bacterial cells import more amino acid(s) into
the cell when the transporter is expressed than unmodified bacteria
of the same bacterial subtype under the same conditions. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding an amino acid transporter. In one embodiment, the
bacterial cell comprises a heterologous gene encoding an amino acid
transporter and a genetic modification that reduces export of an
amino acid, e.g., a genetic mutation in an exporter gene or
promoter.
[0133] In one embodiment, the bacterial cell comprises at least one
gene encoding an amino acid transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding an amino
acid transporter. In some embodiments, the at least one native gene
encoding an amino acid transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding an amino acid transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native amino acid transporter, as well as at
least one copy of at least one heterologous gene encoding anamino
acid transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding an amino acid transporter. In one embodiment, the
bacterial cell comprises multiple copies of the at least one
heterologous gene encoding an amino acid transporter.
[0134] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding an amino acid transporter, wherein
said amino acid transporter comprises an amino acid sequence that
has at least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity to the amino acid sequence of a polypeptide encoded by an
amino acid transporter gene disclosed herein.
[0135] In some embodiments, the amino acid transporter is encoded
by an amino acid transporter gene derived from a bacterial genus or
species, including but not limited to, Bacillus, Campylobacter,
Clostridium, Escherichia, Lactobacillus, Pseudomonas, Salmonella,
Staphylococcus, Bacillus subtilis, Campylobacter jejuni,
Clostridium perfringens, Escherichia coli, Lactobacillus
delbrueckii, Pseudomonas aeruginosa, Salmonella typhimurium, or
Staphylococcus aureus. In some embodiments, the bacterial species
is Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0136] The present disclosure further comprises genes encoding
functional fragments of an amino acid transporter or functional
variants of an amino acid transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of an
amino acid transporter relates to an element having qualitative
biological activity in common with the wild-type amino acid
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
amino acid transporter is one which retains essentially the same
ability to import an amino acid into the bacterial cell as does the
amino acid transporter protein from which the functional fragment
or functional variant was derived. In one embodiment, the
recombinant bacterial cell comprises at least one heterologous gene
encoding a functional fragment of an amino acid transporter. In
another embodiment, the recombinant bacterial cell comprises a
heterologous gene encoding a functional variant of an amino acid
transporter.
[0137] Assays for testing the activity of an amino acid
transporter, a functional variant of an amino acid transporter, or
a functional fragment of an amino acid transporter are well known
to one of ordinary skill in the art. For example, import of an
amino acid may be determined using the methods as described in
Haney et al., J. Bact., 174(1):108-15, 1992; Rahmanian et al., J.
Bact., 116(3):1258-66, 1973; and Ribardo and Hendrixson, J. Bact.,
173(22):6233-43, 2011, the entire contents of each of which are
expressly incorporated by reference herein.
[0138] In one embodiment, the genes encoding the amino acid
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the amino acid transporter have been codon-optimized
for use in Escherichia coli.
[0139] The present disclosure also encompasses genes encoding an
amino acid transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0140] In some embodiments, the at least one gene encoding an amino
acid transporter is mutagenized; mutants exhibiting increased amino
acid import are selected; and the mutagenized at least one gene
encoding an amino acid transporter is isolated and inserted into
the bacterial cell. In some embodiments, the at least one gene
encoding an amino acid transporter is mutagenized; mutants
exhibiting decreased amino acid import are selected; and the
mutagenized at least one gene encoding an amino acid transporter is
isolated and inserted into the bacterial cell. The transporter
modifications described herein may be present on a plasmid or
chromosome.
[0141] In some embodiments, the bacterial cell comprises a
heterologous gene encoding an amino acid transporter operably
linked to a promoter. In one embodiment, the at least one gene
encoding an amino acid transporter is directly operably linked to
the promoter. In another embodiment, the at least one gene encoding
an amino acid transporter is indirectly operably linked to the
promoter.
[0142] In one embodiment, the promoter is not operably linked with
the at least one gene encoding an amino acid transporter in nature.
In some embodiments, the at least one gene encoding the amino acid
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the amino acid
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the amino acid
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the amino acid transporter is controlled by a constitutive
promoter.
[0143] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0144] In one embodiment, the at least one gene encoding an amino
acid transporter is located on a plasmid in the bacterial cell. In
some embodiments, the plasmid is a high copy number plasmid. In
some embodiments, the plasmid is a low copy number plasmid. In
another embodiment, the at least one gene encoding an amino acid
transporter is located in the chromosome of the bacterial cell. In
yet another embodiment, a native copy of the at least one gene
encoding an amino acid transporter is located in the chromosome of
the bacterial cell, and a copy of at least one gene encoding an
amino acid transporter from a different species of bacteria is
located on a plasmid in the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding an
amino acid transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding an amino acid
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding an amino acid transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding an amino acid transporter from a
different species of bacteria is located in the chromosome of the
bacterial cell.
[0145] In some embodiments, the at least one native gene encoding
the amino acid transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the amino acid transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0146] In some embodiments, at least one native gene encoding the
amino acid transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the amino acid transporter in the
bacterial cell is modified, and one or more additional copies of at
least one native gene encoding the transporter are present in the
bacterial cell on a plasmid. In alternate embodiments, the at least
one native gene encoding the transporter is modified, and a copy of
at least one gene encoding the transporter from a different
bacterial species is present in the bacteria on a plasmid.
[0147] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0148] In one embodiment, when the amino acid transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more amino acids into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the amino acid transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more amino acids, into the bacterial
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In yet
another embodiment, when the amino acid transporter is expressed in
the recombinant bacterial cells, the bacterial cells import
two-fold more amino acids into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
amino acid transporter is expressed in the recombinant bacterial
cells, the bacterial cells import three-fold, four-fold, five-fold,
six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold
more amino acid into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0149] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous amino acid transporter and a
second heterologous amino acid transporter. In one embodiment, said
first amino acid transporter is derived from a different organism
than said second amino acid transporter. In some embodiments, said
first amino acid transporter is derived from the same organism as
said second amino acid transporter. In some embodiments, said first
amino acid transporter imports the same amino acid as said second
amino acid transporter. In other embodiment, said first amino acid
transporter imports a different amino acid from said second amino
acid transporter. In some embodiments, said first amino acid
transporter is a wild-type amino acid transporter and said second
amino acid transporter is a mutagenized version of said first amino
acid transporter. In some embodiments, the recombinant bacterial
cells described herein comprise at least a third heterologous amino
acid transporter. In some embodiments, the recombinant bacterial
cells described herein comprise at least four heterologous amino
acid transporters. In some embodiments, the recombinant bacterial
cells described herein comprise at least five heterologous amino
acid transporters or more.
[0150] In one embodiment, the amino acid transporter imports one
amino acid into the bacterial cell. In another embodiment, the
amino acid transporter imports two amino acids into the bacterial
cell. In yet another embodiment, the amino acid transporter imports
three amino acids into the bacterial cell. In another embodiment,
the amino acid transporter imports four or more amino acids into
the cell. In one embodiment, the amino acid transporter is an
arginine transporter. In another embodiment, the amino acid
transporter is an asparagine transporter. In another embodiment,
the amino acid transporter is a serine transporter. In another
embodiment, the amino acid transporter is an transporter of
glycine. In another embodiment, the amino acid transporter is a
tryptophan transporter. In another embodiment, the amino acid
transporter is a methionine transporter. In another embodiment, the
amino acid transporter is a threonine transporter. In another
embodiment, the amino acid transporter is a cysteine transporter.
In another embodiment, the amino acid transporter is a tyrosine
transporter. In another embodiment, the amino acid transporter is a
phenylalanine transporter. In another embodiment, the amino acid
transporter is a glutamic acid transporter. In another embodiment,
the amino acid transporter is a histidine transporter. In another
embodiment, the amino acid transporter is a proline transporter. In
another embodiment, the amino acid transporter is an transporter of
leucine. In another embodiment, the amino acid transporter is an
transporter of isoleucine. In another embodiment, the amino acid
transporter is an transporter of valine. In another embodiment, the
amino acid transporter is a lysine transporter. In another
embodiment, the amino acid transporter is a glutamine transporter.
In another embodiment, the amino acid transporter is an transporter
of aspartic acid. In another embodiment, the amino acid transporter
is an transporter of alanine. In another embodiment, the amino acid
transporter is an transporter of branched chain amino acids.
[0151] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an amino acid transporter
may be used to treat a disease, condition, and/or symptom
associated with amino acid metabolism. In some embodiments,
disclosed herein are methods for reducing, ameliorating, or
eliminating one or more symptom(s) associated with these diseases
or disorders.
[0152] As used herein the terms "disease associated with amino acid
metabolism" or a "disorder associated with amino acid metabolism"
is a disease or disorder involving the abnormal, e.g., increased,
levels of one or more amino acids in a subject. In one embodiment,
a disease or disorder associated with amino acid metabolism is a
cancer, e.g., a cancer described herein. In another embodiment, a
disease or disorder associated with amino acid metabolism is a
metabolic disease. In one embodiment, the cancer is glioma. In
another embodiment, the cancer is breast cancer. In another
embodiment, the cancer is melanoma. In another embodiment, the
cancer is hepatocarcinoma. In another embodiment, the cancer is
acute lymphoblastic leukemia (ALL). In another embodiment, the
cancer is ovarian cancer. In another embodiment, the cancer is
prostate cancer. In another embodiment, the cancer is lymphoblastic
leukemia. In another embodiment, the cancer is non-small cell lung
cancer.
[0153] Multiple distinct transporters of amino acids are well known
in the art and are described in the subsections, below.
[0154] 1. Branched Chain Amino Acid Transporters
[0155] In one embodiment, the amino acid transporter is a branched
chain amino acid transporter. The term "branched chain amino acid"
or "BCAA," as used herein, refers to an amino acid which comprises
a branched side chain. Leucine, isoleucine, and valine are
naturally occurring amino acids comprising a branched side chain.
However, non-naturally occurring, usual, and/or modified amino
acids comprising a branched side chain are also encompassed by the
term branched chain amino acid.
[0156] Branched chain amino acid transporters may be expressed or
modified in the recombinant bacteria described herein in order to
enhance branched chain amino acid transport into the cell.
Specifically, when the transporter of branched chain amino acids is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import more branched chain amino acids into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding an transporter of branched chain amino acids may be used
to import one or more branched chain amino acids into the
bacteria.
[0157] The uptake of branched chain amino acids into bacterial
cells is mediated by proteins well known to those of skill in the
art. For example, two well characterized BCAA transport systems
have been characterized in several bacteria, including Escherichia
coli. BCAAs are transported by two systems into bacterial cells
(i.e., imported), the osmotic-shock-sensitive systems designated
LIV-I and LS (leucine-specific), and by an osmotic-shock resistant
system, BrnQ, formerly known as LIV-II (see Adams et al., J. Biol.
Chem. 265:11436-43 (1990); Anderson and Oxender, J. Bacteriol.
130:384-92 (1977); Anderson and Oxender, J. Bacteriol. 136:168-74
(1978); Haney et al., J. Bacteriol. 174:108-15 (1992); Landick and
Oxender, J. Biol. Chem. 260:8257-61 (1985); Nazos et al., J.
Bacteriol. 166:565-73 (1986); Nazos et al., J. Bacteriol.
163:1196-202 (1985); Oxender et al., Proc. Natl. Acad. Sci. USA
77:1412-16 (1980); Quay et al., J. Bacteriol. 129:1257-65 (1977);
Rahmanian et al., J. Bacteriol. 116:1258-66 (1973); Wood, J. Biol.
Chem. 250:4477-85 (1975); Guardiola et al., J. Bacteriol.
117:393-405 (1974); Guardiola and Iaccarino, J. Bacteriol.
108:1034-44 (1971); Ohnishi et al., Jpn. J. Genet.
63:343-57)(1988); Yamato and Anraku, J. Bacteriol. 144:36-44
(1980); and Yamato et al., J. Bacteriol. 138:24-32 (1979)).
Transport by the BrnQ system is mediated by a single membrane
protein. Transport mediated by the LIV-I system is dependent on the
substrate binding protein LivJ (also known as LIV-BP), while
transport mediated by LS system is mediated by the substrate
binding protein LivK (also known as LS-BP). LivJ is encoded by the
livJ gene, and binds isoleucine, leucine and valine with K.sub.d
values of .about.10.sup.-6 and .about.10.sup.-7 M, while LivK is
encoded by the livK gene, and binds leucine with a K.sub.d value of
.about.10.sup.-6 M (See Landick and Oxender, J. Biol. Chem.
260:8257-61 (1985)). Both LivJ and LivK interact with the inner
membrane components LivHMGF to enable ATP-hydrolysis-coupled
transport of their substrates into the cell, forming the LIV-I and
LS transport systems, respectively. The LIV-I system transports
leucine, isoleucine and valine, and to a lesser extent serine,
threonine and alanine, whereas the LS system only transports
leucine. The six genes encoding the E. coli LIV-I and LS systems
are organized into two transcriptional units, with livKHMGF
transcribed as a single operon, and livJ transcribed separately.
The Escherichia coli liv genes can be grouped according to protein
function, with the livJ and livK genes encoding periplasmic binding
proteins with the binding affinities described above, the livH and
livM genes encoding inner membrane permeases, and the livG and livF
genes encoding cytoplasmic ATPases.
[0158] In one embodiment, the at least one gene encoding an
branched chain amino acid transporter is the brnQ gene. An
exemplary sequence for brnQ is provided below.
TABLE-US-00002 BCAA transporter BrnQ from E. coli: Nucleotide
sequence: atgacccatcaattaagatcgcgcgatatcatcgctctgggctttatgac
atttgcgttgttcgtcggcgcaggtaacattattttccctccaatggtcg
gcttgcaggcaggcgaacacgtctggactgcggcattcggcttcctcatt
actgccgttggcctaccggtattaacggtagtggcgctggcaaaagttgg
cggcggtgttgacagtctcagcacgccaattggtaaagtcgctggcgtac
tgctggcaacagtttgttacctggcggtggggccgctttttgctacgccg
cgtacagctaccgtttcttttgaagtgggcattgcgccgctgacgggtga
ttccgcgctgccgctgtttatttacagcctggtctatttcgctatcgtta
ttctggtttcgctctatccgggcaagctgctggataccgtgggcaacttc
cttgcgccgctgaaaattatcgcgctggtcatcctgtctgttgccgcaat
tatctggccggcgggttctatcagtacggcgactgaggcttatcaaaacg
ctgcgttttctaacggcttcgtcaacggctatctgaccatggatacgctg
ggcgcaatggtgtttggtatcgttattgttaacgcggcgcgttctcgtgg
cgttaccgaagcgcgtctgctgacccgttataccgtctgggctggcctga
tggcgggtgttggtctgactctgctgtacctggcgctgttccgtctgggt
tcagacagcgcgtcgctggtcgatcagtctgcaaacggtgcggcgatcct
gcatgcttacgttcagcatacctttggcggcggcggtagcttcctgctgg
cggcgttaatcttcatcgcctgcctggtcacggcggttggcctgacctgt
gcttgtgcagaattcttcgcccagtacgtaccgctctcttatcgtacgct
ggtgtttatcctcggcggcttctcgatggtggtgtctaacctcggcttga
gccagctgattcagatctctgtaccggtgctgaccgccatttatccgccg
tgtatcgcactggttgtattaagttttacacgctcatggtggcataattc
gtcccgcgtgattgctccgccgatgtttatcagcctgctttttggtattc
tcgacgggatcaaggcatctgcattcagcgatatcttaccgtcctgggcg
cagcgtttaccgctggccgaacaaggtctggcgtggttaatgccaacagt
ggtgatggtggttctggccattatctgggatcgtgcggcaggtcgtcagg
tgacctccagcgctcactaa AA sequence:
MTHQLRSRDIIALGFMTFALFVGAGNIIFPPMVGLQAGEHVWTAAFGFLI
TAVGLPVLTVVALAKVGGGVDSLSTPIGKVAGVLLATVCYLAVGPLFATP
RTATVSFEVGIAPLTGDSALPLFIYSLVYFAIVILVSLYPGKLLDTVGNF
LAPLKIIALVILSVAAIIWPAGSISTATEAYQNAAFSNGFVNGYLTMDTL
GAMVFGIVIVNAARSRGVTEARLLTRYTVWAGLMAGVGLTLLYLALFRLG
SDSASLVDQSANGAAILHAYVQHTFGGGGSFLLAALIFIACLVTAVGLTC
ACAEFFAQYVPLSYRTLVFILGGFSMVVSNLGLSQLIQISVPVLTAIYPP
CIALVVLSFTRSWWHNSSRVIAPPMFISLLFGILDGIKASAFSDILPSWA
QRLPLAEQGLAWLMPTVVMVVLAIIWDRAAGRQVTSSAH
[0159] In one embodiment, the at least one gene encoding an
branched chain amino acid transporter is the livJ gene. In one
embodiment, the at least one gene encoding an branched chain amino
acid transporter is the livH gene. In one embodiment, the at least
one gene encoding an branched chain amino acid transporter is the
livM gene. In one embodiment, the at least one gene encoding an
branched chain amino acid transporter is the livG gene. In one
embodiment, the at least one gene encoding an branched chain amino
acid transporter is the livF gene. In one embodiment, the at least
one gene encoding an branched chain amino acid transporter is the
livKHMGF operon. In one embodiment, the at least one gene encoding
an branched chain amino acid transporter is the livK gene. In
another embodiment, the livKHMGF operon is an Escherichia coli
livKHMGF operon. In another embodiment, the at least one gene
encoding an branched chain amino acid transporter comprises the
livKHMGF operon and the livJ gene. In one embodiment, the bacterial
cell of the invention has been genetically engineered to comprise
at least one heterologous gene encoding a LIV-I system. In one
embodiment, the bacterial cell of the invention has been
genetically engineered to comprise at least one heterologous gene
encoding a LS system. In one embodiment, the bacterial cell of the
invention has been genetically engineered to comprise at least one
heterologous gene encoding a LIV-I system. In one embodiment, the
bacterial cell of the invention has been genetically engineered to
comprise at least one heterologous livJ gene, and at least one
heterologous gene selected from the group consisting of livH, livM,
livG, and livF. In one embodiment, the bacterial cell of the
invention has been genetically engineered to comprise at least one
heterologous livK gene, and at least one heterologous gene selected
from the group consisting of livH, livM, livG, and livF.
[0160] In one embodiment, the branched chain amino acid transporter
gene has at least about 80% identity with the uppercase sequence of
SEQ ID NO:9. Accordingly, in one embodiment, the branched chain
amino acid transporter gene has at least about 90% identity with
the uppercase sequence of SEQ ID NO:9. Accordingly, in one
embodiment, the branched chain amino acid transporter gene has at
least about 95% identity with the uppercase sequence of SEQ ID
NO:9. Accordingly, in one embodiment, the branched chain amino acid
transporter gene has at least about 70%, 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity with the uppercase sequence of SEQ ID NO:9. In another
embodiment, the branched chain amino acid transporter gene
comprises the uppercase sequence of SEQ ID NO:9. In yet another
embodiment the branched chain amino acid transporter gene consists
of the uppercase sequence of SEQ ID NO:9.
[0161] In one embodiment, the branched chain amino acid transporter
gene has at least about 80% identity with the sequence of SEQ ID
NO:10. Accordingly, in one embodiment, the branched chain amino
acid transporter gene has at least about 90% identity with the
sequence of SEQ ID NO:10. Accordingly, in one embodiment, the
branched chain amino acid transporter gene has at least about 95%
identity with the sequence of SEQ ID NO:10. Accordingly, in one
embodiment, the branched chain amino acid transporter gene has at
least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of
SEQ ID NO:10. In another embodiment, the branched chain amino acid
transporter gene comprises the sequence of SEQ ID NO:10. In yet
another embodiment the branched chain amino acid transporter gene
consists of the sequence of SEQ ID NO:10.
[0162] In some embodiments, the branched chain amino acid
transporter is encoded by an branched chain amino acid transporter
gene derived from a bacterial genus or species, including but not
limited to, Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0163] Assays for testing the activity of a branched chain amino
acid transporter, a functional variant of a branched chain amino
acid transporter, or a functional fragment of a branched chain
amino acid transporter are well known to one of ordinary skill in
the art. For example, import of an amino acid may be determined
using the methods as described in Haney et al., J. Bact.,
174(1):108-15, 1992; Rahmanian et al., J. Bact., 116(3):1258-66,
1973; and Ribardo and Hendrixson, J. Bact., 173(22):6233-43, 2011,
the entire contents of each of which are expressly incorporated by
reference herein.
[0164] In one embodiment, when the branched chain amino acid
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import 10% more branched
chain amino acid into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In another embodiment, when the
transporter of branched chain amino acids is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more branched
chain amino acids into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
branched chain amino acid transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cell
imports two-fold more branched chain amino acids into the cell when
the transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the branched chain amino acid transporter is
expressed in the recombinant bacterial cell described herein, the
bacterial cell import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, or ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more branched
chain amino acid into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0165] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a branched chain amino acid
transporter may be used to treat a disease, condition, and/or
symptom associated with the catabolism of a branched chain amino
acid. In some embodiments, disclosed herein are methods for
reducing, ameliorating, or eliminating one or more symptom(s)
associated with these diseases or disorders. In one embodiment, the
disorder involving the catabolism of a branched chain amino acid is
a metabolic disorder involving the abnormal catabolism of a
branched chain amino acid. Metabolic diseases associated with
abnormal catabolism of a branched chain amino acid include maple
syrup urine disease (MSUD), isovaleric acidemia, propionic
acidemia, methylmalonic acidemia, and diabetes ketoacidosis. In one
embodiment, the disease associated with abnormal catabolism of a
branched chain amino acid is isovaleric acidemia. In one
embodiment, the disease associated with abnormal catabolism of a
branched chain amino acid is propionic acidemia. In one embodiment,
the disease associated with abnormal catabolism of a branched chain
amino acid is methylmalonic acidemia. In another embodiment, the
disease associated with abnormal catabolism of a branched chain
amino acid is diabetes.
[0166] 2. Arginine Transporters
[0167] In one embodiment, the amino acid transporter is an arginine
transporter. Arginine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
arginine transport into the cell. Specifically, when the arginine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more arginine into the
cell when the arginine transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
Thus, the genetically engineered bacteria comprising a heterologous
gene encoding an arginine transporter which may be used to import
arginine into the bacteria.
[0168] The uptake of arginine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example,
three different arginine transport systems have been characterized
in several bacteria: the arginine-specific system encoded by the
artPIQM operon and the artJ gene (see, e.g., Wissenbach et al.
(1993) J. Bacteriol. 175(11): 3687-8); the basic amino acid uptake
system, known as LAO (lysine, arginine, ornithine) (see, e.g.,
Rosin et al. (1971) J. Biol. Chem. 246: 3653-62); and the AO
(arginine, ornithine) system (see, e.g., Celis (1977) J. Bacteriol.
130: 1234-43). Transport by the arginine-specific system is
mediated by several proteins encoded by the two transcriptional
units, the artPIQM operon and the artJ gene. In this system, ArtP
(encoded by artP) is an ATPase, ArtQ and ArtM (encoded by artQ and
artM, respectively) are transmembrane proteins, and ArtI and ArtJ
(encoded by artI and artJ, respectively)are arginine-binding
periplasmic proteins. This system has been well characterized in
Escherichia coli (see, e.g., Wissenbach U. (1995) Mol. Microbiol.
17(4): 675-86; Wissenbach et al. (1993) J. Bacteriol. 175(11):
3687-88). In addition, bacterial systems that are homologous and
orthologous of the E. coli arginine-specific system have been
characterized in other bacterial species, including, for example,
Haemophilus influenzae (see, e.g., Mironov et al. (1999) Nucleic
Acids Res. 27(14): 2981-9). The second arginine transport system,
the basic amino acid LAO system, consists of the periplasmic LAO
protein (also referred to herein as ArgT; encoded by argT), which
binds lysine, arginine and ornithine, and the membranous and
membrane-associated proteins of the histidine permease (Q M P
complex), encoded by the hisJQMP operon, resulting in the uptake of
arginine (see, e.g., Oh et al. (1994) J. Biol. Chem. 269(42):
26323-30). Members of the basic amino acid LAO system have been
well characterized in Escherichia coli and Salmonella enterica.
Finally, the third arginine transport system, the AO system,
consists of the binding protein AbpS (encoded by abpS) and the ATP
hydrolase ArgK (encoded by argK) which mediate the ATP-dependent
uptake of arginine (see, e.g., Celis et al. (1998) J. Bacteriol.
180(18): 4828-33).
[0169] In one embodiment, the at least one gene encoding an
arginine transporter is the artJ gene. In one embodiment, the at
least one gene encoding an arginine transporter is the artPIQM
operon. In one embodiment, the at least one gene encoding an
arginine transporter is the artP gene. In one embodiment, the at
least one gene encoding an arginine transporter is the artI gene.
In one embodiment, the at least one gene encoding an arginine
transporter is the artQ gene. In one embodiment, the at least one
gene encoding an arginine transporter is the artM gene. In one
embodiment, the at least one gene encoding an arginine transporter
is the argT gene. In one embodiment, the at least one gene encoding
an arginine transporter is the hisJQMP operon. In one embodiment,
the at least one gene encoding an arginine transporter is the hisJ
gene. In one embodiment, the at least one gene encoding an arginine
transporter is the hisQ gene. In one embodiment, the at least one
gene encoding an arginine transporter is the hisM gene. In one
embodiment, the at least one gene encoding an arginine transporter
is the hisP gene. In one embodiment, the at least one gene encoding
an arginine transporter is the abpS gene. In one embodiment, the at
least one gene encoding an arginine transporter is the argK gene.
In another embodiment, the at least one gene encoding an arginine
transporter comprises the artPIQM operon and the artJ gene. In
another embodiment, the at least one gene encoding an arginine
transporter comprises the hisJQMP operon and the argT gene. In yet
another embodiment, the at least one gene encoding an arginine
transporter comprises the abpS gene and the argK gene.
[0170] In one embodiment, the argT gene has at least about 80%
identity with the sequence of SEQ ID NO:13. Accordingly, in one
embodiment, the argT gene has at least about 90% identity with the
sequence of SEQ ID NO:13. Accordingly, in one embodiment, the argT
gene has at least about 95% identity with the sequence of SEQ ID
NO:13. Accordingly, in one embodiment, the argT gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:13. In another embodiment, the argT gene comprises the
sequence of SEQ ID NO:13. In yet another embodiment the argT gene
consists of the sequence of SEQ ID NO:13.
[0171] In one embodiment, the artP gene has at least about 80%
identity with the sequence of SEQ ID NO:14. Accordingly, in one
embodiment, the artP gene has at least about 90% identity with the
sequence of SEQ ID NO:14. Accordingly, in one embodiment, the artP
gene has at least about 95% identity with the sequence of SEQ ID
NO:14. Accordingly, in one embodiment, the artP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:14. In another embodiment, the artP gene comprises the
sequence of SEQ ID NO:14. In yet another embodiment the artP gene
consists of the sequence of SEQ ID NO:14.
[0172] In one embodiment, the artI gene has at least about 80%
identity with the sequence of SEQ ID NO:15. Accordingly, in one
embodiment, the artI gene has at least about 90% identity with the
sequence of SEQ ID NO:15. Accordingly, in one embodiment, the artI
gene has at least about 95% identity with the sequence of SEQ ID
NO:15. Accordingly, in one embodiment, the artI gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:15. In another embodiment, the artI gene comprises the
sequence of SEQ ID NO:15. In yet another embodiment the artI gene
consists of the sequence of SEQ ID NO:15.
[0173] In one embodiment, the artQ gene has at least about 80%
identity with the sequence of SEQ ID NO:16. Accordingly, in one
embodiment, the artQ gene has at least about 90% identity with the
sequence of SEQ ID NO:16. Accordingly, in one embodiment, the artQ
gene has at least about 95% identity with the sequence of SEQ ID
NO:16. Accordingly, in one embodiment, the artQ gene has at least
about 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:16. In another embodiment, the artQ gene comprises the
sequence of SEQ ID NO:16. In yet another embodiment the artQ gene
consists of the sequence of SEQ ID NO:16.
[0174] In one embodiment, the artM gene has at least about 80%
identity with the sequence of SEQ ID NO:17. Accordingly, in one
embodiment, the artM gene has at least about 90% identity with the
sequence of SEQ ID NO:17. Accordingly, in one embodiment, the artM
gene has at least about 95% identity with the sequence of SEQ ID
NO:17. Accordingly, in one embodiment, the artM gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:17. In another embodiment, the artM gene comprises the
sequence of SEQ ID NO:17. In yet another embodiment the artM gene
consists of the sequence of SEQ ID NO:17.
[0175] In one embodiment, the artJ gene has at least about 80%
identity with the sequence of SEQ ID NO:18. Accordingly, in one
embodiment, the artJ gene has at least about 90% identity with the
sequence of SEQ ID NO:18. Accordingly, in one embodiment, the artJ
gene has at least about 95% identity with the sequence of SEQ ID
NO:18. Accordingly, in one embodiment, the artJ gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:18. In another embodiment, the artJ gene comprises the
sequence of SEQ ID NO:18. In yet another embodiment the artJ gene
consists of the sequence of SEQ ID NO:18.
[0176] In some embodiments, the arginine transporter is encoded by
an arginine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia, Haemophilus,
Salmonella, Escherichia coli, Haemophilus influenza, Salmonella
enterica, or Salmonella typhimurium. In some embodiments, the
bacterial species is Escherichia coli. In some embodiments, the
bacterial species is Escherichia coli strain Nissle.
[0177] Assays for testing the activity of an arginine transporter,
a functional variant of an arginine transporter, or a functional
fragment of arginine transporter are well known to one of ordinary
skill in the art. For example, import of arginine may be determined
using the methods as described in Sakanaka et al (2015) J. Biol.
Chem. 290(35): 21185-98, the entire contents of each of which are
expressly incorporated by reference herein.
[0178] In one embodiment, when the arginine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more arginine into the bacterial cell
when the arginine transporter is expressed than unmodified bacteria
of the same bacterial subtype under the same conditions. In another
embodiment, when the arginine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more arginine
into the bacterial cell when the arginine transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the arginine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more arginine
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In yet another embodiment, when the arginine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more arginine
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same
conditions.
[0179] 3. Lysine Transporters
[0180] In one embodiment, the amino acid transporter is a lysine
transporter. Lysine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
lysine transport into the cell. Specifically, when the transporter
of lysine is expressed in the recombinant bacterial cells described
herein, the bacterial cells import more lysine into the cell when
the lysine transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a lysine transporter which may be used to import lysine
into the bacteria.
[0181] The uptake of lysine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, LysP
is a lysine-specific permease originally identified in E. coli,
that has now been further characterized in other bacterial species
(Steffes et al. (1992) J. Bacteriol. 174: 3242-9; Trip et al.
(2013) J. Bacteriol. 195(2): 340-50; Nji et al. (2014) Acta
Crystallogr. F Struct. Biol. Commun. 70(Pt 10): 1362-7). Another
lysine transporter, YsvH, has been described in Bacillus, having
similarities to the lysine permease LysI of Corynebacterium
glutamicum (Rodionov et al. (2003) Nucleic Acids Res. 31(23):
6748-57).
[0182] In one embodiment, the at least one gene encoding a lysine
transporter is the lysP gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous lysP gene. In one embodiment, the at least
one gene encoding a lysine transporter is the Escherichia coli lysP
gene. In one embodiment, the at least one gene encoding a lysine
transporter is the Lactococcus lactis lysP gene. In one embodiment,
the at least one gene encoding a lysine transporter is the
Pseudomonas aeruginosa lysP gene. In one embodiment, the at least
one gene encoding a lysine transporter is the Klebsiella pneumoniae
lysP gene.
[0183] In one embodiment, the lysP gene has at least about 80%
identity with the sequence of SEQ ID NO:26. Accordingly, in one
embodiment, the lysP gene has at least about 90% identity with the
sequence of SEQ ID NO:26. Accordingly, in one embodiment, the lysP
gene has at least about 95% identity with the sequence of SEQ ID
NO:26. Accordingly, in one embodiment, the lysP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:26. In another embodiment, the lysP gene comprises the
sequence of SEQ ID NO:26. In yet another embodiment the lysP gene
consists of the sequence of SEQ ID NO:26.
[0184] In one embodiment, the at least one gene encoding a lysine
transporter is the ysvH gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous ysvH gene. In one embodiment, the at least
one gene encoding a lysine transporter is the Bacillus subtilis
ysvH gene. In one embodiment, the at least one gene encoding a
lysine transporter is the Bacillus cereus ysvH gene. In one
embodiment, the at least one gene encoding a lysine transporter is
the Bacillus stearothermophilus ysvH gene.
[0185] In one embodiment, the at least one gene encoding a lysine
transporter is the Corynebacterium glutamicum (see, e.g.,
Seep-Feldhaus et al. (1991) Mol. Microbiol. 5(12): 2995-3005, the
entire contents of which are incorporated herein by reference).
[0186] n one embodiment, the ysvH gene has at least about 80%
identity with the sequence of SEQ ID NO:25. Accordingly, in one
embodiment, the ysvH gene has at least about 90% identity with the
sequence of SEQ ID NO:25. Accordingly, in one embodiment, the ysvH
gene has at least about 95% identity with the sequence of SEQ ID
NO:25. Accordingly, in one embodiment, the ysvH gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:25. In another embodiment, the ysvH gene comprises the
sequence of SEQ ID NO:25. In yet another embodiment the ysvH gene
consists of the sequence of SEQ ID NO:25.
[0187] In some embodiments, the transporter of lysine is encoded by
a lysine transporter gene derived from a bacterial genus or
species, including but not limited to, Bacillus subtilis, Bacillus
cereus, Bacillus stearothermophilus, Corynebacterium glutamicum,
Escherichia coli, Lactococcus lactis, Pseudomonas aeruginosa, and
Klebsiella pneumoniae. In some embodiments, the bacterial species
is Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0188] Assays for testing the activity of a lysine transporter, a
functional variant of a lysine transporter, or a functional
fragment of a lysine transporter are well known to one of ordinary
skill in the art. For example, import of lysine may be determined
using the methods as described in Steffes et al. (1992) J.
Bacteriol. 174: 3242-9, the entire contents of each of which are
expressly incorporated by reference herein.
[0189] In one embodiment, when the lysine transporter is expressed
in the recombinant bacterial cells described herein, the bacterial
cells import 10% more lysine into the bacterial cell when the
lysine transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the lysine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more lysine
into the bacterial cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the lysine transporter
is expressed in the recombinant bacterial cells described herein,
the bacterial cells import two-fold more lysine into the cell when
the transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the transporter of lysine is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import three-fold, four-fold, five-fold, six-fold, seven-fold,
eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold,
thirty-fold, fourty-fold, or fifty-fold, more lysine into the cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions.
[0190] 4. Asparagine Transporters
[0191] In one embodiment, the amino acid transporter is an
asparagine transporter. Asparagine transporters may be expressed or
modified in the recombinant bacteria described herein in order to
enhance asparagine transport into the cell. Specifically, when the
asparagine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import more asparagine
into the cell when the asparagine transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. Thus, the genetically engineered bacteria comprising a
heterologous gene encoding an asparagine transporter which may be
used to import asparagine into the bacteria.
[0192] The uptake of asparagine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, two
distinct systems for asparagine uptake, distinguishable on the
basis of their specificity for asparagine have been identified in
E. coli (see, e.g., Willis and Woolfolk (1975) J. Bacteriol. 123:
937-945). The bacterial gene ansP encodes an asparagine permease
responsible for asparagine uptake in many bacteria (see, e.g.,
Jennings et al. (1995) Microbiology 141: 141-6; Ortuno-Olea and
Duran-Vargas (2000) FEMS Microbiol. Lett. 189(2): 177-82; Barel et
al. (2015) Front. Cell. Infect. Microbiol. 5: 9; and Gouzy et al.
(2014) PLoS Pathog. 10(2): e1003928).
[0193] In one embodiment, the at least one gene encoding an
asparagine transporter is the ansP gene. In one embodiment, the
bacterial cell described herein has been genetically engineered to
comprise at least one heterologous ansP gene. In one embodiment,
the at least one gene encoding an asparagine transporter is the
Escherichia coli ansP gene. In one embodiment, the at least one
gene encoding an asparagine transporter is the Francisella
tularensis ansP gene. In one embodiment, the at least one gene
encoding an asparagine transporter is the Mycobacterium bovis ansP2
gene. In one embodiment, the at least one gene encoding an
asparagine transporter is the Salmonella enterica ansP gene. In one
embodiment, the at least one gene encoding an asparagine
transporter is the Yersinia pestis ansP gene.
[0194] In one embodiment, the ansP2 gene has at least about 80%
identity with the sequence of SEQ ID NO:29. Accordingly, in one
embodiment, the ansP2 gene has at least about 90% identity with the
sequence of SEQ ID NO:29. Accordingly, in one embodiment, the ansP2
gene has at least about 95% identity with the sequence of SEQ ID
NO:29. Accordingly, in one embodiment, the ansP2 gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:29. In another embodiment, the ansP2 gene comprises the
sequence of SEQ ID NO:29. In yet another embodiment the ansP2 gene
consists of the sequence of SEQ ID NO:29.
[0195] In some embodiments, the asparagine transporter is encoded
by an asparagine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia, Francisella,
Mycobacterium, Salmonella, Yersinia, Escherichia coli, Francisella
tularensis, Mycobacterium tuberculosis, Salmonella enterica, or
Yersinia pestis. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0196] Assays for testing the activity of an asparagine
transporter, a functional variant of an asparagine transporter, or
a functional fragment of asparagine transporter are well known to
one of ordinary skill in the art. For example, import of asparagine
may be determined using the methods as described in Jennings et al.
(1995) Microbiology 141: 141-6, the entire contents of each of
which are expressly incorporated by reference herein.
[0197] In one embodiment, when the transporter of an asparagine is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more asparagine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the asparagine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
asparagine into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
asparagine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import two-fold more
asparagine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the asparagine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more asparagine into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0198] 5. Serine Transporters
[0199] In one embodiment, the amino acid transporter is a serine
transporter. Serine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
serine transport into the cell. Specifically, when the serine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more serine into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a serine transporter which may be used to import serine
into the bacteria.
[0200] The uptake of serine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, SdaC
(encoded by the sdaC gene; also known as DcrA) is an inner membrane
threonine-insensitive serine transporter that was originally
identified in Escherichia coli (Shao et al. (1994) Eur. J. Biochem.
222: 901-7). Additional serine transporters that have been
identified include the Na.sup.+/serine symporter, SstT (encoded by
the sstT gene), the leucine-isoleucine-valine transporter LIV-1,
which transports serine slowly, and the H.sup.+/serine-threonine
symporter TdcC (encoded by the tdcC gene) (see, e.g., Ogawa et al.
(1998) J. Bacteria 180: 6749-52; Ogawa et al. (1997) J. Biochem.
122(6): 1241-5).
[0201] In one embodiment, the at least one gene encoding a serine
transporter is the sdaC gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous sdaC gene. In one embodiment, the at least
one gene encoding a serine transporter is the Escherichia coli sdaC
gene. In one embodiment, the at least one gene encoding a serine
transporter is the Campylobacter jejuni sdaC gene.
[0202] In one embodiment, the sdaC gene has at least about 80%
identity with the sequence of SEQ ID NO:35. Accordingly, in one
embodiment, the sdaC gene has at least about 90% identity with the
sequence of SEQ ID NO:35. Accordingly, in one embodiment, the sdaC
gene has at least about 95% identity with the sequence of SEQ ID
NO:35. Accordingly, in one embodiment, the sdaC gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:35. In another embodiment, the sdaC gene comprises the
sequence of SEQ ID NO:35. In yet another embodiment the sdaC gene
consists of the sequence of SEQ ID NO:35.
[0203] In one embodiment, the at least one gene encoding a serine
transporter is the sstT gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous sstT gene. In one embodiment, the at least
one gene encoding a serine transporter is the Escherichia coli sstT
gene.
[0204] In one embodiment, the at least one gene encoding a serine
transporter is the tdcC gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous tdcC gene. In one embodiment, the at least
one gene encoding a serine transporter is the Escherichia coli tdcC
gene.
[0205] In some embodiments, the serine transporter is encoded by a
serine transporter gene derived from a bacterial genus or species,
including but not limited to, Campylobacter, Campylobacter jejuni,
Escherichia, and Escherichia coli In some embodiments, the
bacterial species is Escherichia coli. In some embodiments, the
bacterial species is Escherichia coli strain Nissle.
[0206] Assays for testing the activity of a serine transporter, a
functional variant of a serine transporter, or a functional
fragment of transporter of serine are well known to one of ordinary
skill in the art. For example, import of serine may be determined
using the methods as described in Hama et al. (1987) Biochim.
Biophys. Acta 905: 231-9, the entire contents of each of which are
expressly incorporated by reference herein.
[0207] In one embodiment, when the transporter of a serine is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more serine into the bacterial cell when
the transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the serine transporter is expressed in the recombinant
bacterial cells described herein, the bacterial cells import 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more serine into the
bacterial cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In yet another embodiment, when the serine transporter is expressed
in the recombinant bacterial cells described herein, the bacterial
cells import two-fold more serine into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the serine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import three-fold, four-fold, five-fold, six-fold, seven-fold,
eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold,
thirty-fold, fourty-fold, or fifty-fold, more serine into the cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions.
[0208] 6. Glutamine Transporters
[0209] In one embodiment, the amino acid transporter is a glutamine
transporter. Glutamine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
glutamine transport into the cell. Specifically, when the glutamine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more glutamine into
the cell when the transporter is expressed than unmodified bacteria
of the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a glutamine transporter which may be used to import
glutamine into the bacteria.
[0210] The uptake of glutamine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, a
glutamine permease glnHPQ operon has been identified in Escherichia
coli (Nohno et al., Mol. Gen. Genet. 205(2):260-269, 1986).
[0211] In one embodiment, the at least one gene encoding a
glutamine transporter is the glnHPQ operon. In one embodiment, the
bacterial cell described herein has been genetically engineered to
comprise at least one heterologous gene from the glnHPQ operon. In
one embodiment, the bacterial cell described herein has been
genetically engineered to comprise at least one heterologous glnH
gene. In one embodiment, the bacterial cell described herein has
been genetically engineered to comprise at least one heterologous
glnP gene. In one embodiment, the bacterial cell described herein
has been genetically engineered to comprise at least one
heterologous glnQ gene.
[0212] In one embodiment, the glnHPQ operon has at least about 80%
identity with the sequence of SEQ ID NO:41. Accordingly, in one
embodiment, the glnHPQ operon has at least about 90% identity with
the sequence of SEQ ID NO:41. Accordingly, in one embodiment, the
glnHPQ operon has at least about 95% identity with the sequence of
SEQ ID NO:41. Accordingly, in one embodiment, the glnHPQ operon has
at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the
sequence of SEQ ID NO:41. In another embodiment, the glnHPQ operon
comprises the sequence of SEQ ID NO:41. In yet another embodiment
the glnHPQ operon consists of the sequence of SEQ ID NO:41.
[0213] In one embodiment, the glnH gene has at least about 80%
identity with the sequence of SEQ ID NO:42. Accordingly, in one
embodiment, the glnH gene has at least about 90% identity with the
sequence of SEQ ID NO:42. Accordingly, in one embodiment, the glnH
gene has at least about 95% identity with the sequence of SEQ ID
NO:42. Accordingly, in one embodiment, the glnH gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:42. In another embodiment, the glnH gene comprises the
sequence of SEQ ID NO:42. In yet another embodiment the glnH gene
consists of the sequence of SEQ ID NO:42.
[0214] In one embodiment, the glnP gene has at least about 80%
identity with the sequence of SEQ ID NO:43. Accordingly, in one
embodiment, the glnP gene has at least about 90% identity with the
sequence of SEQ ID NO:43. Accordingly, in one embodiment, the glnP
gene has at least about 95% identity with the sequence of SEQ ID
NO:43. Accordingly, in one embodiment, the glnP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:43. In another embodiment, the glnP gene comprises the
sequence of SEQ ID NO:43. In yet another embodiment the glnP gene
consists of the sequence of SEQ ID NO:43.
[0215] In one embodiment, the glnQ gene has at least about 80%
identity with the sequence of SEQ ID NO:44. Accordingly, in one
embodiment, the glnQ gene has at least about 90% identity with the
sequence of SEQ ID NO:44. Accordingly, in one embodiment, the glnQ
gene has at least about 95% identity with the sequence of SEQ ID
NO:44. Accordingly, in one embodiment, the glnQ gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:44. In another embodiment, the glnQ gene comprises the
sequence of SEQ ID NO:44. In yet another embodiment the glnQ gene
consists of the sequence of SEQ ID NO:44.
[0216] In some embodiments, the glutamine transporter is encoded by
a glutamine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia coli. In some
embodiments, the bacterial species is Escherichia coli strain
Nissle.
[0217] Assays for testing the activity of a glutamine transporter,
a functional variant of a glutamine transporter, or a functional
fragment of transporter of glutamine are well known to one of
ordinary skill in the art. For example, import of glutamine may be
determined using the methods as described in Nohno et al., Mol.
Gen. Genet., 205(2):260-269, 1986, the entire contents of which are
expressly incorporated by reference herein.
[0218] In one embodiment, when the glutamine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more glutamine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the glutamine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
glutamine into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the glutamine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more
glutamine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the glutamine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more glutamine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions.
[0219] 7. Tryptophan Transporters
[0220] In one embodiment, the amino acid transporter is a
tryptophan transporter. Tryptophan transporters may be expressed or
modified in the recombinant bacteria described herein in order to
enhance tryptophan transport into the cell. Specifically, when the
tryptophan transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import more tryptophan
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
Thus, the genetically engineered bacteria comprising a heterologous
gene encoding a tryptophan transporter which may be used to import
tryptophan into the bacteria.
[0221] The uptake of tryptophan into bacterial cells is mediated by
proteins well known to those of skill in the art. For example,
three different tryptophan transporters, distinguishable on the
basis of their affinity for tryptophan have been identified in E.
coli (see, e.g., Yanofsky et al. (1991) J. Bacteriol. 173:
6009-17). The bacterial genes mtr, aroP, and tnaB encode tryptophan
permeases responsible for tryptophan uptake in bacteria. High
affinity permease, Mtr, is negatively regulated by the trp
repressor and positively regulated by the TyR product (see, e.g.,
Yanofsky et al. (1991) J. Bacteria 173: 6009-17 and Heatwole et al.
(1991) J. Bacteriol. 173: 3601-04), while AroP is negatively
regulated by the tyR product (Chye et al. (1987) J. Bacteria
169:386-93).
[0222] In one embodiment, the at least one gene encoding a
tryptophan transporter is a gene selected from the group consisting
of mtr, aroP and tnaB. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous gene selected from the group consisting of
mtr, aroP and tnaB. In one embodiment, the at least one gene
encoding a tryptophan transporter is the Escherichia coli mtr gene.
In one embodiment, the at least one gene encoding a tryptophan
transporter is the Escherichia coli aroP gene. In one embodiment,
the at least one gene encoding a tryptophan transporter is the
Escherichia coli tnaB gene.
[0223] In one embodiment, the mtr gene has at least about 80%
identity with the sequence of SEQ ID NO:46. Accordingly, in one
embodiment, the mtr gene has at least about 90% identity with the
sequence of SEQ ID NO:46. Accordingly, in one embodiment, the mtr
gene has at least about 95% identity with the sequence of SEQ ID
NO:46. Accordingly, in one embodiment, the mtr gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:46. In another embodiment, the mtr gene comprises the
sequence of SEQ ID NO:46. In yet another embodiment the mtr gene
consists of the sequence of SEQ ID NO:46.
[0224] In one embodiment, the tnaB gene has at least about 80%
identity with the sequence of SEQ ID NO:47. Accordingly, in one
embodiment, the tnaB gene has at least about 90% identity with the
sequence of SEQ ID NO:47. Accordingly, in one embodiment, the tnaB
gene has at least about 95% identity with the sequence of SEQ ID
NO:47. Accordingly, in one embodiment, the tnaB gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:47. In another embodiment, the tnaB gene comprises the
sequence of SEQ ID NO:47. In yet another embodiment the tnaB gene
consists of the sequence of SEQ ID NO:47.
[0225] In one embodiment, the aroP gene has at least about 80%
identity with the sequence of SEQ ID NO:48. Accordingly, in one
embodiment, the aroP gene has at least about 90% identity with the
sequence of SEQ ID NO:48. Accordingly, in one embodiment, the aroP
gene has at least about 95% identity with the sequence of SEQ ID
NO:48. Accordingly, in one embodiment, the aroP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:48. In another embodiment, the aroP gene comprises the
sequence of SEQ ID NO:48. In yet another embodiment the aroP gene
consists of the sequence of SEQ ID NO:48.
[0226] In some embodiments, the tryptophan transporter is encoded
by a tryptophan transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia,
Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or
Corynebacterium glutamicum. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0227] Assays for testing the activity of a tryptophan transporter,
a functional variant of a tryptophan transporter, or a functional
fragment of transporter of tryptophan are well known to one of
ordinary skill in the art. For example, import of tryptophan may be
determined using the methods as described in Shang et al. (2013) J.
Bacteriol. 195:5334-42, the entire contents of each of which are
expressly incorporated by reference herein.
[0228] In one embodiment, when the tryptophan transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more tryptophan into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the tryptophan transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
tryptophan into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
tryptophan transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import two-fold more
tryptophan into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the tryptophan
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more tryptophan into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0229] 8. Methionine Transporters
[0230] In one embodiment, the amino acid transporter is a
methionine transporter. Methionine transporters may be expressed or
modified in the recombinant bacteria described herein in order to
enhance methionine transport into the cell. Specifically, when the
methionine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import more methionine
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
Thus, the genetically engineered bacteria comprising a heterologous
gene encoding a methionine transporter which may be used to import
methionine into the bacteria.
[0231] The uptake of methionine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, a
methionine transporter operon has been identified in
Corynebacterium glutamicum (Trotschel et al., J. Bacteriology,
187(11):3786-3794, 2005). In addition, the high affinity MetD ABC
transporter system has been characterized in Escherichia coli
(Kadaba et al. (2008) Science 5886: 250-253; Kadner and Watson
(1974) J. Bacteriol. 119: 401-9). The MetD transporter system is
capable of mediating the translocation of several substrates across
the bacterial membrane, including methionine. The metD system of
Escherichia coli consists of MetN (encoded by metN), which
comprises the ATPase domain, Metl (encoded by meti), which
comprises the transmembrane domain, and MetQ (encoded by metQ), the
cognate binding protein which is located in the periplasm.
Orthologues of the genes encoding the E. coli metD transporter
system have been identified in multiple organisms including, e.g.,
Yersinia pestis, Vibrio cholerae, Pasteurella multocida,
Haemophilus influenza, Agrobacterium tumefaciens, Sinorhizobium
meliloti, Brucella meliloti, and Mesorhizobium loti (Merlin et al.
(2002) J. Bacteriol. 184: 5513-7).
[0232] In one embodiment, the at least one gene encoding a
methionine transporter is a metP gene, a metN gene, a metI gene, or
a metQ gene from Corynebacterium glutamicum, Escherichia coli, and
Bacillus subtilis (Trotschel et al., J. Bacteriology,
187(11):3786-3794, 2005).
[0233] In one embodiment, the metP gene has at least about 80%
identity with the sequence of SEQ ID NO:59. Accordingly, in one
embodiment, the metP gene has at least about 90% identity with the
sequence of SEQ ID NO:59. Accordingly, in one embodiment, the metP
gene has at least about 95% identity with the sequence of SEQ ID
NO:59. Accordingly, in one embodiment, the metP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:59. In another embodiment, the metP gene comprises the
sequence of SEQ ID NO:59. In yet another embodiment the metP gene
consists of the sequence of SEQ ID NO:59.
[0234] In one embodiment, the metN gene has at least about 80%
identity with the sequence of SEQ ID NO:60. Accordingly, in one
embodiment, the metN gene has at least about 90% identity with the
sequence of SEQ ID NO:60. Accordingly, in one embodiment, the metN
gene has at least about 95% identity with the sequence of SEQ ID
NO:60. Accordingly, in one embodiment, the metN gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:60. In another embodiment, the metN gene comprises the
sequence of SEQ ID NO:60. In yet another embodiment the metN gene
consists of the sequence of SEQ ID NO:60.
[0235] In one embodiment, the metI gene has at least about 80%
identity with the sequence of SEQ ID NO:61. Accordingly, in one
embodiment, the metI gene has at least about 90% identity with the
sequence of SEQ ID NO:61. Accordingly, in one embodiment, the metI
gene has at least about 95% identity with the sequence of SEQ ID
NO:61. Accordingly, in one embodiment, the metI gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:61. In another embodiment, the metI gene comprises the
sequence of SEQ ID NO:61. In yet another embodiment the metI gene
consists of the sequence of SEQ ID NO:61.
[0236] In one embodiment, the metQ gene has at least about 80%
identity with the sequence of SEQ ID NO:62. Accordingly, in one
embodiment, the metQ gene has at least about 90% identity with the
sequence of SEQ ID NO:62. Accordingly, in one embodiment, the metQ
gene has at least about 95% identity with the sequence of SEQ ID
NO:62. Accordingly, in one embodiment, the metQ gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:62. In another embodiment, the metQ gene comprises the
sequence of SEQ ID NO:62. In yet another embodiment the metQ gene
consists of the sequence of SEQ ID NO:62.
[0237] In some embodiments, the methionine transporter is encoded
by a methionine transporter gene derived from a bacterial genus or
species, including but not limited to, Corynebacterium glutamicum,
Escherichia coli, and Bacillus subtilis. In some embodiments, the
bacterial species is Escherichia coli strain Nissle.
[0238] Assays for testing the activity of a methionine transporter,
a functional variant of a methionine transporter, or a functional
fragment of a methionine transporter are well known to one of
ordinary skill in the art. For example, import of methionine may be
determined using the methods as described in Trotschel et al., J.
Bacteriology, 187(11):3786-3794, 2005, the entire contents of which
are expressly incorporated by reference herein.
[0239] In one embodiment, when the methionine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more methionine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the methionine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
methionine into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
methionine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import two-fold more
methionine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the methionine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more methionine into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0240] 9. Threonine Transporters
[0241] In one embodiment, the amino acid transporter is a threonine
transporter. Threonine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
threonine transport into the cell. Specifically, when the threonine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more threonine into
the cell when the transporter is expressed than unmodified bacteria
of the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a threonine transporter which may be used to import
threonine into the bacteria.
[0242] The uptake of threonine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, the
threonine transporter TdcC has been identified (Wook Lee et al.,
Nature Chemical Biology, 8:536-546, 2012). Additional
serine/threonine transporters have been identified and are
disclosed in the serine section herein.
[0243] In one embodiment, the at least one gene encoding a
threonine transporter is the tdcC gene. In one embodiment, the
bacterial cell described herein has been genetically engineered to
comprise at least one heterologous tdcC gene. In one embodiment,
the at least one gene encoding a threonine transporter is the
Escherichia coli tdcC gene. In one embodiment, the at least one
gene encoding a threonine transporter is the Salmonella typhimurium
tdcC gene.
[0244] In one embodiment, the tdcC gene has at least about 80%
identity with the sequence of SEQ ID NO:69. Accordingly, in one
embodiment, the tdcC gene has at least about 90% identity with the
sequence of SEQ ID NO:69. Accordingly, in one embodiment, the tdcC
gene has at least about 95% identity with the sequence of SEQ ID
NO:69. Accordingly, in one embodiment, the tdcC gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:69. In another embodiment, the tdcC gene comprises the
sequence of SEQ ID NO:69. In yet another embodiment the tdcC gene
consists of the sequence of SEQ ID NO:69.
[0245] In some embodiments, the threonine transporter is encoded by
a threonine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia coli or
Salmonella typhimurium. In some embodiments, the bacterial species
is Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0246] Assays for testing the activity of a threonine transporter,
a functional variant of a threonine transporter, or a functional
fragment of transporter of threonine are well known to one of
ordinary skill in the art. For example, import of threonine may be
determined using the methods as described in Wook Lee et al. (2012)
Nature Chemical Biology, 8:536-546, the entire contents of which
are expressly incorporated by reference herein.
[0247] In one embodiment, when the threonine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more threonine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the threonine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
threonine into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the threonine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more
threonine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the threonine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more threonine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions.
[0248] 10. Cysteine Transporters
[0249] In one embodiment, the amino acid transporter is a cysteine
transporter. Cysteine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
cysteine transport into the cell. Specifically, when the cysteine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more cysteine into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a cysteine transporter which may be used to import
cysteine into the bacteria so that any gene encoding a cysteine
catabolism enzyme expressed in the organism can catabolize the
cysteine to treat a disease associated with cysteine, such as
cancer.
[0250] The uptake of cysteine into bacterial cells is mediated by
proteins well known to those of skill in the art.
[0251] In some embodiments, the cysteine transporter is encoded by
a cysteine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia coli. In some
embodiments, the bacterial species is Escherichia coli. In some
embodiments, the bacterial species is Escherichia coli strain
Nissle.
[0252] Assays for testing the activity of a cysteine transporter, a
functional variant of a cysteine transporter, or a functional
fragment of transporter of cysteine are well known to one of
ordinary skill in the art.
[0253] In one embodiment, when the transporter of a cysteine is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more cysteine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the cysteine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more cysteine
into the bacterial cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the cysteine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more cysteine
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In yet another embodiment, when the cysteine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold, more cysteine
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same
conditions.
[0254] 11. Tyrosine Transporters
[0255] In one embodiment, the amino acid transporter is a tyrosine
transporter. Tyrosine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
tyrosine transport into the cell. Specifically, when the tyrosine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more tyrosine into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a tyrosine transporter which may be used to import
tyrosine into the bacteria.
[0256] The uptake of tyrosine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, a
tyrosine transporter TyrP has been identified in Lactobacillus
brevis (Wolken et al., J. Bacteriol., 188(6): 2198-2206, 2006) and
Escherichia coli.
[0257] In one embodiment, the at least one gene encoding a tyrosine
transporter is the tyrP gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous tyrP gene. In one embodiment, the at least
one gene encoding a tyrosine transporter is the Escherichia coli
tyrP gene. In one embodiment, the at least one gene encoding a
tyrosine transporter is the Lactobacillus brevi tyrP gene.
[0258] In one embodiment, the tyrP gene has at least about 80%
identity with the sequence of SEQ ID NO:87. Accordingly, in one
embodiment, the tyrP gene has at least about 90% identity with the
sequence of SEQ ID NO:87. Accordingly, in one embodiment, the tyrP
gene has at least about 95% identity with the sequence of SEQ ID
NO:87. Accordingly, in one embodiment, the tyrP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:87. In another embodiment, the tyrP gene comprises the
sequence of SEQ ID NO:87. In yet another embodiment the tyrP gene
consists of the sequence of SEQ ID NO:87.
[0259] In some embodiments, the tyrosine transporter is encoded by
a tyrosine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia coli or
Lactobacillus brevis. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0260] Assays for testing the activity of a tyrosine transporter, a
functional variant of a tyrosine transporter, or a functional
fragment of a tyrosine transporter are well known to one of
ordinary skill in the art. For example, import of tyrosine may be
determined using the methods as described in Wolken et al., J.
Bacteriol., 188(6):2198-2206, 2006, the entire contents of which
are expressly incorporated by reference herein.In one embodiment,
when the tyrosine transporter is expressed in the recombinant
bacterial cells described herein, the bacterial cells import 10%
more tyrosine into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In another embodiment, when the tyrosine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more tyrosine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In yet another
embodiment, when the tyrosine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import two-fold more tyrosine into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
tyrosine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import three-fold,
four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or
fifty-fold, more tyrosine into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions.
[0261] 12. Phenylalanine Transporters
[0262] In one embodiment, the amino acid transporter is a
phenylalanine transporter. Phenylalanine transporters may be
expressed or modified in the recombinant bacteria described herein
in order to enhance phenylalanine transport into the cell.
Specifically, when the phenylalanine transporter is expressed in
the recombinant bacterial cells described herein, the bacterial
cells import more phenylalanine into the cell when the transporter
is expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. Thus, the genetically engineered
bacteria comprising a heterologous gene encoding a phenylalanine
transporter which may be used to import phenylalanine into the
bacteria.
[0263] The uptake of phenylalanine into bacterial cells is mediated
by proteins well known to those of skill in the art. For example, a
phenylalanine transporter PheP has been identified (Pi et al.
(1991) J. Bacteriol. 173(12): 3622-9; Pi et al. (1996) J.
Bacteriol. 178(9): 2650-5; Pi et al. (1998) J. Bacteriol. 180(21):
5515-9; and Horsburgh et al. (2004) Infect. Immun. 72(5):
3073-3076). Additional phenylalanine transporters have been
identified and are known in the art.
[0264] In one embodiment, the at least one gene encoding a
phenylalanine transporter is the pheP gene. In one embodiment, the
bacterial cell described herein has been genetically engineered to
comprise at least one heterologous pheP gene. In one embodiment,
the at least one gene encoding a phenylalanine transporter is the
Escherichia coli pheP gene. In one embodiment, the at least one
gene encoding a phenylalanine transporter is the Staphylococcus
aureus pheP gene. "Phenylalanine transporter" is used to refer to a
membrane transport protein that is capable of transporting
phenylalanine into bacterial cells (see, e.g., Pi et al., 1991). In
Escherichia coli, the pheP gene encodes a high affinity
phenylalanine-specific permease responsible for phenylalanine
transport (Pi et al., 1998). In some embodiments, the phenylalanine
transporter is encoded by a pheP gene derived from a bacterial
species, including but not limited to, Acinetobacter calcoaceticus,
Salmonella enterica, and Escherichia coli. Other phenylalanine
transporters include Aageneral amino acid permease, encoded by the
aroP gene, transports three aromatic amino acids, including
phenylalanine, with high affinity, and is thought, together with
PheP, responsible for the lion share of phenylalanine import.
Additionally, a low level of phenylalanine transport activity has
been traced to the activity of the LIV-I/LS system, which is a
branched-chain amino acid transporter consisting of two periplasmic
binding proteins, the LIV-binding protein (LIV-I system) and
LS-binding protein (LS system), and membrane components, LivHMGF.
In some embodiments, the phenylalanine transporter is encoded by a
aroP gene derived from a bacterial species. In some embodiments,
the phenylalanine transporter is encoded by LIV-binding protein and
LS-binding protein and LivHMGF genes derived from a bacterial
species. In some embodiments, the genetically engineered bacteria
comprise more than one type of phenylalanine transporter, selected
from pheP, aroP, and the LIV-I/LS system.
[0265] In one embodiment, the pheP gene has at least about 80%
identity with the sequence of SEQ ID NO:98. Accordingly, in one
embodiment, the pheP gene has at least about 90% identity with the
sequence of SEQ ID NO:98. Accordingly, in one embodiment, the pheP
gene has at least about 95% identity with the sequence of SEQ ID
NO:98. Accordingly, in one embodiment, the pheP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:98. In another embodiment, the pheP gene comprises the
sequence of SEQ ID NO:98. In yet another embodiment the pheP gene
consists of the sequence of SEQ ID NO:98.
[0266] In some embodiments, the phenylalanine transporter is
encoded by a phenylalanine transporter gene derived from a
bacterial genus or species, including but not limited to,
Escherichia coli or Staphylococcus aureus. In some embodiments, the
bacterial species is Escherichia coli. In some embodiments, the
bacterial species is Escherichia coli strain Nissle.
[0267] Assays for testing the activity of a phenylalanine
transporter, a functional variant of a phenylalanine transporter,
or a functional fragment of a phenylalanine transporter are well
known to one of ordinary skill in the art. For example, import of
phenylalanine may be determined using the methods as described in
Pi et al. (1998) J. Bacterial. 180(21): 5515-9, the entire contents
of which are expressly incorporated by reference herein.
[0268] In one embodiment, when the phenylalanine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more phenylalanine into the bacterial
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In another
embodiment, when the phenylalanine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
phenylalanine into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
phenylalanine transporter is expressed in the recombinant bacterial
cells described herein, the bacterial cells import two-fold more
phenylalanine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the phenylalanine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more phenylalanine into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0269] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a phenylalanie transporter
may be used to treat a disease, condition, and/or symptom
associated with cancer, e.g., a cancer described herein. In some
embodiments, the recombinant bacterial cells described herein may
be used to reduce, ameliorate, or eliminate one or more symptom(s)
associated with a cancer.
[0270] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a phenylalanine transporter
may be used to treat a disease, condition, and/or symptom
associated with hyperphenylalaninemia. In some embodiments, the
recombinant bacterial cells described herein may be used to reduce,
ameliorate, or eliminate one or more symptom(s) associated with
hyperphenylalaninemia. In some embodiments, the disease is selected
from the group consisting of: phenylketonuria, classical or typical
phenylketonuria, atypical phenylketonuria, permanent mild
hyperphenylalaninemia, nonphenylketonuric hyperphenylalaninemia,
phenylalanine hydroxylase deficiency, cofactor deficiency,
dihydropteridine reductase deficiency, tetrahydropterin synthase
deficiency, and Segawa's disease. In some embodiments,
hyperphenylalaninemia is secondary to other conditions, e.g., liver
diseases. In some embodiments, the invention provides methods for
reducing, ameliorating, or eliminating one or more symptom(s)
associated with these diseases, including but not limited to
neurological deficits, mental retardation, encephalopathy,
epilepsy, eczema, reduced growth, microcephaly, tremor, limb
spasticity, and/or hypopigmentation. In some embodiments, the
subject to be treated is a human patient.
[0271] It was discovered that PAL1 and PAL3 expressed on a
high-copy plasmid and a low-copy plasmid in genetically engineered
E. coli Nissle metabolized and reduced phenylalanine to similar
levels, and the rate-limiting step of phenylalanine metabolism was
phenylalanine availability. Thus, in some embodiments for the
treatment of PKU, it is advantageous to increase phenylalanine
transport into the cell, thereby enhancing phenylalanine
metabolism. Unexpectedly, even low-copy PAL plasmids are capable of
almost completely eliminating Phe from a test sample when expressed
in conjunction with pheP. Furthermore, there may be additional
advantages to using a low-copy PAL-expressing plasmid in
conjunction with pheP in order to enhance the stability of PAL
expression while maintaining high phenylalanine metabolism, and to
reduce negative selection pressure on the transformed bacterium. In
alternate embodiments, the phenylalanine transporter is used in
conjunction with the high-copy plasmid.
[0272] The genetically engineered bacteria further comprise a gene
encoding a phenylalanine transporter. Phenylalanine transporters
may be expressed or modified in the genetically engineered bacteria
of the invention in order to enhance phenylalanine transport into
the cell.
[0273] PheP is a membrane transport protein that is capable of
transporting phenylalanine into bacterial cells (see, e.g., Pi et
al., 1991). In some embodiments, the native pheP gene in the
genetically modified bacteria of the invention is not modified. In
some embodiments, the genetically engineered bacteria of the
invention comprise multiple copies of the native pheP gene. In some
embodiments, the genetically engineered bacteria of the invention
comprise multiple copies of a non-native pheP gene. In some
embodiments, the genetically engineered bacteria of the invention
comprise a pheP gene that is controlled by its native promoter, an
inducible promoter, a promoter that is stronger than the native
promoter, e.g., the GlnRS promoter or the P(Bla) promoter, or a
constitutive promoter. In some embodiments, expression of the pheP
gene is controlled by a different promoter than the promoter that
controls expression of the gene encoding the
phenylalanine-metabolizing enzyme and/or the transcriptional
regulator. In some embodiments, expression of the pheP gene is
controlled by the same promoter that controls expression of the
phenylalanine-metabolizing enzyme and/or the transcriptional
regulator. In some embodiments, the pheP gene and the
phenylalanine-metabolizing enzyme and/or the transcriptional
regulator are divergently transcribed from a promoter region. In
some embodiments, expression of each of the genes encoding PheP,
the phenylalanine-metabolizing enzyme, and the transcriptional
regulator is controlled by a different promoter. In some
embodiments, expression of the genes encoding PheP, the
phenylalanine-metabolizing enzyme, and the transcriptional
regulator is controlled by the same promoter.
[0274] In some embodiments, the native pheP gene in the genetically
modified bacteria is not modified, and one or more additional
copies of the native pheP gene are inserted into the genome under
the control of the same inducible promoter that controls expression
of PAL, e.g., the FNR promoter, or a different inducible promoter
than the one that controls expression of PAL, or a constitutive
promoter. In alternate embodiments, the native pheP gene is not
modified, and a copy of a non-native pheP gene from a different
bacterial species is inserted into the genome under the control of
the same inducible promoter that controls expression of PAL, e.g.,
the FNR promoter, or a different inducible promoter than the one
that controls expression of PAL, or a constitutive promoter.
[0275] In some embodiments, the native pheP gene in the genetically
modified bacteria is not modified, and one or more additional
copies of the native pheP gene are present in the bacteria on a
plasmid and under the control of the same inducible promoter that
controls expression of PAL, e.g., the FNR promoter, or a different
inducible promoter than the one that controls expression of the
PME, or a constitutive promoter. In alternate embodiments, the
native pheP gene is not modified, and a copy of a non-native pheP
gene from a different bacterial species is present in the bacteria
on a plasmid and under the control of the same inducible promoter
that controls expression of PAL, e.g., the FNR promoter, or a
different inducible promoter than the one that controls expression
of PAL, or a constitutive promoter.
[0276] In some embodiments, the native pheP gene is mutagenized,
mutants exhibiting increased phenylalanine transport are selected,
and the mutagenized pheP gene is isolated and inserted into the
genetically engineered bacteria (see, e.g., Pi et al., 1996; Pi et
al., 1998). The phenylalanine transporter modifications described
herein may be present on a plasmid or chromosome.
[0277] In some embodiments, the genetically engineered bacterium is
E. coli Nissle, and the native pheP gene in E. coli Nissle is not
modified; one or more additional copies the native E. coli Nissle
pheP genes are inserted into the E. coli Nissle genome under the
control of the same inducible promoter that controls expression of
PAL, e.g., the FNR promoter, or a different inducible promoter than
the one that controls expression of PAL, or a constitutive
promoter. In an alternate embodiment, the native pheP gene in E.
coli Nissle is not modified, and a copy of a non-native pheP gene
from a different bacterium is inserted into the E. coli Nissle
genome under the control of the same inducible promoter that
controls expression of PAL, e.g., the FNR promoter, or a different
inducible promoter than the one that controls expression of PAL, or
a constitutive promoter. In some embodiments, the genetically
engineered bacterium is E. coli Nissle, and the native pheP gene in
E. coli Nissle is not modified; one or more additional copies the
native E. coli Nissle pheP genes are present in the bacterium on a
plasmid and under the control of the same inducible promoter that
controls expression of PAL, e.g., the FNR promoter, or a different
inducible promoter than the one that controls expression of PAL, or
a constitutive promoter. In an alternate embodiment, the native
pheP gene in E. coli Nissle is not modified, and a copy of a
non-native pheP gene from a different bacterium, are present in the
bacterium on a plasmid and under the control of the same inducible
promoter that controls expression of PAL, e.g., the FNR promoter,
or a different inducible promoter than the one that controls
expression of PAL, or a constitutive promoter.
[0278] It has been reported that Escherichia coli has five distinct
transport systems (AroP, Mtr, PheP, TnaB, and TyrP) for the
accumulation of aromatic amino acids. A general amino acid
permease, encoded by the aroP gene, transports three aromatic amino
acids, including phenylalanine, with high affinity, and is thought,
together with PheP, responsible for the lion share of phenylalanine
import. Additionally, a low level of accumulation of phenylalanine
was observed in an aromatic amino acid transporter-deficient E.
coli strain (.DELTA.aroP .DELTA.pheP .DELTA.mtr .DELTA.tna
.DELTA.tyrP), and was traced to the activity of the LIV-I/LS
system, which is a branched-chain amino acid transporter consisting
of two periplasmic binding proteins, the LIV-binding protein (LIV-I
system) and LS-binding protein (LS system), and membrane
components, LivHMGF (Koyanagi et al., and references therein;
Identification of the LIV-I/LS System as the Third Phenylalanine
Transporter in Escherichia coli K-12).
[0279] In some embodiments, the genetically engineered bacteria
comprise an aroP gene. In some embodiments, the genetically
engineered bacterium is E. coli Nissle, and the native aroP gene in
E. coli Nissle is not modified; one or more additional copies the
native E. coli Nissle aroP genes are present in the bacterium on a
plasmid and under the control of the same inducible promoter that
controls expression of the PME, e.g., the FNR promoter, or the
araBAD promoter, a different inducible promoter than the one that
controls expression of the PME, or a constitutive promoter. In an
alternate embodiment, the native aroP gene in E. coli Nissle is not
modified, and a copy of a non-native aroP gene from a different
bacterium, are present in the bacterium on a plasmid and under the
control of the same inducible promoter that controls expression of
the PME, e.g., the FNR promoter or the AraBAD promoter, or a
different inducible promoter than the one that controls expression
of the PME, or a constitutive promoter.
[0280] In other embodiments, the genetically engineered bacteria
comprise AroP and PheP, under the control of the same or different
inducible or constitutive promoters.
[0281] In some embodiments, the pheP gene is expressed on a
chromosome. In some embodiments, expression from the chromosome may
be useful for increasing stability of expression of pheP. In some
embodiments, the pheP gene is integrated into the bacterial
chromosome at one or more integration sites in the genetically
engineered bacteria. In some embodiments, the pheP gene is inserted
into the bacterial genome at one or more of the following insertion
sites in E. coli Nissle: malE/K, araC/BAD, lacZ, agaI/rsmI, thyA,
and malP/T. Any suitable insertion site may be used (see, e.g., The
insertion site may be anywhere in the genome, e.g., in a gene
required for survival and/or growth, such as thyA (to create an
auxotroph); in an active area of the genome, such as near the site
of genome replication; and/or in between divergent promoters in
order to reduce the risk of unintended transcription, such as
between AraB and AraC of the arabinose operon.
[0282] In some embodiments, the genetically engineered bacterium
comprises multiple mechanisms of action and/or one or more
auxotrophies. In certain embodiments, the bacteria are genetically
engineered to comprise five copies of PAL under the control of an
oxygen level-dependent promoter (e.g., P.sub.fnrS-PAL3) inserted at
different integration sites on the chromosome (e.g., malE/K,
yicS/nepI, malP/T, agaI/rsmI, and cea), and one copy of a
phenylalanine transporter gene under the control of an oxygen
level-dependent promoter (e.g., P.sub.fnrS-pheP) inserted at a
different integration site on the chromosome (e.g., lacZ). In a
more specific aspect, the bacteria are genetically engineered to
further include a kanamycin resistance gene, and a thyA auxotrophy,
in which the thyA gene is deleted and/or replaced with an unrelated
gene.
[0283] 13. Glutamic Acid Transporters
[0284] In one embodiment, the amino acid transporter is a glutamic
transporter. Glutamic acid transporters may be expressed or
modified in the recombinant bacteria described herein in order to
enhance glutamic acid transport into the cell. Specifically, when
the glutamic acid transporter is expressed in the recombinant
bacterial cells described herein, the bacterial cells import more
glutamic acid into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. Thus, the genetically engineered bacteria comprising a
heterologous gene encoding a glutamic acid transporter which may be
used to import glutamic acid into the bacteria.
[0285] The uptake of glutamic acid into bacterial cells is mediated
by proteins well known to those of skill in the art. For example, a
Natcoupled symporter GltT for glutamic acid uptake has been
identified in Bacillus subtilis (see, e.g., Zaprasis et al. (2015)
App. Env. Microbiol. 81:250-9). The bacterial gene gltT encodes a
glutamic acid transporter responsible for glutamic acid uptake in
many bacteria (see, e.g., Jan Slotboom et al. (1999) Microb. Mol.
Biol. Rev.63:293-307; Takahashi et al. (2015) Inf. Imm. 83:3555-67;
Ryan et al. (2007) Nat. Struct. Mol. Biol. 14:365-71; and Tolner et
al. (1992) Mol. Microbiol. 6:2845-56).
[0286] In one embodiment, the at least one gene encoding a glutamic
acid transporter is the gltT gene. In one embodiment, the bacterial
cell described herein has been genetically engineered to comprise
at least one heterologous gltT gene. In one embodiment, the at
least one gene encoding a glutamic acid transporter is the
Escherichia coli gltP gene. In one embodiment, the at least one
gene encoding a glutamic acid transporter is the Bacillus subtilis
gltT gene. In one embodiment, the at least one gene encoding a
glutamic acid transporter is the Mycobacterium tuberculosis dctA
gene. In one embodiment, the at least one gene encoding a glutamic
acid transporter is the Salmonella typhimurium dctA gene. In one
embodiment, the at least one gene encoding a glutamic acid
transporter is the Caenorhabditis elegans gltT gene.
[0287] In one embodiment, the gltT gene has at least about 80%
identity with the sequence of SEQ ID NO:91. Accordingly, in one
embodiment, the gltT gene has at least about 90% identity with the
sequence of SEQ ID NO:91. Accordingly, in one embodiment, the gltT
gene has at least about 95% identity with the sequence of SEQ ID
NO:91. Accordingly, in one embodiment, the gltT gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:91. In another embodiment, the gltT gene comprises the
sequence of SEQ ID NO:91. In yet another embodiment the gltT gene
consists of the sequence of SEQ ID NO:91.
[0288] In some embodiments, the glutamic acid transporter is
encoded by a glutamic acid transporter gene derived from a
bacterial genus or species, including but not limited to,
Escherichia, Bacillus, Chlamydia, Mycobacterium, Salmonella,
Escherichia coli, Mycobacterium tuberculosis, Salmonella
typhimurium, or Caenorhabditis elegans (see, e.g., Jan Slotboom et
al. (1999) Microbiol. Mol. Biol. Rev. 63:293-307) In some
embodiments, the bacterial species is Escherichia coli. In some
embodiments, the bacterial species is Escherichia coli strain
Nissle.
[0289] Assays for testing the activity of a glutamic acid
transporter, a functional variant of a glutamic acid transporter,
or a functional fragment of transporter of glutamic acid are well
known to one of ordinary skill in the art. For example, import of
glutamic acid may be determined using the methods as described in
Zaprasis et al. (2015) App. Env. Microbiol. 81:250-9, the entire
contents of each of which are expressly incorporated by reference
herein.
[0290] In one embodiment, when the glutamic acid transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more glutamic acid into the bacterial
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In another
embodiment, when the glutamic acid transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more glutamic
acid into the bacterial cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the glutamic acid
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more glutamic
acid into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the glutamic acid
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more glutamic acid into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0291] 14. Histidine Transporters
[0292] In one embodiment, the amino acid transporter is a histidine
transporter. Histidine transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
histidine transport into the cell. Specifically, when the histidine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more histidine into
the cell when the transporter is expressed than unmodified bacteria
of the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a histidine transporter which may be used to import
histidine into the bacteria.
[0293] The uptake of histidine into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, a
histidine transport system is encoded by the hisJQMP operon and the
artJ gene (see, e.g., Caldara et al. (2007) J. Mol. Biol. 373(2):
251-67). Transport by the histidine transport system is mediated by
several proteins regulated by the ArgR-L-arginine DNA-binding
transcriptional dual regulator. ArgR complexed with L-arginine
represses the transcription of several genes involved in transport
of histidine. In this system, HisJ (encoded by hisJ) is a histidine
ABC transporter-periplasmic binding protein, HisQ and HisM (encoded
by hisQ and hisM respectively) are the lysine/arginine/ornithine
ABC transporter/histidine ABC transporter-membrane subunits, HisP
(encoded by hisP) is a lysine/arginine/ornithine ABC
transporter/histidine ABC transporter-ATP binding subunit. This
system has been well characterized in Escherichia coli. In
addition, bacterial systems that are homologous and orthologous to
the E. coli histidine-specific system have been characterized in
other bacterial species, including, for example, Pseudomonas
fluorescens (see, e.g., Bender (2012) Microbiol. Mol. Biol. Reviews
76: 565-584). The membranous and membrane-associated proteins of
the histidine permease (Q M P complex), encoded by the hisJQMP
operon mediate the uptake of histidine (see, e.g., Oh et al. (1994)
J. Biol. Chem. 269(42): 26323-30).
[0294] In one embodiment, the at least one gene encoding a
histidine transporter comprises the hisJQMP operon. In one
embodiment, the at least one gene encoding a histidine transporter
comprises the hisJ gene. In one embodiment, the at least one gene
encoding a histidine transporter comprises the hisQ gene. In one
embodiment, the at least one gene encoding a histidine transporter
comprises the hisM gene. In one embodiment, the at least one gene
encoding a histidine transporter comprises the hisP gene.
[0295] In one embodiment, the hisJ gene has at least about 80%
identity with the sequence of SEQ ID NO:94. Accordingly, in one
embodiment, the hisJ gene has at least about 90% identity with the
sequence of SEQ ID NO:94. Accordingly, in one embodiment, the hisJ
gene has at least about 95% identity with the sequence of SEQ ID
NO:94. Accordingly, in one embodiment, the hisJ gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:94. In another embodiment, the hisJ gene comprises the
sequence of SEQ ID NO:94. In yet another embodiment, the hisJ gene
consists of the sequence of SEQ ID NO:94.
[0296] In one embodiment, the hisQ gene has at least about 80%
identity with the sequence of SEQ ID NO:95. Accordingly, in one
embodiment, the hisQ gene has at least about 90% identity with the
sequence of SEQ ID NO:95. Accordingly, in one embodiment, the hisQ
gene has at least about 95% identity with the sequence of SEQ ID
NO:95. Accordingly, in one embodiment, the hisQ gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:95. In another embodiment, the hisQ gene comprises the
sequence of SEQ ID NO:95. In yet another embodiment, the hisQ gene
consists of the sequence of SEQ ID NO:95.
[0297] In one embodiment, the hisM gene has at least about 80%
identity with the sequence of SEQ ID NO:103. Accordingly, in one
embodiment, the hisM gene has at least about 90% identity with the
sequence of SEQ ID NO:103. Accordingly, in one embodiment, the hisM
gene has at least about 95% identity with the sequence of SEQ ID
NO:103. Accordingly, in one embodiment, the hisM gene nhas at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:103. In another embodiment, the hisM gene comprises the
sequence of SEQ ID NO:103. In yet another embodiment, the hisM gene
consists of the sequence of SEQ ID NO:103.
[0298] In one embodiment, the hisP gene has at least about 80%
identity with the sequence of SEQ ID NO:96. Accordingly, in one
embodiment, the hisP gene has at least about 90% identity with the
sequence of SEQ ID NO:96. Accordingly, in one embodiment, the hisP
gene has at least about 95% identity with the sequence of SEQ ID
NO:96. Accordingly, in one embodiment, the hisP gene nhas at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:96. In another embodiment, the hisP gene comprises the
sequence of SEQ ID NO:96. In yet another embodiment, the hisP gene
consists of the sequence of SEQ ID NO:96.
[0299] In some embodiments, the histidine transporter is encoded by
a histidine transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia and Pseudomonas
In some embodiments, the bacterial species is Escherichia coli. In
some embodiments, the bacterial species is Escherichia coli strain
Nissle.
[0300] Assays for testing the activity of a histidine transporter,
a functional variant of a histidine transporter, or a functional
fragment of a histidine transporter are well known to one of
ordinary skill in the art. For example, import of histidine may be
determined using the methods described in Liu et al. (1997) J.
Biol. Chem. 272: 859-866 and Shang et al. (2013) J. Bacteriology.
195(23): 5334-5342, the entire contents of each of which are
expressly incorporated by reference herein.
[0301] In one embodiment, when the histidine transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import 10% more histidine into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In another
embodiment, when the histidine transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
histidine into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the histidine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import two-fold more
histidine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the histidine
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold,
more histidine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions.
[0302] 15. Proline Transporters
[0303] In one embodiment, the amino acid transporter is a proline
transporter. Proline transporters may be expressed or modified in
the recombinant bacteria described herein in order to enhance
proline transport into the cell. Specifically, when the proline
transporter is expressed in the recombinant bacterial cells
described herein, the bacterial cells import more proline into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. Thus, the
genetically engineered bacteria comprising a heterologous gene
encoding a proline transporter which may be used to import proline
into the bacteria.
[0304] The uptake of proline into bacterial cells is mediated by
proteins well known to those of skill in the art. The proline
utilization operon (put) allows bacterial cells to transport and
use proline. The put operon consists of two genes putA and putP. In
bacteria, there are two distinct systems for proline uptake,
proline porter I (PPI) and proline porter II (PPII) (see, e.g.,
Grothe (1986) J. Bacteriol. 166: 253-259). The bacterial gene putP
encodes a proline transporter responsible for proline uptake in
many bacteria (see, e.g., Ostrovsky et al. (1993) Proc. Natl. Acad.
Sci. 90: 429-8; Grothe (1986) J. Bacteriol. 166: 253-259). The putA
gene expresses a polypeptide that has proline dehydrogenase (EC
1.5.99.8) activity and pyrroline-5-carboxylate (P5C) (EC 1.5.1.12)
activity (see, e.g., Menzel and Roth (1981) J. Biol. Chem.
256:9755-61). In the absence of proline, putA remains in the
cytoplasm and represses put gene expression. In the presence of
proline, putA binds to the membrane relieving put repression
allowing put gene expression (see, e.g., Ostrovsky et al. (1993)
Proc. Natl. Acad. Sci. 90: 429-8).
[0305] In one embodiment, the at least one gene encoding a proline
transporter is the putP gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous putP gene. In one embodiment, the at least
one gene encoding a proline transporter is the Escherichia coli
putP gene. In one embodiment, the at least one gene encoding a
proline transporter is the Salmonella typhimurium putP gene. In one
embodiment, the at least one gene encoding a proline transporter is
the Escherichia coli putP gene.
[0306] In one embodiment, the putP gene has at least about 80%
identity with the sequence of SEQ ID NO:98. Accordingly, in one
embodiment, the putP gene has at least about 90% identity with the
sequence of SEQ ID NO:98. Accordingly, in one embodiment, the putP
gene has at least about 95% identity with the sequence of SEQ ID
NO:98. Accordingly, in one embodiment, the putP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:98. In another embodiment, the putP gene comprises the
sequence of SEQ ID NO:98. In yet another embodiment the putP gene
consists of the sequence of SEQ ID NO:98.
[0307] In some embodiments, the proline transporter is encoded by a
proline transporter gene derived from a bacterial genus or species,
including but not limited to, Escherichia, Salmonella, Escherichia
coli or Salmonella typhimurium. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0308] Assays for testing the activity of a proline transporter, a
functional variant of a proline transporter, or a functional
fragment of a proline transporter are well known to one of ordinary
skill in the art. For example, import of proline may be determined
using the methods as described in Moses et al. (2012) Journal of
Bacteriology 194: 745-58 and Hoffman et al. (2012) App. and Enviro.
Microbiol. 78: 5753-62), the entire contents of each of which are
expressly incorporated by reference herein.
[0309] In one embodiment, when the proline transporter is expressed
in the recombinant bacterial cells described herein, the bacterial
cells import 10% more proline into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the proline transporter is expressed in the recombinant
bacterial cells described herein, the bacterial cells import 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more proline into the
bacterial cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In yet another embodiment, when the proline transporter is
expressed in the recombinant bacterial cells described herein, the
bacterial cells import two-fold more proline into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the proline transporter is expressed in the
recombinant bacterial cells described herein, the bacterial cells
import three-fold, four-fold, five-fold, six-fold, seven-fold,
eight-fold, nine-fold, ten-fold, fifteen-fold, twenty-fold,
thirty-fold, fourty-fold, or fifty-fold, more proline into the cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions.
B. Nucleoside Transporters
[0310] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a nucleoside
transporter. In one embodiment, the nucleoside transporter is a
purine nucleoside transporter. In one embodiment, the nucleoside
transporter is a pyrimidine nucleoside transporter. In one
embodiment, the nucleoside transporter transports at least one
nucleoside selected from the group consisting of adenosine,
guanosine, uridine, inosine, xanthosine, thymidine and cytidine,
into the cell.
[0311] The uptake of nucleosides into bacterial cells is mediated
by proteins well known to those of skill in the art. For example,
many bacteria scavenge nucleosides from the environment for the
synthesis of nuceotides and deoxynucleotides. In some bacterial
species, e.g., Escherichia coli, nucleosides can be used as the
sole source of nitrogen and carbon for growth (see, e.g., Neuhard
and Nygaard "Biosynthesis and conversion of nucleotides, purines
and pyrimidines," in: Neidhardt F C, Ingraham J L, Low K B,
Magasanik B, Schaechter M, Umbarger H E, editors. Escherichia coli
and Salmonella typhimurium: Cellular and Molecular Biology.
Washington DC: ASM Press; 1987. pp. 445-473). Two evolutionarily
unrelated cation-linked transporter families have been identified:
the concentrative nucleoside transporter (CNT) family and the
nucleoside:H.sup.+ Symporter (NHS) family, both of which are
responsible for nucleoside uptake (see, e.g., Cabrita et al. (2002)
Biochem. Cell Biol. 80(5): 623-38, the contents of which is herein
incorporated by reference in its entirety).
[0312] Passive transport of nucleosides across the outer membrane
of some Gram-negative bacteria, e.g., Salmonella enterica, and into
the periplasm can be mediated by the Tsx porin, encoded by the tsx
gene (see, e.g., Bucarey et al. (20005) Infect. Immun. 73(10):
6210-9).
[0313] Active transport of nucleosides across the inner membrance
is mediated by the nucleoside permeases NupC and NupG, encoded by
the nupC and nupG genes, respectively. NupG can facilitate the
uptake of all tested purine and pyrimidine nucleosides while NupC
has specificity towards the pyrimidine nucleosides and their
deoxyderivatives. Both permeases are powered by proton motive
force. E. coli mutants defective in both the nupC and nupG genes
cannot grow with nucleosides as their single carbon source. Both
permeases are proton-linked but they differ in their selectivity.
NupG is capable of transporting a wide range of nucleosides and
deoxynucleosides; in contrast, NupC does not transport guanosine or
deoxyguanosine. Homologs of NupG from E. coli are found in a wide
range of bacteria, including human gut pathogens such as Salmonella
typhimurium, organisms associated with periodontal disease such as
Porphyromonas gingivalis and Prevotella intermedia, and plant
pathogens of the genus Erwinia (see, e.g., Vaziri et al. (2013)
Mol. Membr. Biol. 30(1-2): 114-128, the contents of which is herein
incorporated by reference in its entirety).
[0314] An additional nucleoside transporter, the xanthosine
permease, XapB, having 58% identity to NupG was identified in
Escherichia coli Norholm and Dandanell (2001) J. Bacteriol.
183(16): 4900-4. XapB exhibits similar specificity to NupG, since
it appears to be able to transport all nucelosides except
guanosine. Putative bacterial transporters from the CNT superfamily
and transporters from the NupG/XapB family include those listed in
the tables 2 and 3 below. In addition, codB (GenBank P25525,
Escherichia coli) was identified based on homology to a yeast
transporter family termed the uracil/allantoin transportor family
(Cabrita et al. (2002)).
TABLE-US-00003 TABLE 2 Putative CNT family transporters Name
GenBank Accession No. Organism BH1446 BAB05165 Bacillus halodurans
BsNupC CAA57663 Bacillus subtilis BsyutK CAB15208 B. subtilis
BsyxjA CAB15938 B. subtilis CcCNT (CC2089) AAK24060 Caulobacter
crescentus yeiJ AAC75222 E. coli yeiM AAC75225 E. coli HI0519
AAC22177 Haemophilus influenzae HP1180 (NupC) AAD08224 Helicobacter
pylori SA0600 (NupC) BAB41833 Staphylococcus aureus SAV0645 (NupC)
BAB56807 S. aureus SpNupC AAK34582 Streptococcus pyogenes VC2352
(NupC) AAF95495 Vibrio cholerae VC1953 (NupC) AAF95101 V. cholera
VCA0179 AAF96092 V. cholera
TABLE-US-00004 TABLE 3 Bacterial transporters from the NupG/XapB
family Protein (gene name) GenBank Accession No. Organism yegT
P76417 Escherichia coli NupG P09452 E. coli XapB P45562 E. coli
CC1628 AAK23606 Caulobacter crescentus
[0315] In one embodiment, the nucleoside transporter is a
nucleoside permease (e.g., NupC or NupG). In one embodiment, the
nucleoside transporter is a adenosine permease. In one embodiment,
the nucleoside transporter is a guanosine permease. In one
embodiment, the nucleoside transporter is a uridine permease. In
one embodiment, the nucleoside transporter is a inosine permease.
In one embodiment, the nucleoside transporter is a xanthosine
permease. In one embodiment, the nucleoside transporter is a
thymidine permease. In one embodiment, the nucleoside transporter
is a cytidine permease.
[0316] In one embodiment, the nucleoside transporter is a
nucleoside porin (e.g., Tsx). In one embodiment, the nucleoside
transporter is a sodium-dependent nucleoside transporter. In one
embodiment, the nucleoside transporter is a xanthosine transporter
(e.g., XapB).
[0317] Nucleoside transporters may be expressed or modified in the
bacteria in order to enhance nucleoside transport into the cell.
Specifically, when the nucleoside transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
nucleoside(s) into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a nucleoside transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a nucleoside transporter and a genetic modification that
reduces export of a nucleoside, e.g., a genetic mutation in an
exporter gene or promoter.
[0318] In one embodiment, the bacterial cell comprises at least one
gene encoding a nucleoside transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a
nucleoside transporter. In some embodiments, the at least one
native gene encoding a nucleoside transporter is not modified. In
another embodiment, the bacterial cell comprises more than one copy
of at least one native gene encoding a nucleoside transporter. In
yet another embodiment, the bacterial cell comprises a copy of at
least one gene encoding a native nucleoside transporter, as well as
at least one copy of at least one heterologous gene encoding
annucleoside transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a nucleoside transporter. In one embodiment, the bacterial
cell comprises multiple copies of the at least one heterologous
gene encoding a nucleoside transporter.
[0319] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a nucleoside transporter, wherein said
nucleoside transporter comprises a nucleoside sequence that has at
least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity
to the nucleoside sequence of a polypeptide encoded by a nucleoside
transporter gene disclosed herein.
[0320] In some embodiments, the nucleoside transporter is encoded
by a nucleoside transporter gene derived from a bacterial genus or
species, including but not limited to, Bacillus halodurans,
Bacillus subtilis, Caulobacter crescentus, Escherichia coli,
Haemoophilus influenzae, Helicobacter pylori, Pseudomonas, Bacillus
subtilis, Escherichia coli, Prevotella intermedia, Porphytomonas
gingivalis, Salmonella typhimurium, Salmonella enterica, or Vibrio
cholera. In some embodiments, the bacterial species is Escherichia
coli. In some embodiments, the bacterial species is Escherichia
coli strain Nissle.
[0321] The present disclosure further comprises genes encoding
functional fragments of a nucleoside transporter or functional
variants of a nucleoside transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
nucleoside transporter relates to an element having qualitative
biological activity in common with the wild-type nucleoside
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
nucleoside transporter is one which retains essentially the same
ability to import a nucleoside into the bacterial cell as does the
nucleoside transporter protein from which the functional fragment
or functional variant was derived. In one embodiment, the
recombinant bacterial cell comprises at least one heterologous gene
encoding a functional fragment of a nucleoside transporter. In
another embodiment, the recombinant bacterial cell comprises a
heterologous gene encoding a functional variant of a nucleoside
transporter.
[0322] Assays for testing the activity of a nucleoside transporter,
a functional variant of a nucleoside transporter, or a functional
fragment of a nucleoside transporter are well known to one of
ordinary skill in the art. For example, import of a nucleoside may
be determined using, e.g., a .sup.14C-labeled nucleoside uptake
assay as described in Norholm and Dandanell (2001) J. Bacteriol.
183(16): 4900-4, the entire contents of each of which are expressly
incorporated by reference herein.
[0323] In one embodiment, the genes encoding the nucleoside
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the nucleoside transporter have been codon-optimized
for use in Escherichia coli.
[0324] The present disclosure also encompasses genes encoding a
nucleoside transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0325] In some embodiments, the at least one gene encoding a
nucleoside transporter is mutagenized; mutants exhibiting increased
nucleoside import are selected; and the mutagenized at least one
gene encoding a nucleoside transporter is isolated and inserted
into the bacterial cell. In some embodiments, the at least one gene
encoding a nucleoside transporter is mutagenized; mutants
exhibiting decreased nucleoside import are selected; and the
mutagenized at least one gene encoding a nucleoside transporter is
isolated and inserted into the bacterial cell. The transporter
modifications described herein may be present on a plasmid or
chromosome.
[0326] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a nucleoside transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding a
nucleoside transporter is directly operably linked to the promoter.
In another embodiment, the at least one gene encoding a nucleoside
transporter is indirectly operably linked to the promoter.
[0327] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a nucleoside transporter in nature.
In some embodiments, the at least one gene encoding the nucleoside
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the nucleoside
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the nucleoside
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the nucleoside transporter is controlled by a constitutive
promoter.
[0328] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0329] In one embodiment, the at least one gene encoding a
nucleoside transporter is located on a plasmid in the bacterial
cell. In some embodiments, the plasmid is a high copy number
plasmid. In some embodiments, the plasmid is a low copy number
plasmid. In another embodiment, the at least one gene encoding a
nucleoside transporter is located in the chromosome of the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a nucleoside transporter is located in the
chromosome of the bacterial cell, and a copy of at least one gene
encoding a nucleoside transporter from a different species of
bacteria is located on a plasmid in the bacterial cell. In yet
another embodiment, a native copy of the at least one gene encoding
a nucleoside transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding a nucleoside
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a nucleoside transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a nucleoside transporter from a
different species of bacteria is located in the chromosome of the
bacterial cell.
[0330] In some embodiments, the at least one native gene encoding
the nucleoside transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the nucleoside transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0331] In some embodiments, at least one native gene encoding the
nucleoside transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the nucleoside transporter in the
bacterial cell is modified, and one or more additional copies of at
least one native gene encoding the transporter are present in the
bacterial cell on a plasmid. In alternate embodiments, the at least
one native gene encoding the transporter is modified, and a copy of
at least one gene encoding the transporter from a different
bacterial species is present in the bacteria on a plasmid.
[0332] In one embodiment, the nucleoside transporter is encoded by
a tsx gene, e.g., a tsx gene disclosed herein. In one embodiment,
the tsx gene has at least about 80% identity with the sequence of
SEQ ID NO:107. Accordingly, in one embodiment, the tsx gene has at
least about 90% identity with the sequence of SEQ ID NO:107.
Accordingly, in one embodiment, the tsx gene has at least about 95%
identity with the sequence of SEQ ID NO:107. Accordingly, in one
embodiment, the tsx gene has at least about 70%, 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity with the sequence of SEQ ID NO:107. In another
embodiment, the tsx gene comprises the sequence of SEQ ID NO:107.
In yet another embodiment the tsx gene consists of the sequence of
SEQ ID NO:107.
[0333] In one embodiment, the tsx gene has at least about 80%
identity with the sequence of SEQ ID NO:108. Accordingly, in one
embodiment, the tsx gene has at least about 90% identity with the
sequence of SEQ ID NO:108. Accordingly, in one embodiment, the tsx
gene has at least about 95% identity with the sequence of SEQ ID
NO:108. Accordingly, in one embodiment, the tsx gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:108. In another embodiment, the tsx gene comprises the
sequence of SEQ ID NO:108. In yet another embodiment the tsx gene
consists of the sequence of SEQ ID NO:108.
[0334] In one embodiment, the nucleoside transporter is encoded by
a BH1446 gene, e.g., a BH1446 gene disclosed herein. In one
embodiment, the BH1446 gene has at least about 80% identity with
the sequence of SEQ ID NO:109. Accordingly, in one embodiment, the
BH1446 gene has at least about 90% identity with the sequence of
SEQ ID NO:109. Accordingly, in one embodiment, the BH1446 gene has
at least about 95% identity with the sequence of SEQ ID NO:109.
Accordingly, in one embodiment, the BH1446 gene has at least about
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID
NO:109. In another embodiment, the BH1446 gene comprises the
sequence of SEQ ID NO:109. In yet another embodiment the BH1446
gene consists of the sequence of SEQ ID NO:109.
[0335] In one embodiment, the nucleoside transporter is encoded by
a nupC gene, e.g., a nupC gene disclosed herein. In one embodiment,
the nupC gene is a nupC gene from Bacillus subtilis. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:110. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:110. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:110. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:110. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:110.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:110.
[0336] In one embodiment, the nupC gene is a nupC gene from
Helicobacter pylori. In one embodiment, the nupC gene has at least
about 80% identity with the sequence of SEQ ID NO:117. Accordingly,
in one embodiment, the nupC gene has at least about 90% identity
with the sequence of SEQ ID NO:117. Accordingly, in one embodiment,
the nupC gene has at least about 95% identity with the sequence of
SEQ ID NO:117. Accordingly, in one embodiment, the nupC gene has at
least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of
SEQ ID NO:117. In another embodiment, the nupC gene comprises the
sequence of SEQ ID NO:117. In yet another embodiment the nupC gene
consists of the sequence of SEQ ID NO:117.
[0337] In one embodiment, the nupC (also referred to herein as
SA0600) gene is a nupC gene from Staphylococcus aureus. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:118. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:118. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:118. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:118. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:118.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:118.
[0338] In one embodiment, the nupC (also referred to herein as
SAV0645) gene is a nupC gene from Staphylococcus aureus. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:119. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:119. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:119. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:119. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:119.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:119.
[0339] In one embodiment, the nupC (also referred to herein as
spNupC) gene is a nupC gene from Streptococcus pyogenes. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:120. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:120. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:120. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:120. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:120.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:120.
[0340] In one embodiment, the nupC (also referred to herein as
VC2352) gene is a nupC gene from Vibrio cholerae. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:121. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:121. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:121. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:121. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:121.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:121.
[0341] In one embodiment, the nupC (also referred to herein as
VC1953) gene is a nupC gene from Vibrio cholerae. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:122. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:122. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:122. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:122. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:122.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:122.
[0342] In one embodiment, the nupC (also referred to herein as
VCA0179) gene is a nupC gene from Vibrio cholerae. In one
embodiment, the nupC gene has at least about 80% identity with the
sequence of SEQ ID NO:123. Accordingly, in one embodiment, the nupC
gene has at least about 90% identity with the sequence of SEQ ID
NO:123. Accordingly, in one embodiment, the nupC gene has at least
about 95% identity with the sequence of SEQ ID NO:123. Accordingly,
in one embodiment, the nupC gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:123. In another
embodiment, the nupC gene comprises the sequence of SEQ ID NO:123.
In yet another embodiment the nupC gene consists of the sequence of
SEQ ID NO:123.
[0343] In one embodiment, the nucleoside transporter is encoded by
a yutK gene, e.g., a yutK gene disclosed herein. In one embodiment,
the yutK gene is a yutK gene from Bacillus subtilis. In one
embodiment, the yutK gene has at least about 80% identity with the
sequence of SEQ ID NO:111. Accordingly, in one embodiment, the yutK
gene has at least about 90% identity with the sequence of SEQ ID
NO:111. Accordingly, in one embodiment, the yutK gene has at least
about 95% identity with the sequence of SEQ ID NO:111. Accordingly,
in one embodiment, the yutK gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:111. In another
embodiment, the yutK gene comprises the sequence of SEQ ID NO:111.
In yet another embodiment the yutK gene consists of the sequence of
SEQ ID NO:111.
[0344] In one embodiment, the nucleoside transporter is encoded by
a yxjA gene, e.g., a yxjA gene disclosed herein. In one embodiment,
the yxjA gene is a yxjA gene from Bacillus subtilis. In one
embodiment, the yxjA gene has at least about 80% identity with the
sequence of SEQ ID NO:112. Accordingly, in one embodiment, the yxjA
gene has at least about 90% identity with the sequence of SEQ ID
NO:112. Accordingly, in one embodiment, the yxjA gene has at least
about 95% identity with the sequence of SEQ ID NO:112. Accordingly,
in one embodiment, the yxjA gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:112. In another
embodiment, the yxjA gene comprises the sequence of SEQ ID NO:112.
In yet another embodiment the yxjA gene consists of the sequence of
SEQ ID NO:112.
[0345] In one embodiment, the nucleoside transporter is encoded by
a sodium-dependent nucleoside transporter gene, e.g., a
sodium-dependent nucleoside transporter gene disclosed herein. In
one embodiment, the sodium-dependent nucleoside transporter gene is
a CC2089 (also referred to herein as CcCNT) gene. In one
embodiment, the sodium-dependent nucleoside transporter gene is a
CC2089 gene from Caulobacter crescentus. In one embodiment, the
sodium-dependent nucleoside transporter gene has at least about 80%
identity with the sequence of SEQ ID NO:113. Accordingly, in one
embodiment, the sodium-dependent nucleoside transporter gene has at
least about 90% identity with the sequence of SEQ ID NO:113.
Accordingly, in one embodiment, the sodium-dependent nucleoside
transporter gene has at least about 95% identity with the sequence
of SEQ ID NO:113. Accordingly, in one embodiment, the
sodium-dependent nucleoside transporter gene has at least about
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID
NO:113. In another embodiment, the sodium-dependent nucleoside
transporter gene comprises the sequence of SEQ ID NO:113. In yet
another embodiment the sodium-dependent nucleoside transporter gene
consists of the sequence of SEQ ID NO:113.
[0346] In one embodiment, the nucleoside transporter is encoded by
a yeiJ gene, e.g., a yeiJ gene disclosed herein. In one embodiment,
the yeiJ gene is a yeiJ gene from Escherichia coli. In one
embodiment, the yeiJ gene has at least about 80% identity with the
sequence of SEQ ID NO:114. Accordingly, in one embodiment, the yeiJ
gene has at least about 90% identity with the sequence of SEQ ID
NO:114. Accordingly, in one embodiment, the yeiJ gene has at least
about 95% identity with the sequence of SEQ ID NO:114. Accordingly,
in one embodiment, the yeiJ gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:114. In another
embodiment, the yeiJ gene comprises the sequence of SEQ ID NO:114.
In yet another embodiment the yeiJ gene consists of the sequence of
SEQ ID NO:114.
[0347] In one embodiment, the nucleoside transporter is encoded by
a yeiM gene, e.g., a yeiM gene disclosed herein. In one embodiment,
the yeiM gene is a yeiM gene from Escherichia coli. In one
embodiment, the yeiM gene has at least about 80% identity with the
sequence of SEQ ID NO:115. Accordingly, in one embodiment, the yeiM
gene has at least about 90% identity with the sequence of SEQ ID
NO:115. Accordingly, in one embodiment, the yeiM gene has at least
about 95% identity with the sequence of SEQ ID NO:115. Accordingly,
in one embodiment, the yeiM gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:115. In another
embodiment, the yeiM gene comprises the sequence of SEQ ID NO:115.
In yet another embodiment the yeiM gene consists of the sequence of
SEQ ID NO:115.
[0348] In one embodiment, the nucleoside transporter is encoded by
a HI0519 gene, e.g., a HI0519 gene disclosed herein. In one
embodiment, the HI0519 gene is a HI0519 gene from Haemophilus
influenzae. In one embodiment, the HI0519 gene has at least about
80% identity with the sequence of SEQ ID NO:116. Accordingly, in
one embodiment, the HI0519 gene has at least about 90% identity
with the sequence of SEQ ID NO:116. Accordingly, in one embodiment,
the HI0519 gene has at least about 95% identity with the sequence
of SEQ ID NO:116. Accordingly, in one embodiment, the HI0519 gene
has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the
sequence of SEQ ID NO:116. In another embodiment, the HI0519 gene
comprises the sequence of SEQ ID NO:116. In yet another embodiment
the HI0519 gene consists of the sequence of SEQ ID NO:116.
[0349] In one embodiment, the nucleoside transporter is encoded by
a yegT gene, e.g., a yegT gene disclosed herein. In one embodiment,
the yegT gene is a yegT gene from Escherichia coli. In one
embodiment, the yegT gene has at least about 80% identity with the
sequence of SEQ ID NO:124. Accordingly, in one embodiment, the yegT
gene has at least about 90% identity with the sequence of SEQ ID
NO:124. Accordingly, in one embodiment, the yegT gene has at least
about 95% identity with the sequence of SEQ ID NO:124. Accordingly,
in one embodiment, the yegT gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:124. In another
embodiment, the yegT gene comprises the sequence of SEQ ID NO:124.
In yet another embodiment the yegT gene consists of the sequence of
SEQ ID NO:124.
[0350] In one embodiment, the nucleoside transporter is encoded by
a nupG gene, e.g., a nupG gene disclosed herein. In one embodiment,
the nupG gene is a nupG gene from Escherichia coli. In one
embodiment, the nupG gene has at least about 80% identity with the
sequence of SEQ ID NO:125. Accordingly, in one embodiment, the nupG
gene has at least about 90% identity with the sequence of SEQ ID
NO:125. Accordingly, in one embodiment, the nupG gene has at least
about 95% identity with the sequence of SEQ ID NO:125. Accordingly,
in one embodiment, the nupG gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:125. In another
embodiment, the nupG gene comprises the sequence of SEQ ID NO:125.
In yet another embodiment the nupG gene consists of the sequence of
SEQ ID NO:125.
[0351] In one embodiment, the nucleoside transporter is encoded by
a xapB gene, e.g., a xapB gene disclosed herein. In one embodiment,
the xapB gene is a xapB gene from Escherichia coli. In one
embodiment, the xapB gene has at least about 80% identity with the
sequence of SEQ ID NO:126. Accordingly, in one embodiment, the xapB
gene has at least about 90% identity with the sequence of SEQ ID
NO:126. Accordingly, in one embodiment, the xapB gene has at least
about 95% identity with the sequence of SEQ ID NO:126. Accordingly,
in one embodiment, the xapB gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:126. In another
embodiment, the xapB gene comprises the sequence of SEQ ID NO:126.
In yet another embodiment the xapB gene consists of the sequence of
SEQ ID NO:126.
[0352] In one embodiment, the nucleoside transporter is encoded by
a CC1628 gene, e.g., a CC1628 gene disclosed herein. In one
embodiment, the CC1628 gene is a CC1628 gene from Caulobacter
crescentus. In one embodiment, the CC1628 gene has at least about
80% identity with the sequence of SEQ ID NO:127. Accordingly, in
one embodiment, the CC1628 gene has at least about 90% identity
with the sequence of SEQ ID NO:127. Accordingly, in one embodiment,
the CC1628 gene has at least about 95% identity with the sequence
of SEQ ID NO:127. Accordingly, in one embodiment, the CC1628 gene
has at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the
sequence of SEQ ID NO:127. In another embodiment, the CC1628 gene
comprises the sequence of SEQ ID NO:127. In yet another embodiment
the CC1628 gene consists of the sequence of SEQ ID NO:127.
[0353] In one embodiment, the nucleoside transporter is a cytosine
permease, e.g., CodB. In one embodiment, the nucleoside transporter
is encoded by a codB gene, e.g., a codB gene disclosed herein. In
one embodiment, the codB gene is a codB gene from Escherichia coli.
In one embodiment, the codB gene has at least about 80% identity
with the sequence of SEQ ID NO:128. Accordingly, in one embodiment,
the codB gene has at least about 90% identity with the sequence of
SEQ ID NO:128. Accordingly, in one embodiment, the codB gene has at
least about 95% identity with the sequence of SEQ ID NO:128.
Accordingly, in one embodiment, the codB gene has at least about
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID
NO:128. In another embodiment, the codB gene comprises the sequence
of SEQ ID NO:128. In yet another embodiment the codB gene consists
of the sequence of SEQ ID NO:128.
[0354] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0355] In one embodiment, when the nucleoside transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more nucleosides into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the nucleoside transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more nucleosides, into the bacterial
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In yet
another embodiment, when the nucleoside transporter is expressed in
the recombinant bacterial cells, the bacterial cells import
two-fold more nucleosides into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
nucleoside transporter is expressed in the recombinant bacterial
cells, the bacterial cells import three-fold, four-fold, five-fold,
six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold
more nucleoside into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0356] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous nucleoside transporter and a
second heterologous nucleoside transporter. For, example, in one
embodiment, the recombinant bacterial cell comprises at least one
outer membrance nucleoside transporter, e.g., tsx, and at least one
inner membrane nucleoside transporter, e.g., nupC and/or nupG. In
one embodiment, said first nucleoside transporter is derived from a
different organism than said second nucleoside transporter. In some
embodiments, said first nucleoside transporter is derived from the
same organism as said second nucleoside transporter. In some
embodiments, said first nucleoside transporter imports the same
nucleoside as said second nucleoside transporter. In other
embodiment, said first nucleoside transporter imports a different
nucleoside from said second nucleoside transporter. In some
embodiments, said first nucleoside transporter is a wild-type
nucleoside transporter and said second nucleoside transporter is a
mutagenized version of said first nucleoside transporter. In some
embodiments, the recombinant bacterial cells described herein
comprise at least a third heterologous nucleoside transporter. In
some embodiments, the recombinant bacterial cells described herein
comprise at least four heterologous nucleoside transporters. In
some embodiments, the recombinant bacterial cells described herein
comprise at least five heterologous nucleoside transporters or
more.
[0357] In one embodiment, the nucleoside transporter imports one
nucleoside into the bacterial cell. In another embodiment, the
nucleoside transporter imports two nucleosides into the bacterial
cell. In yet another embodiment, the nucleoside transporter imports
three nucleosides into the bacterial cell. In another embodiment,
the nucleoside transporter imports four or more nucleosides into
the cell. In one embodiment, the nucleoside transporter is an outer
membrane nucleoside transporter. In one embodiment, the nucleoside
transporter is an inner membrane nucleoside transporter. In one
embodiment, the nucleoside transporter is an adenosine transporter.
In another embodiment, the nucleoside transporter is an guanosine
transporter. In another embodiment, the nucleoside transporter is
an uridine transporter. In another embodiment, the amino acid
transporter is a inosine transporter. In another embodiment, the
amino acid transporter is a xanthosine transporter. In another
embodiment, the amino acid transporter is a thymidine transporter.
In one embodiment, the nucleoside transporter is an cytidine
transporter.
[0358] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a nucleoside transporter,
e.g., an adenosine transporter, may be used to treat a disease,
condition, and/or symptom associated with cancer, e.g., a cancer
described herein. In some embodiments, the recombinant bacterial
cells described herein may be used to reduce, ameliorate, or
eliminate one or more symptom(s) associated with a cancer.
[0359] For example, an important barrier to successful cancer
immunotherapy is that tumors employ a number of mechanisms to
facilitate immune escape, including the production of
anti-inflammatory cytokines, the recruitment of regulatory immune
subsets, and the production of immunosuppressive metabolites. One
such immunosuppressive pathway is the production of extracellular
adenosine, a potent immunosuppressive molecule, by CD73. The
purinergic system regulates and refines immune cell functions, such
as cell-to-cell interactions, cytokine and chemokine secretion,
surface antigen shedding, intracellular pathogen removal, and
generating reactive oxygen species. Extracellular ATP, released by
damaged or dying cells and bacteria, promotes the recruitment of
immune phagocytes and activates P2X7R, a coactivator of the NLRP3
inflammasome, which then triggers the production of proinflammatory
cytokines, such as IL-1.beta. and IL-18. The catabolism of
extracellular ATP into ADP, AMP and adenosine is controlled by
glycosylphosphatidylinositol (GPI-) anchored ectonucleotidases and
membrane-bound kinases. CD39 (ecto-nucleoside triphosphate
diphosphohydrolase 1, E-NTPDase1) hydrolyzes ATP into AMP, which is
then dephosphorylated into adenosine by CD73 (ecto-5'-nucleotidase,
Ecto5'NTase). Thus, CD39 and CD73 act in concert to convert
proinflammatory ATP into immunosuppressive adenosine. Notably, the
activity of CD39 is reversible by the actions of NDP kinase and
adenylate kinase, whereas the activity of CD73 is virtually
irreversible. Thus, CD73 represents a crucial checkpoint in the
conversion of an ATP-driven proinflammatory environment to an
anti-inflammatory milieu induced by adenosine. Stated another way,
CD73 negatively regulates the proinflammatory effects of
extracellular adenosine triphosphate (ATP).
[0360] In the tumor setting, CD39 and CD73 generate increased
adenosine levels characteristic of the tumor microenvironment. High
expression and activity of CD39 and CD73 has been observed in
several blood or solid tumors. In addition, CD39- and
CD73-expressing cancer exosomes can also raise adenosine levels
within the tumor microenvironment. The CD39/CD73 complex
participates in the process of tumor immunoescape, by inhibiting
the activation, clonal expansion, and homing of tumor-specific T
cells (in particular, T helper and cytotoxic T cells), impairing
tumor cell killing by cytolytic effector T lymphocytes, and
inducing the suppressive capabilities of Treg and Th17 cells, and
enhancing the conversion of type 1 macrophages into tumor-promoting
type 2 macrophages (reviewed in Antonioli et al., Trends Mol Med.
2013 Jun; 19(6): 355-367. CD39 and CD73 in immunity and
inflammation). Myeloid-derived suppressor cells (MDSCs), also
appear to promote tumor growth by a CD39-mediated mechanism.
[0361] Beside its immunoregulatory roles, the ectonucleotidase
pathway contributes directly to the modulation of cancer cell
growth, differentiation, invasion, migration, metastasis, and tumor
angiogenesis. Agents targeting these enzymes show anti-tumor
efficacy and a favorable tolerability profile in several murine
models of malignancy (Anonioli et al., 2013). In some embodiments,
the genetically engineered bacteria comprise a means for removing
excess adenosine from the tumor microenvironment. Many bacteria
scavenge low concentrations of nucleosides from the environment for
synthesis of nucleotides and deoxynucleotides by salvage pathways
of synthesis. Additionally, in Escherichia coli, nucleosides can be
used as the sole source of nitrogen and carbon for growth (Neuhard
J, Nygaard P. Biosynthesis and conversion of nucleotides, purines
and pyrimidines. In: Neidhardt F C, Ingraham J L, Low K B,
Magasanik B, Schaechter M, Umbarger H E, editors. Escherichia coli
and Salmonella typhimurium: Cellular and molecular biology.
Washington DC: ASM Press; 1987. pp. 445-473). Two evolutionarily
unrelated cation-linked transporter families, the Concentrative
Nucleoside Transporter (CNT) family and the Nucleoside:H+ Symporter
(NHS) family, are responsible for nucleoside uptake (see e.g.,
Cabrita et al., Biochem. Cell Biol. Vol. 80, 2002. Molecular
biology and regulation of nucleoside and nucleobase transporter
proteins in eukaryotes and prokaryotes), the contents of which is
herein incorporated by reference in its entirety. NupC and NupG,
are the transporter family members in E. coli. Mutants defective in
both the nupC and nupG genes cannot grow with nucleosides as a
single carbon source. Both of these transporters are proton-linked
but they differ in their selectivity. NupG is capable of
transporting a wide range of nucleosides and deoxynucleosides; in
contrast, NupC does not transport guanosine or deoxyguanosine.
Homologs of NupG from E. coli are found in a wide range of
eubacteria, including human gut pathogens such as Salmonella
typhimurium, organisms associated with periodontal disease such as
Porphyromonas gingivalis and Prevotella intermedia, and plant
pathogens in the genus Erwinia (As described in Vaziri et al., Mol
Membr Biol. 2013 Mar; 30(1-2): 114-128. Use of molecular modelling
to probe the mechanism of the nucleoside transporter NupG, the
contents of which is herein incorporated by reference in its
entirety). Putative bacterial transporters from the CNT superfamily
and transporters from the NupG/XapB family include those listed
herein. In addition, codB (GenBank P25525, Escherichia coli) was
identified based on homology to a yeast transporter family termed
the uracil/allantoin transpertor family (Cabrita et al.,
supra).
[0362] Thus, the genetically engineered bacteria comprise a means
for metabolizing or degrading adenosine. In some embodiments, the
genetically engineered bacteria comprise one or more gene sequences
encoding one or more enzymes that are capable of converting
adenosine to urate. In some embodiments, the genetically engineered
bacteria comprise sequence(s) encoding add, xapA, deoD, xdhA, xdhB,
and xdhC genes from E. Coli. In some embodiments, the genetically
engineered bacteria or genetically engineered oncolytic virus
further comprise a means for importing adenosine into the
engineered bacteria from the tumor microenvironment. In some
embodiments, the genetically engineered bacteria comprise sequence
for encoding a nucleoside transporter. In some embodiments, the
genetically engineered bacteria for encoding an adenosine
transporter. In certain embodiments, genetically engineered
bacteria for encoding E. coli Nucleoside Permease nupG or nupC. In
some embodiments, the genetically engineered bacteria comprise
sequence(s) encoding add, xapA, deoD, xdhA, xdhB, and xdhC genes
from E. Coli and comprise sequence encoding a nucleoside or
adenosine transporter. In some embodiments, the genetically
engineered bacteria comprise sequence(s) encoding add, xapA, deoD,
xdhA, xdhB, and xdhC genes from E. Coli and comprise sequence
encoding nupG or nupC. An exemplary engineered bacteria is shown in
FIG. 34.
C. Kynurenine Transporters
[0363] The catabolism of the essential amino acid tryptophan is a
central pathway maintaining the immunosuppressive microenvironment
in many types of cancers. Tumor cells or myeloid cells in the tumor
microenvironment express high levels of indoleamine-2,3-dioxygenase
1 (IDO1), which is the first and rate-limiting enzyme in the
degradation of tryptophan. This enzymatic activity results in the
depletion of tryptophan in the local microenvironment and
subsequent inhibition of T cell responses, which results in
immunosuppression (as T cells are particularly sensitive to low
tryptophan levels). More recent preclinical studies suggest an
alternative route of tryptophan degradation in tumors via the
enzyme TRP-2,3-dioxygenase 2 (TDO). Thus, tumor cells may express
and catabolize tryptophan via TDO instead of or in addition to
IDO1.
[0364] In addition, several studies have proposed that
immunosuppression by tryptophan degradation is not solely a
consequence of lowering local tryptophan levels but also of
accumulating high levels of tryptophan metabolites. Preclinical
studies and analyses of human tumor tissue have demonstrated that T
cell responses are inhibited by tryptophan metabolites, primarily
by binding to the aryl hydrocarbon receptor (AHR), a cytoplasmic
transcription factor. These studies show that binding of the
tryptophan metabolite kynurenine to the aryl hydrocarbon receptor
results in reprograming the differentiation of naive CD4+T-helper
(Th) cells favoring a regulatory T cells phenotype (Treg) while
suppressing the differentiation into interleukin-17
(IL-17)-producing Th (Th17) cells. Activation of the aryl hydrogen
receptor also results in promoting a tolerogenic phenotype on
dendritic cells. As discussed above, studies have shown that the
binding of kynurenine to the aryl hydrocarbon receptor results in
the production of regulatory T cells (Tregs). Thus, in some
embodiments, the engineered microbe has a mechanism for importing
(transporting) Kynurenine from the local environment into the cell.
Thus, in some embodiments, the genetically engineered bacteria
comprise gene sequence(s) encoding a kynureninase transporter. In
some embodiments, the genetically engineered bacteria comprise one
or more copies of aroP, tnaB or mtr gene.
[0365] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a kynurenine
transporter. In one embodiment, the kynurenine transporter
transports kynurenine into the cell.
[0366] The uptake of kynurenine into bacterial cells is mediated by
proteins well known to those of skill in the art. In one
embodiment, the at least one gene encoding a transporter is a gene
selected from the group consisting of mtr, aroP and tnaB. In one
embodiment, the bacterial cell described herein has been
genetically engineered to comprise at least one heterologous gene
selected from the group consisting of mtr, aroP and tnaB. In one
embodiment, the at least one gene encoding a kynurenine transporter
is the Escherichia coli mtr gene. In one embodiment, the at least
one gene encoding a kynurenine transporter is the Escherichia coli
aroP gene. In one embodiment, the at least one gene encoding a
kynurenine transporter is the Escherichia coli tnaB gene. In some
embodiments, the kynurenine transporter is encoded by a kynurenine
transporter gene derived from a bacterial genus or species,
including but not limited to, Escherichia, Corynebacterium,
Sacharomyces, Escherichia coli, Saccharomyces cerevisiae or
Corynebacterium glutamicum. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0367] Assays for testing the activity of a kynurenine transporter,
a functional variant of a kynurenine transporter, or a functional
fragment of transporter of kynurenine are well known to one of
ordinary skill in the art.
[0368] Kynurenine transporters may be expressed or modified in the
bacteria in order to enhance kynurenine transport into the cell.
Specifically, when the kynurenine transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
kynurenine(s) into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a kynurenine transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a kynurenine transporter and a genetic modification that
reduces export of a kynurenine, e.g., a genetic mutation in an
exporter gene or promoter.
[0369] In one embodiment, the bacterial cell comprises at least one
gene encoding a kynurenine transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a
kynurenine transporter. In some embodiments, the at least one
native gene encoding a kynurenine transporter is not modified. In
another embodiment, the bacterial cell comprises more than one copy
of at least one native gene encoding a kynurenine transporter. In
yet another embodiment, the bacterial cell comprises a copy of at
least one gene encoding a native kynurenine transporter, as well as
at least one copy of at least one heterologous gene encoding a
kynurenine transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a kynurenine transporter. In one embodiment, the bacterial
cell comprises multiple copies of the at least one heterologous
gene encoding a kynurenine transporter.
[0370] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a kynurenine transporter, wherein said
kynurenine transporter comprises a kynurenine sequence that has at
least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity
to the kynurenine sequence of a polypeptide encoded by a kynurenine
transporter gene disclosed herein.
[0371] The present disclosure further comprises genes encoding
functional fragments of a kynurenine transporter or functional
variants of a kynurenine transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
kynurenine transporter relates to an element having qualitative
biological activity in common with the wild-type kynurenine
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
kynurenine transporter is one which retains essentially the same
ability to import a kynurenine into the bacterial cell as does the
kynurenine transporter protein from which the functional fragment
or functional variant was derived. In one embodiment, the
recombinant bacterial cell comprises at least one heterologous gene
encoding a functional fragment of a kynurenine transporter. In
another embodiment, the recombinant bacterial cell comprises a
heterologous gene encoding a functional variant of a kynurenine
transporter.
[0372] In one embodiment, the genes encoding the kynurenine
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the kynurenine transporter have been codon-optimized
for use in Escherichia coli.
[0373] The present disclosure also encompasses genes encoding a
kynurenine transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0374] In some embodiments, the at least one gene encoding a
kynurenine transporter is mutagenized; mutants exhibiting increased
kynurenine import are selected; and the mutagenized at least one
gene encoding a kynurenine transporter is isolated and inserted
into the bacterial cell. In some embodiments, the at least one gene
encoding a kynurenine transporter is mutagenized; mutants
exhibiting decreased kynurenine import are selected; and the
mutagenized at least one gene encoding a kynurenine transporter is
isolated and inserted into the bacterial cell. The transporter
modifications described herein may be present on a plasmid or
chromosome.
[0375] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a kynurenine transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding a
kynurenine transporter is directly operably linked to the promoter.
In another embodiment, the at least one gene encoding a kynurenine
transporter is indirectly operably linked to the promoter.
[0376] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a kynurenine transporter in nature.
In some embodiments, the at least one gene encoding the kynurenine
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the kynurenine
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the kynurenine
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the kynurenine transporter is controlled by a constitutive
promoter.
[0377] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0378] In one embodiment, the at least one gene encoding a
kynurenine transporter is located on a plasmid in the bacterial
cell. In some embodiments, the plasmid is a high copy number
plasmid. In some embodiments, the plasmid is a low copy number
plasmid. In another embodiment, the at least one gene encoding a
kynurenine transporter is located in the chromosome of the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a kynurenine transporter is located in the
chromosome of the bacterial cell, and a copy of at least one gene
encoding a kynurenine transporter from a different species of
bacteria is located on a plasmid in the bacterial cell. In yet
another embodiment, a native copy of the at least one gene encoding
a kynurenine transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding a kynurenine
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a kynurenine transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a kynurenine transporter from a
different species of bacteria is located in the chromosome of the
bacterial cell.
[0379] In some embodiments, the at least one native gene encoding
the kynurenine transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the kynurenine transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0380] In some embodiments, at least one native gene encoding the
kynurenine transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the kynurenine transporter in the
bacterial cell is modified, and one or more additional copies of at
least one native gene encoding the transporter are present in the
bacterial cell on a plasmid. In alternate embodiments, the at least
one native gene encoding the transporter is modified, and a copy of
at least one gene encoding the transporter from a different
bacterial species is present in the bacteria on a plasmid.
[0381] In one embodiment, the mtr gene has at least about 80%
identity with the sequence of SEQ ID NO:46. Accordingly, in one
embodiment, the mtr gene has at least about 90% identity with the
sequence of SEQ ID NO:46. Accordingly, in one embodiment, the mtr
gene has at least about 95% identity with the sequence of SEQ ID
NO:46. Accordingly, in one embodiment, the mtr gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:46. In another embodiment, the mtr gene comprises the
sequence of SEQ ID NO:46. In yet another embodiment the mtr gene
consists of the sequence of SEQ ID NO:46.
[0382] In one embodiment, the tnaB gene has at least about 80%
identity with the sequence of SEQ ID NO:47. Accordingly, in one
embodiment, the tnaB gene has at least about 90% identity with the
sequence of SEQ ID NO:47. Accordingly, in one embodiment, the tnaB
gene has at least about 95% identity with the sequence of SEQ ID
NO:47. Accordingly, in one embodiment, the tnaB gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:47. In another embodiment, the tnaB gene comprises the
sequence of SEQ ID NO:47. In yet another embodiment the tnaB gene
consists of the sequence of SEQ ID NO:47.
[0383] In one embodiment, the aroP gene has at least about 80%
identity with the sequence of SEQ ID NO:48. Accordingly, in one
embodiment, the aroP gene has at least about 90% identity with the
sequence of SEQ ID NO:48. Accordingly, in one embodiment, the aroP
gene has at least about 95% identity with the sequence of SEQ ID
NO:48. Accordingly, in one embodiment, the aroP gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:48. In another embodiment, the aroP gene comprises the
sequence of SEQ ID NO:48. In yet another embodiment the aroP gene
consists of the sequence of SEQ ID NO:48.
[0384] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0385] In one embodiment, when the kynurenine transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more kynurenine into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the kynurenine transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more kynurenine, into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In yet another
embodiment, when the kynurenine transporter is expressed in the
recombinant bacterial cells, the bacterial cells import two-fold
more kynurenine into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the kynurenine
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold more
kynurenine into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions.
[0386] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous kynurenine transporter and a
second heterologous kynurenine transporter. In one embodiment, said
first kynurenine transporter is derived from a different organism
than said second kynurenine transporter. In some embodiments, said
first kynurenine transporter is derived from the same organism as
said second kynurenine transporter. In some embodiments, said first
kynurenine transporter imports the same kynurenine as said second
kynurenine transporter. In other embodiment, said first kynurenine
transporter imports a different kynurenine from said second
kynurenine transporter. In some embodiments, said first kynurenine
transporter is a wild-type kynurenine transporter and said second
kynurenine transporter is a mutagenized version of said first
kynurenine transporter. In some embodiments, the recombinant
bacterial cells described herein comprise at least a third
heterologous kynurenine transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least four
heterologous kynurenine transporters. In some embodiments, the
recombinant bacterial cells described herein comprise at least five
heterologous kynurenine transporters or more.
[0387] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a kynurenine transporter
may be used to treat a disease, condition, and/or symptom
associated with cancer, e.g., a cancer described herein. In some
embodiments, the recombinant bacterial cells described herein may
be used to reduce, ameliorate, or eliminate one or more symptom(s)
associated with a cancer.
[0388] Means for optimizing kynurenine uptake are provided in the
Example section.
D. Prostaglandin E2 Transporters
[0389] Prostaglandin E2 (PGE2) is overproduced in many tumors,
where it aids in cancer progression. PGE2 is a pleiotropic molecule
involved in numerous biological processes, including angiogenesis,
apoptosis, inflammation, and immune suppression. PGE2 is
synthesized from arachidonic acid by cyclooxygenase 2 (COX-2).
COX-2, converts arachidonic acid (AA) to prostaglandin endoperoxide
H2 (PGH2). PHG2 is then converted to PHE2 by prostaglandin E
synthase (PGES), of which there are three forms. PGE2 can be
catabolized into biologically inactive 15-keto-PGs by 15-PGDH and
carbonyl reductase or secreted by the transporter MRP4.
[0390] MDSCs are thought to play a key role in the PGE2 production
in the tumor environment. Tumor derived factors induce COX2, PGES1,
and MRP4 and downregulate the expression of 15-PGDH in MDSCs, and
is associated with MDSC suppressive activity. Inhibition of PGE2
through COX-2 inhibitors show promise as cancer treatments, but
systemic administration is associated with serious side effects,
and in the case of the COX-2 inhibitor celecoxib, resistance to
tumor prevention has been observed.
[0391] In addition to inhibition of PGE production, the degradation
of PGE2 by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is
another way to reduce PGE2 levels in tumors. A lack of
prostaglandin dehydrogenase prevents catabolism of prostaglandin
E2, which helps cancer cells both to evade the immune system and
circumvent drug treatment. Recent studies have demonstrated that
15-PGDH delivered locally to the tumor microenvironment can effect
an antitumor immune response. For example, injection of an
adenovirus encoding 15-PGDH into mouse tumors comprising
non-lymphocyte white blood cells expressing CD1 1b (which have
increased PGE2 levels, higher COX-2 expression and significantly
reduced expression of 15-PGDH as compared with cells from outside
the tumor), resulted in significantly slowed tumor growth. These
studies further showed that 15-PGDH expression was highest in tumor
cells but also significant in tumor-associated CD1 1b cells, where
it produced a four-fold reduction in PGE2 secretion. This was
associated with reduced secretion of immunosuppressive cytokines by
the CD1 1b cells which resulted in a switch in their fate,
promoting their differentiation into dendritic cells. These studies
show that overproduction of PGE2 in tumors contributes to immune
evasion by preventing maturation of antigen-presenting cells, and
that evasion can be overcome by enforced expression of 15-PGDH.
(Eruslanov et al., Volume 88, November 2010 Journal of Leukocyte
Biology; Tumor-mediated induction of myeloid-derived suppressor
cells and M2-polarized macrophages by altering intracellular PGE2
catabolism in myeloid cells).
[0392] Other studies confirm the benefit of local PGE2 catabolism
in cancer treatment. Celecoxib, a non-steroidal anti-inflammatory
COX-2 inhibitor used to treat pain and inflammation, reduces the
recurrence of colon adenomas but does not work in some patients who
have low levels of 15-PGDH. These results correspond with studies
which show that in mice, gene knockout of 15-PGDH confers
near-complete resistance to the ability of celecoxib to prevent
colon tumors. These and other studies highlight the potential
importance of reducing PGE2 levels in cancer, either through
inhibition of synthesis or promotion of catalysis or both.
[0393] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a prostaglandin
E2 (PGE2) transporter. In one embodiment, the PGE2 transporter
transports PGE2 into the cell.
[0394] The uptake of PGE2 into bacterial cells is mediated by
proteins well known to those of skill in the art.
[0395] In some embodiments, the PGE2 transporter is encoded by a
PGE2 transporter gene derived from a bacterial genus or species,
including but not limited to, Escherichia, Corynebacterium,
Escherichia coli, Saccharomyces cerevisiae or Corynebacterium
glutamicum. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0396] Assays for testing the activity of a PGE2 transporter, a
functional variant of a PGE2 transporter, or a functional fragment
of transporter of PGE2 are well known to one of ordinary skill in
the art. For example, import of PGE2 may be determined using the
methods as described in, the entire contents of each of which are
expressly incorporated by reference herein.
[0397] PGE2 transporters may be expressed or modified in the
bacteria in order to enhance PGE2 transport into the cell.
Specifically, when the PGE2 transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more PGE2
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In one embodiment, the bacterial cell comprises a heterologous gene
encoding a PGE2 transporter. In one embodiment, the bacterial cell
comprises a heterologous gene encoding a PGE2 transporter and a
genetic modification that reduces export of a PGE2, e.g., a genetic
mutation in an exporter gene or promoter.
[0398] In one embodiment, the bacterial cell comprises at least one
gene encoding a PGE2 transporter from a different organism, e.g., a
different species of bacteria. In one embodiment, the bacterial
cell comprises at least one native gene encoding a PGE2
transporter. In some embodiments, the at least one native gene
encoding a PGE2 transporter is not modified. In another embodiment,
the bacterial cell comprises more than one copy of at least one
native gene encoding a PGE2 transporter. In yet another embodiment,
the bacterial cell comprises a copy of at least one gene encoding a
native PGE2 transporter, as well as at least one copy of at least
one heterologous gene encoding a PGE2 transporter from a different
bacterial species. In one embodiment, the bacterial cell comprises
at least one, two, three, four, five, or six copies of the at least
one heterologous gene encoding a PGE2 transporter. In one
embodiment, the bacterial cell comprises multiple copies of the at
least one heterologous gene encoding a PGE2 transporter.
[0399] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a PGE2 transporter, wherein said PGE2
transporter comprises a PGE2 sequence that has at least 70%, 75%,
80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the PGE2
sequence of a polypeptide encoded by a PGE2 transporter gene
disclosed herein.
[0400] The present disclosure further comprises genes encoding
functional fragments of a PGE2 transporter or functional variants
of a PGE2 transporter. As used herein, the term "functional
fragment thereof" or "functional variant thereof" of a PGE2
transporter relates to an element having qualitative biological
activity in common with the wild-type PGE2 transporter from which
the fragment or variant was derived. For example, a functional
fragment or a functional variant of a mutated PGE2 transporter is
one which retains essentially the same ability to import PGE2 into
the bacterial cell as does the PGE2 transporter protein from which
the functional fragment or functional variant was derived. In one
embodiment, the recombinant bacterial cell comprises at least one
heterologous gene encoding a functional fragment of a PGE2
transporter. In another embodiment, the recombinant bacterial cell
comprises a heterologous gene encoding a functional variant of a
PGE2 transporter.
[0401] In one embodiment, the genes encoding the PGE2 transporter
have been codon-optimized for use in the host organism, e.g., a
bacterial cell disclosed herein. In one embodiment, the genes
encoding the PGE2 transporter have been codon-optimized for use in
Escherichia coli.
[0402] The present disclosure also encompasses genes encoding a
PGE2 transporter comprising amino acids in its sequence that are
substantially the same as an amino acid sequence described herein
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0403] In some embodiments, the at least one gene encoding a PGE2
transporter is mutagenized; mutants exhibiting increased PGE2
import are selected; and the mutagenized at least one gene encoding
a PGE2 transporter is isolated and inserted into the bacterial
cell. In some embodiments, the at least one gene encoding a PGE2
transporter is mutagenized; mutants exhibiting decreased PGE2
import are selected; and the mutagenized at least one gene encoding
a PGE2 transporter is isolated and inserted into the bacterial
cell. The transporter modifications described herein may be present
on a plasmid or chromosome.
[0404] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a PGE2 transporter operably linked to a
promoter. In one embodiment, the at least one gene encoding a PGE2
transporter is directly operably linked to the promoter. In another
embodiment, the at least one gene encoding a PGE2 transporter is
indirectly operably linked to the promoter.
[0405] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a PGE2 transporter in nature. In
some embodiments, the at least one gene encoding the PGE2
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the PGE2 transporter is
controlled by an inducible promoter. In some embodiments, the at
least one gene encoding the PGE2 transporter is controlled by a
promoter that is stronger than its native promoter. In some
embodiments, the at least one gene encoding the PGE2 transporter is
controlled by a constitutive promoter.
[0406] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0407] In one embodiment, the at least one gene encoding a PGE2
transporter is located on a plasmid in the bacterial cell. In some
embodiments, the plasmid is a high copy number plasmid. In some
embodiments, the plasmid is a low copy number plasmid. In another
embodiment, the at least one gene encoding a PGE2 transporter is
located in the chromosome of the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a PGE2
transporter is located in the chromosome of the bacterial cell, and
a copy of at least one gene encoding a PGE2 transporter from a
different species of bacteria is located on a plasmid in the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a PGE2 transporter is located on a plasmid
in the bacterial cell, and a copy of at least one gene encoding a
PGE2 transporter from a different species of bacteria is located on
a plasmid in the bacterial cell. In yet another embodiment, a
native copy of the at least one gene encoding a PGE2 transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a PGE2 transporter from a different
species of bacteria is located in the chromosome of the bacterial
cell.
[0408] In some embodiments, the at least one native gene encoding
the PGE2 transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the PGE2 transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0409] In some embodiments, at least one native gene encoding the
PGE2 transporter in the bacterial cell is not modified, and one or
more additional copies of at least one native gene encoding the
transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the PGE2 transporter in the bacterial cell
is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0410] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0411] In one embodiment, when the PGE2 transporter is expressed in
the recombinant bacterial cells, the bacterial cells import 10%
more PGE2 into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In another embodiment, when the PGE2 transporter
is expressed in the recombinant bacterial cells, the bacterial
cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
PGE2, into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the PGE2
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import two-fold more PGE2 into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the PGE2 transporter is expressed in the
recombinant bacterial cells, the bacterial cells import three-fold,
four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or
fifty-fold more PGE2 into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions.
[0412] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous PGE2 transporter and a second
heterologous PGE2 transporter. In one embodiment, said first PGE2
transporter is derived from a different organism than said second
PGE2 transporter. In some embodiments, said first PGE2 transporter
is derived from the same organism as said second PGE2 transporter.
In some embodiments, said first PGE2 transporter is a wild-type
PGE2 transporter and said second PGE2 transporter is a mutagenized
version of said first PGE2 transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least a
third heterologous PGE2 transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least four
heterologous PGE2 transporters. In some embodiments, the
recombinant bacterial cells described herein comprise at least five
heterologous PGE2 transporters or more.
[0413] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a PGE2 transporter may be
used to treat a disease, condition, and/or symptom associated with
cancer, e.g., a cancer described herein. In some embodiments, the
recombinant bacterial cells described herein may be used to reduce,
ameliorate, or eliminate one or more symptom(s) associated with a
cancer.
E. Lactic Acid Transporters
[0414] The anti-cancer immune response is influenced by the
environmental pH; an acidic pH has been shown to inhibit the
function of immune cells. Lowering the environmental pH to 6.0-6.5,
as can be found in tumour masses, has been reported to lead to loss
of T-cell function of human and murine tumour-infiltrating
lymphocytes (eg impairment of cytolytic activity and cytokine
secretion); the T-cell function could be completely restored by
buffering the pH at physiological values. The primary cause
responsible for the acidic pH and pH-dependent T-cell
function-suppressive effect in a tumour micro-environment has been
identified as lactic acid (as reviewed in Chio et al., J Pathol.
2013 Aug; 230(4): 350-355. Cancer-generated lactic acid: a
regulatory, immunosuppressive metabolite?), the contents of which
is herein incorporated by reference in its entirety. It has also
been demonstrated that cancer-generated lactic acid and the
resultant acidification of the micro-environment increase the
expression of ARG1 in tumour-associated macrophages, characteristic
of the M2 helper phenotype.
[0415] In some embodiments, the genetically engineered bacterium
are able to import lactic acid from the tumor microenvironment. In
one embodiment, the recombinant bacterial cell of the invention
comprises a heterologous gene encoding a substrate transporter,
wherein the substrate transporter is a lactic acid transporter. In
one embodiment, the lactic acid transporter transports lactic acid
into the cell.
[0416] The uptake of lactic acid into bacterial cells is mediated
by proteins well known to those of skill in the art.
[0417] In some embodiments, the lactic acid transporter is encoded
by a lactic acid transporter gene derived from a bacterial genus or
species, including but not limited to, Escherichia,
Corynebacterium, Escherichia coli, Saccharomyces cerevisiae or
Corynebacterium glutamicum. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0418] Assays for testing the activity of a lactic acid
transporter, a functional variant of a lactic acid transporter, or
a functional fragment of transporter of lactic acid are well known
to one of ordinary skill in the art.
[0419] lactic acid transporters may be expressed or modified in the
bacteria in order to enhance lactic acid transport into the cell.
Specifically, when the lactic acid transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more lactic
acid into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a lactic acid transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a lactic acid transporter and a genetic modification that
reduces export of a lactic acid, e.g., a genetic mutation in an
exporter gene or promoter.
[0420] In one embodiment, the bacterial cell comprises at least one
gene encoding a lactic acid transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a lactic
acid transporter. In some embodiments, the at least one native gene
encoding a lactic acid transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding a lactic acid transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native lactic acid transporter, as well as at
least one copy of at least one heterologous gene encoding a lactic
acid transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a lactic acid transporter. In one embodiment, the
bacterial cell comprises multiple copies of the at least one
heterologous gene encoding a lactic acid transporter.
[0421] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a lactic acid transporter, wherein
said lactic acid transporter comprises a lactic acid sequence that
has at least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity to the lactic acid sequence of a polypeptide encoded by a
lactic acid transporter gene disclosed herein.
[0422] The present disclosure further comprises genes encoding
functional fragments of a lactic acid transporter or functional
variants of a lactic acid transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
lactic acid transporter relates to an element having qualitative
biological activity in common with the wild-type lactic acid
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
lactic acid transporter is one which retains essentially the same
ability to import lactic acid into the bacterial cell as does the
lactic acid transporter protein from which the functional fragment
or functional variant was derived. In one embodiment, the
recombinant bacterial cell comprises at least one heterologous gene
encoding a functional fragment of a lactic acid transporter. In
another embodiment, the recombinant bacterial cell comprises a
heterologous gene encoding a functional variant of a lactic acid
transporter.
[0423] In one embodiment, the genes encoding the lactic acid
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the lactic acid transporter have been
codon-optimized for use in Escherichia coli.
[0424] The present disclosure also encompasses genes encoding a
lactic acid transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0425] In some embodiments, the at least one gene encoding a lactic
acid transporter is mutagenized; mutants exhibiting increased
lactic acid import are selected; and the mutagenized at least one
gene encoding a lactic acid transporter is isolated and inserted
into the bacterial cell. In some embodiments, the at least one gene
encoding a lactic acid transporter is mutagenized; mutants
exhibiting decreased lactic acid import are selected; and the
mutagenized at least one gene encoding a lactic acid transporter is
isolated and inserted into the bacterial cell. The transporter
modifications described herein may be present on a plasmid or
chromosome.
[0426] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a lactic acid transporter operably
linked to a promoter. In one embodiment, the at least one gene
encoding a lactic acid transporter is directly operably linked to
the promoter. In another embodiment, the at least one gene encoding
a lactic acid transporter is indirectly operably linked to the
promoter.
[0427] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a lactic acid transporter in nature.
In some embodiments, the at least one gene encoding the lactic acid
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the lactic acid
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the lactic acid
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the lactic acid transporter is controlled by a
constitutive promoter.
[0428] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0429] In one embodiment, the at least one gene encoding a lactic
acid transporter is located on a plasmid in the bacterial cell. In
some embodiments, the plasmid is a high copy number plasmid. In
some embodiments, the plasmid is a low copy number plasmid. In
another embodiment, the at least one gene encoding a lactic acid
transporter is located in the chromosome of the bacterial cell. In
yet another embodiment, a native copy of the at least one gene
encoding a lactic acid transporter is located in the chromosome of
the bacterial cell, and a copy of at least one gene encoding a
lactic acid transporter from a different species of bacteria is
located on a plasmid in the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a
lactic acid transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding a lactic acid
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a lactic acid transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a lactic acid transporter from a
different species of bacteria is located in the chromosome of the
bacterial cell.
[0430] In some embodiments, the at least one native gene encoding
the lactic acid transporter in the recombinant bacterial cell is
not modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the lactic acid transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0431] In some embodiments, at least one native gene encoding the
lactic acid transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the lactic acid transporter in the
bacterial cell is modified, and one or more additional copies of at
least one native gene encoding the transporter are present in the
bacterial cell on a plasmid. In alternate embodiments, the at least
one native gene encoding the transporter is modified, and a copy of
at least one gene encoding the transporter from a different
bacterial species is present in the bacteria on a plasmid.
[0432] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0433] In one embodiment, when the lactic acid transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more lactic acid into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the lactic acid transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more lactic acid, into the bacterial
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In yet
another embodiment, when the lactic acid transporter is expressed
in the recombinant bacterial cells, the bacterial cells import
two-fold more lactic acid into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
lactic acid transporter is expressed in the recombinant bacterial
cells, the bacterial cells import three-fold, four-fold, five-fold,
six-fold, seven-fold, eight-fold, nine-fold, ten-fold,
fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or fifty-fold
more lactic acid into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions.
[0434] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous lactic acid transporter and a
second heterologous lactic acid transporter. In one embodiment,
said first lactic acid transporter is derived from a different
organism than said second lactic acid transporter. In some
embodiments, said first lactic acid transporter is derived from the
same organism as said second lactic acid transporter. In some
embodiments, said first lactic acid transporter is a wild-type
lactic acid transporter and said second lactic acid transporter is
a mutagenized version of said first lactic acid transporter. In
some embodiments, the recombinant bacterial cells described herein
comprise at least a third heterologous lactic acid transporter. In
some embodiments, the recombinant bacterial cells described herein
comprise at least four heterologous lactic acid transporters. In
some embodiments, the recombinant bacterial cells described herein
comprise at least five heterologous lactic acid transporters or
more.
[0435] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding a lactic acid transporter
may be used to treat a disease, condition, and/or symptom
associated with cancer, e.g., a cancer described herein. In some
embodiments, the recombinant bacterial cells described herein may
be used to reduce, ameliorate, or eliminate one or more symptom(s)
associated with a cancer.
E. Propionate Transporters
[0436] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a propionate
transporter. In one embodiment, the propionate transporter
transports propionate into the cell.
[0437] The uptake of propionate into bacterial cells typically
occurs via passive diffusion (see, for example, Kell et al., 1981,
Biochem. Biophys. Res. Commun., 9981-8). However, the active import
of propionate is also mediated by proteins well known to those of
skill in the art. For example, a bacterial transport system for the
update of propionate in Corynebacterium glutamicum named MctC
(monocarboxylic acid transporter) is known (see, for example,
Jolkver et al. (2009) J. Bacteriol. 191(3): 940-8). The putP_6
propionate transporter from Virgibacillus species (UniProt
A0A024QGU1) has also been identified.
[0438] Propionate transporters, may be expressed or modified in the
bacteria of the invention in order to enhance propionate transport
into the cell. Specifically, when the propionate transporter is
expressed in the recombinant bacterial cells of the invention, the
bacterial cells import more propionate into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. Thus, the genetically
engineered bacteria comprising a heterologous gene encoding an
propionate transporter may be used to import propionate into the
bacteria and can be used to treat diseases associated with the
catabolism of propionate, such as organic acidurias (including PA
and MMA) and vitamin B.sub.12 deficiencies. In one embodiment, the
bacterial cell of the invention comprises a heterologous gene
encoding an propionate transporter.
[0439] The uptake of propionate into bacterial cells is mediated by
proteins well known to those of skill in the art. In one
embodiment, the at least one gene encoding a propionate transporter
is a gene selected from the group consisting of metC, PutP_6, mctB,
mctC, dip0780, dip0791, ce0909, ce0910, ce1091, ce1092, sco 1822,
sco1823, sco1218, sco1219, ce1091, sco5827, m_5160, m_5161, m_5165,
m_5166, nfa 17930, nfa 17940, nfa 17950, nfa 17960, actP, yjcH,
ywcB, and ywcA. In one embodiment, the bacterial cell described
herein has been genetically engineered to comprise at least one
heterologous gene selected from the group consisting of metC,
PutP_6, mctB, mctC, dip0780, dip0791, ce0909, ce0910, ce1091,
ce1092, sco1822, sco1823, sco1218, sco1219, ce 1091, sco5827,
m_5160, m_5161, m_5165, m_5166, nfa 17930, nfa 17940, nfa 17950,
nfa 17960, actP, yjcH, ywcB, and ywcA. In one embodiment, the at
least one gene encoding a propionate transporter is the metC gene.
In one embodiment, the at least one gene encoding a propionate
transporter is the putP_6 gene.
[0440] In some embodiments, the propionate transporter is encoded
by a propionate transporter gene derived from a bacterial genus or
species, including but not limited to, Bacillus, Campylobacter,
Clostridium, Corynebacterium, Escherichia, Lactobacillus,
Mycobacterium, Pseudomonas, Salmonella, Staphylococcus,
Streptomyces, Bacillus subtilis, Campylobacter jejuni, Clostridium
perfringens, Escherichia coli, Lactobacillus delbrueckii,
Mycobacterium smegmatis, Nocardia farcinica, Pseudomonas
aeruginosa, Salmonella typhimurium, Virgibacillus, or
Staphylococcus aureus. In some embodiments, the propionate
transporter gene is derived from Virgibacillus. In some
embodiments, the propionate transporter gene is derived from
Corynebacterium. In one embodiment, the propionate transporter gene
is derived from Corynebacterium glutamicum. In another embodiment,
the propionate transporter gene is derived from Corynebacterium
diphtheria. In another embodiment, the propionate transporter gene
is derived from Corynebacterium efficiens. In another embodiment,
the propionate transporter gene is derived from Streptomyces
coelicolor. In another embodiment, the propionate transporter gene
is derived from Mycobacterium smegmatis. In another embodiment, the
propionate transporter gene is derived from Nocardia farcinica. In
another embodiment, the propionate transporter gene is derived from
E. coli. In another embodiment, the propionate transporter gene is
derived from B. subtilis. In some embodiments, the bacterial
species is Escherichia coli. In some embodiments, the bacterial
species is Escherichia coli strain Nissle.
[0441] Assays for testing the activity of a propionate transporter,
a functional variant of a propionate transporter, or a functional
fragment of transporter of propionate are well known to one of
ordinary skill in the art. For example, propionate import can be
assessed by expressing the protein, functional variant, or fragment
thereof, in a recombinant bacterial cell that lacks an endogenous
propionate transporter. Propionate import can also be assessed
using mass spectrometry. Propionate import can also be expressed
using gas chromatography. For example, samples can be injected into
a Perkin Elmer Autosystem XL Gas Chromatograph containing a Supelco
packed column, and the analysis can be performed according to
manufacturing instructions (see, for example, Supelco I (1998)
Analyzing fatty acids by packed column gas chromatography, Bulletin
856B:2014). Alternatively, samples can be analyzed for propionate
import using high-pressure liquid chromatography (HPLC). For
example, a computer-controlled Waters HPLC system equipped with a
model 600 quaternary solvent delivery system, and a model 996
photodiode array detector, and components of the sample can be
resolved with an Aminex HPX-87H (300 by 7.8 mm) organic acid
analysis column (Bio-Rad Laboratories) (see, for example, Palacios
et al., 2003, J. Bacteriol., 185(9):2802-2810).
[0442] Propionate transporters may be expressed or modified in the
bacteria in order to enhance propionate transport into the cell.
Specifically, when the propionate transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
propionate into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a propionate transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a propionate transporter and a genetic modification that
reduces export of a propionate, e.g., a genetic mutation in an
exporter gene or promoter.
[0443] In one embodiment, the bacterial cell comprises at least one
gene encoding a propionate transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a
propionate transporter. In some embodiments, the at least one
native gene encoding a propionate transporter is not modified. In
another embodiment, the bacterial cell comprises more than one copy
of at least one native gene encoding a propionate transporter. In
yet another embodiment, the bacterial cell comprises a copy of at
least one gene encoding a native propionate transporter, as well as
at least one copy of at least one heterologous gene encoding a
propionate transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a propionate transporter. In one embodiment, the bacterial
cell comprises multiple copies of the at least one heterologous
gene encoding a propionate transporter.
[0444] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a propionate transporter, wherein said
propionate transporter comprises a propionate sequence that has at
least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity
to the propionate sequence of a polypeptide encoded by a propionate
transporter gene disclosed herein.
[0445] The present disclosure further comprises genes encoding
functional fragments of a propionate transporter or functional
variants of a propionate transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
propionate transporter relates to an element having qualitative
biological activity in common with the wild-type propionate
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
propionate transporter is one which retains essentially the same
ability to import propionate into the bacterial cell as does the
propionate transporter protein from which the functional fragment
or functional variant was derived. In one embodiment, the
recombinant bacterial cell comprises at least one heterologous gene
encoding a functional fragment of a propionate transporter. In
another embodiment, the recombinant bacterial cell comprises a
heterologous gene encoding a functional variant of a propionate
transporter.
[0446] In one embodiment, the genes encoding the propionate
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the propionate transporter have been codon-optimized
for use in Escherichia coli.
[0447] The present disclosure also encompasses genes encoding a
propionate transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0448] In some embodiments, the at least one gene encoding a
propionate transporter is mutagenized; mutants exhibiting increased
propionate import are selected; and the mutagenized at least one
gene encoding a propionate transporter is isolated and inserted
into the bacterial cell. In some embodiments, the at least one gene
encoding a propionate transporter is mutagenized; mutants
exhibiting decreased propionate import are selected; and the
mutagenized at least one gene encoding a propionate transporter is
isolated and inserted into the bacterial cell. The transporter
modifications described herein may be present on a plasmid or
chromosome.
[0449] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a propionate transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding a
propionate transporter is directly operably linked to the promoter.
In another embodiment, the at least one gene encoding a propionate
transporter is indirectly operably linked to the promoter.
[0450] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a propionate transporter in nature.
In some embodiments, the at least one gene encoding the propionate
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the propionate
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the propionate
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the propionate transporter is controlled by a constitutive
promoter.
[0451] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0452] In one embodiment, the at least one gene encoding a
propionate transporter is located on a plasmid in the bacterial
cell. In some embodiments, the plasmid is a high copy number
plasmid. In some embodiments, the plasmid is a low copy number
plasmid. In another embodiment, the at least one gene encoding a
propionate transporter is located in the chromosome of the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a propionate transporter is located in the
chromosome of the bacterial cell, and a copy of at least one gene
encoding a propionate transporter from a different species of
bacteria is located on a plasmid in the bacterial cell. In yet
another embodiment, a native copy of the at least one gene encoding
a propionate transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding a propionate
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a propionate transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a propionate transporter from a
different species of bacteria is located in the chromosome of the
bacterial cell.
[0453] In some embodiments, the at least one native gene encoding
the propionate transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the propionate transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0454] In some embodiments, at least one native gene encoding the
propionate transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the propionate transporter in the
bacterial cell is modified, and one or more additional copies of at
least one native gene encoding the transporter are present in the
bacterial cell on a plasmid. In alternate embodiments, the at least
one native gene encoding the transporter is modified, and a copy of
at least one gene encoding the transporter from a different
bacterial species is present in the bacteria on a plasmid.
[0455] In one embodiment, the propionate transporter is MctC. In
one embodiment, the mctC gene has at least about 80% identity to
SEQ ID NO:129. Accordingly, in one embodiment, the mctC gene has at
least about 90% identity to SEQ ID NO:129. Accordingly, in one
embodiment, the mctC gene has at least about 95% identity to SEQ ID
NO:12. Accordingly, in one embodiment, the mctC gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:129. In
another embodiment, the mctC gene comprises the sequence of SEQ ID
NO:129. In yet another embodiment the mctC gene consists of the
sequence of SEQ ID NO:129.
[0456] In another embodiment, the propionate transporter is PutP_6.
In one embodiment, the putP_6 gene has at least about 80% identity
to SEQ ID NO:130. Accordingly, in one embodiment, the putP_6 gene
has at least about 90% identity to SEQ ID NO:130. Accordingly, in
one embodiment, the putP_6 gene has at least about 95% identity to
SEQ ID NO:130. Accordingly, in one embodiment, the putP_6 gene has
at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID
NO:130. In another embodiment, the putP_6 gene comprises the
sequence of SEQ ID NO:130. In yet another embodiment the putP_6
gene consists of the sequence of SEQ ID NO:130.
[0457] Other propionate transporter genes are known to those of
ordinary skill in the art. See, for example, Jolker et al., J.
Bacteriol., 2009, 191(3):940-948. In one embodiment, the propionate
transporter comprises the mctBC genes from C. glutamicum. In
another embodiment, the propionate transporter comprises the
dip0780 and dip0791 genes from C. diphtheria. In another
embodiment, the propionate transporter comprises the ce0909 and
ce0910 genes from C. efficiens. In another embodiment, the
propionate transporter comprises the ce1091 and ce1092 genes from
C. efficiens. In another embodiment, the propionate transporter
comprises the sco1822 and sco1823 genes from S. coelicolor. In
another embodiment, the propionate transporter comprises the
sco1218 and sco1219 genes from S. coelicolor. In another
embodiment, the propionate transporter comprises the ce1091 and
sco5827 genes from S. coelicolor. In another embodiment, the
propionate transporter comprises the m_5160, m_5161, m_5165, and
m_5166 genes from M. smegmatis. In another embodiment, the
propionate transporter comprises the nfa 17930, nfa 17940, nfa
17950, and nfa 17960 genes from N. farcinica. In another
embodiment, the propionate transporter comprises the actP and yjcH
genes from E. coli. In another embodiment, the propionate
transporter comprises the ywcB and ywcA genes from B. subtilis.
[0458] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0459] In one embodiment, when the propionate transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more propionate into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the propionate transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more propionate, into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In yet another
embodiment, when the propionate transporter is expressed in the
recombinant bacterial cells, the bacterial cells import two-fold
more propionate into the cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the propionate
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold more
propionate into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions.
[0460] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous propionate transporter and a
second heterologous propionate transporter. In one embodiment, said
first propionate transporter is derived from a different organism
than said second propionate transporter. In some embodiments, said
first propionate transporter is derived from the same organism as
said second propionate transporter. In some embodiments, said first
propionate transporter is a wild-type propionate transporter and
said second propionate transporter is a mutagenized version of said
first propionate transporter. In some embodiments, the recombinant
bacterial cells described herein comprise at least a third
heterologous propionate transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least four
heterologous propionate transporters. In some embodiments, the
recombinant bacterial cells described herein comprise at least five
heterologous propionate transporters or more.
[0461] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an propionate transporter
may be used to treat a disease, condition, and/or symptom
associated with the catabolism of propionate in a subject. In some
embodiments, the recombinant bacterial cells described herein may
be used to reduce, ameliorate, or eliminate one or more symptom(s)
associated with these diseases or disorders. In one embodiment, the
disorder associated with the catabolism of propionate is a
metabolic disorder involving the abnormal catabolism of propionate.
Metabolic diseases associated with abnormal catabolism of
propionate include propionic acidemia (PA) and methylmalonic
acidemia (MMA), as well as severe nutritional vitamin B.sub.12
deficiencies. In one embodiment, the disease associated with
abnormal catabolism of propionate is propionic acidemia. In one
embodiment, the disease associated with abnormal catabolism of
propionate is methylmalonic acidemia. In another embodiment, the
disease associated with abnormal catabolism of propionate is a
vitamin B.sub.12 deficiency.
G. Bile Salt Acid Transporters
[0462] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a bile salt
transporter. In one embodiment, the bile salt transporter
transports bile salt into the cell.
[0463] The uptake of bile salt into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, the
uptake of bile salts into the Lactobacillus and Bifidobacterium has
been found to occur via the bile salt transporters CbsT1 and CbsT2
(see, e.g., Elkins et al., Microbiology, 147(Pt. 12):3403-3412
(2001), the entire contents of which are expressly incorporated
herein by reference). Other proteins that mediate the import of
bile salts into cells are well known to those of skill in the
art.
[0464] In one embodiment, the at least one gene encoding a bile
salt transporter is a cbsT1 or a cbsT2 gene. In one embodiment, the
bacterial cell described herein has been genetically engineered to
comprise at least one heterologous gene selected from a cbsT1 or a
cbsT2 gene. In one embodiment, the at least one gene encoding a
bile salt transporter is the cbsT1 gene. In one embodiment, the at
least one gene encoding a bile salt transporter is the cbsT2 gene.
In one embodiment, the bile acid transporter is the bile acid
sodium symporter ASBT.sub.NM (NMB0705 gene of Neisseria
meningitides).
[0465] In some embodiments, the bile salt transporter is encoded by
a bile salt transporter gene derived from a bacterial genus or
species, including but not limited to, Lactobacillus, for example,
Lactobacillus johnsonni (e.g., Lactobacillus johnsonni strain
100-100). In some embodiments, the bacterial species is Escherichia
coli. In some embodiments, the bacterial species is Escherichia
coli strain Nissle.
[0466] Assays for testing the activity of an transporter of a bile
salt, a functional variant of an transporter of a bile salt, or a
functional fragment of an transporter of a bile salt are well known
to one of ordinary skill in the art. For example, bile salt import
can be assessed as described in Elkins et al. (2001) Microbiology,
147:3403-3412, the entire contents of which are expressly
incorporated herein by reference.
[0467] Bile salt transporters may be expressed or modified in the
bacteria in order to enhance bile salt transport into the cell.
Specifically, when the bile salt transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more bile
salt into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a bile salt transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a bile salt transporter and a genetic modification that
reduces export of a bile salt, e.g., a genetic mutation in an
exporter gene or promoter.
[0468] In one embodiment, the bacterial cell comprises at least one
gene encoding a bile salt transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a bile
salt transporter. In some embodiments, the at least one native gene
encoding a bile salt transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding a bile salt transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native bile salt transporter, as well as at
least one copy of at least one heterologous gene encoding a bile
salt transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a bile salt transporter. In one embodiment, the bacterial
cell comprises multiple copies of the at least one heterologous
gene encoding a bile salt transporter.
[0469] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a bile salt transporter, wherein said
bile salt transporter comprises a bile salt sequence that has at
least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity
to the bile salt sequence of a polypeptide encoded by a bile salt
transporter gene disclosed herein.
[0470] The present disclosure further comprises genes encoding
functional fragments of a bile salt transporter or functional
variants of a bile salt transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
bile salt transporter relates to an element having qualitative
biological activity in common with the wild-type bile salt
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
bile salt transporter is one which retains essentially the same
ability to import bile salt into the bacterial cell as does the
bile salt transporter protein from which the functional fragment or
functional variant was derived. In one embodiment, the recombinant
bacterial cell comprises at least one heterologous gene encoding a
functional fragment of a bile salt transporter. In another
embodiment, the recombinant bacterial cell comprises a heterologous
gene encoding a functional variant of a bile salt transporter.
[0471] In one embodiment, the genes encoding the bile salt
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the bile salt transporter have been codon-optimized
for use in Escherichia coli.
[0472] The present disclosure also encompasses genes encoding a
bile salt transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0473] In some embodiments, the at least one gene encoding a bile
salt transporter is mutagenized; mutants exhibiting increased bile
salt import are selected; and the mutagenized at least one gene
encoding a bile salt transporter is isolated and inserted into the
bacterial cell. In some embodiments, the at least one gene encoding
a bile salt transporter is mutagenized; mutants exhibiting
decreased bile salt import are selected; and the mutagenized at
least one gene encoding a bile salt transporter is isolated and
inserted into the bacterial cell. The transporter modifications
described herein may be present on a plasmid or chromosome.
[0474] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a bile salt transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding a
bile salt transporter is directly operably linked to the promoter.
In another embodiment, the at least one gene encoding a bile salt
transporter is indirectly operably linked to the promoter.
[0475] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a bile salt transporter in nature.
In some embodiments, the at least one gene encoding the bile salt
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the bile salt
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the bile salt
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the bile salt transporter is controlled by a constitutive
promoter.
[0476] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0477] In one embodiment, the at least one gene encoding a bile
salt transporter is located on a plasmid in the bacterial cell. In
some embodiments, the plasmid is a high copy number plasmid. In
some embodiments, the plasmid is a low copy number plasmid. In
another embodiment, the at least one gene encoding a bile salt
transporter is located in the chromosome of the bacterial cell. In
yet another embodiment, a native copy of the at least one gene
encoding a bile salt transporter is located in the chromosome of
the bacterial cell, and a copy of at least one gene encoding a bile
salt transporter from a different species of bacteria is located on
a plasmid in the bacterial cell. In yet another embodiment, a
native copy of the at least one gene encoding a bile salt
transporter is located on a plasmid in the bacterial cell, and a
copy of at least one gene encoding a bile salt transporter from a
different species of bacteria is located on a plasmid in the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a bile salt transporter is located in the
chromosome of the bacterial cell, and a copy of the at least one
gene encoding a bile salt transporter from a different species of
bacteria is located in the chromosome of the bacterial cell.
[0478] In some embodiments, the at least one native gene encoding
the bile salt transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the bile salt transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0479] In some embodiments, at least one native gene encoding the
bile salt transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the bile salt transporter in the bacterial
cell is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid. In one embodiment,
the bile salt transporter is the bile salt transporter CbsT1. In
one embodiment, the cbsT1 gene has at least about 80% identity to
SEQ ID NO:131. Accordingly, in one embodiment, the cbsT1 gene has
at least about 90% identity to SEQ ID NO:131. Accordingly, in one
embodiment, the cbsT1 gene has at least about 95% identity to SEQ
ID NO:131. Accordingly, in one embodiment, the cbsT1 gene has at
least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:131. In
another embodiment, the cbsT1 gene comprises the sequence of SEQ ID
NO:131. In yet another embodiment the cbsT1 gene consists of the
sequence of SEQ ID NO:131.
[0480] In one embodiment, the bile salt transporter is the bile
salt transporter CbsT2. In one embodiment, the cbsT2 gene has at
least about 80% identity to SEQ ID NO:132. Accordingly, in one
embodiment, the cbsT2 gene has at least about 90% identity to SEQ
ID NO:132. Accordingly, in one embodiment, the cbsT2 gene has at
least about 95% identity to SEQ ID NO:132. Accordingly, in one
embodiment, the cbsT2 gene has at least about 70%, 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identity to SEQ ID NO:132. In another embodiment, the cbsT2
gene comprises the sequence of SEQ ID NO:132. In yet another
embodiment the cbsT2 gene consists of the sequence of SEQ ID
NO:132.
[0481] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0482] In one embodiment, when the bile salt transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more bile salt into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the bile salt transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more bile salt, into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In yet another
embodiment, when the bile salt transporter is expressed in the
recombinant bacterial cells, the bacterial cells import two-fold
more bile salt into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the bile salt
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold more bile salt
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same
conditions.
[0483] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous bile salt transporter and a
second heterologous bile salt transporter. In one embodiment, said
first bile salt transporter is derived from a different organism
than said second bile salt transporter. In some embodiments, said
first bile salt transporter is derived from the same organism as
said second bile salt transporter. In some embodiments, said first
bile salt transporter imports the same bile salt as said second
bile salt transporter. In other embodiment, said first bile salt
transporter imports a different bile salt from said second bile
salt transporter. In some embodiments, said first bile salt
transporter is a wild-type bile salt transporter and said second
bile salt transporter is a mutagenized version of said first bile
salt transporter. In some embodiments, the recombinant bacterial
cells described herein comprise at least a third heterologous bile
salt transporter. In some embodiments, the recombinant bacterial
cells described herein comprise at least four heterologous bile
salt transporters. In some embodiments, the recombinant bacterial
cells described herein comprise at least five heterologous bile
salt transporters or more.
[0484] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an bile salt transporter
may be used to treat a disease, condition, and/or symptom
associated with bile salts. In some embodiments, the recombinant
bacterial cells described herein may be used to reduce, ameliorate,
or eliminate one or more symptom(s) associated with these diseases
or disorders. In some embodiments, the disease or disorder
associated with bile salts is cardiovascular disease, metabolic
disease, liver disease, such as cirrhosis or NASH, gastrointestinal
cancer, and/or C. difficile infection. In some embodiments, the
disclosure provides methods for reducing, ameliorating, or
eliminating one or more symptom(s) associated with these diseases,
including but not limited to chest pain, heart failure, or weight
gain. In some embodiments, the disease is secondary to other
conditions, e.g., cardiovascular disease or liver disease.
H. Ammonia Transporters
[0485] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is an ammonia
transporter. In one embodiment, the ammonia transporter transports
ammonia into the cell.
[0486] The uptake of ammonia into bacterial cells is mediated by
proteins well known to those of skill in the art. For example,
theammonium/methylammonium transport B (AmtB) protein is a membrane
transport protein that transports ammonia into bacterial cells. In
one embodiment, the at least one gene encoding an ammonia
transporter is an amtB gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous an amtB gene.
[0487] In some embodiments, the ammonia transporter is encoded by
an ammonia transporter gene derived from a bacterial genus or
species, including but not limited to, Corynebacterium, e.g.,
Corynebacterium glutamicum, Escherichia, e.g., Escherichia coli,
Streptomyces, e.g., Streptomyces coelicolor, or Ruminococcus, e.g.,
Ruminococcus albus. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0488] Assays for testing the activity of an ammonia transporter, a
functional variant of an ammonia transporter, or a functional
fragment of transporter of ammonia are well known to one of
ordinary skill in the art. For example, import of ammonia may be
determined using a methylammonium uptake assay, as described in
Soupene et al. (1998) Proc. Natl. Acad. Sci. U.S.A.95(12): 7030-4,
the entire contents of each of which are expressly incorporated by
reference herein.
[0489] Ammonia transporters may be expressed or modified in the
bacteria in order to enhance ammonia transport into the cell.
Specifically, when the ammonia transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
ammonia into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding an ammonia transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding an ammonia transporter and a genetic modification that
reduces export of a ammonia, e.g., a genetic mutation in an
exporter gene or promoter.
[0490] In one embodiment, the bacterial cell comprises at least one
gene encoding an ammonia transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding an
ammonia transporter. In some embodiments, the at least one native
gene encoding an ammonia transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding an ammonia transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native ammonia transporter, as well as at least
one copy of at least one heterologous gene encoding an ammonia
transporter from a different bacterial species. In one embodiment,
the bacterial cell comprises at least one, two, three, four, five,
or six copies of the at least one heterologous gene encoding an
ammonia transporter. In one embodiment, the bacterial cell
comprises multiple copies of the at least one heterologous gene
encoding an ammonia transporter.
[0491] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding an ammonia transporter, wherein said
ammonia transporter comprises an ammonia sequence that has at least
70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to
the ammonia sequence of a polypeptide encoded by an ammonia
transporter gene disclosed herein.
[0492] The present disclosure further comprises genes encoding
functional fragments of an ammonia transporter or functional
variants of an ammonia transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of an
ammonia transporter relates to an element having qualitative
biological activity in common with the wild-type ammonia
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
ammonia transporter is one which retains essentially the same
ability to import ammonia into the bacterial cell as does the
ammonia transporter protein from which the functional fragment or
functional variant was derived. In one embodiment, the recombinant
bacterial cell comprises at least one heterologous gene encoding a
functional fragment of an ammonia transporter. In another
embodiment, the recombinant bacterial cell comprises a heterologous
gene encoding a functional variant of an ammonia transporter.
[0493] In one embodiment, the genes encoding the ammonia
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the ammonia transporter have been codon-optimized
for use in Escherichia coli.
[0494] The present disclosure also encompasses genes encoding an
ammonia transporter comprising amino acids in its sequence that are
substantially the same as an amino acid sequence described herein
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0495] In some embodiments, the at least one gene encoding an
ammonia transporter is mutagenized; mutants exhibiting increased
ammonia import are selected; and the mutagenized at least one gene
encoding an ammonia transporter is isolated and inserted into the
bacterial cell. In some embodiments, the at least one gene encoding
an ammonia transporter is mutagenized; mutants exhibiting decreased
ammonia import are selected; and the mutagenized at least one gene
encoding an ammonia transporter is isolated and inserted into the
bacterial cell. The transporter modifications described herein may
be present on a plasmid or chromosome.
[0496] In some embodiments, the bacterial cell comprises a
heterologous gene encoding an ammonia transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding an
ammonia transporter is directly operably linked to the promoter. In
another embodiment, the at least one gene encoding an ammonia
transporter is indirectly operably linked to the promoter.
[0497] In one embodiment, the promoter is not operably linked with
the at least one gene encoding an ammonia transporter in nature. In
some embodiments, the at least one gene encoding the ammonia
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the ammonia transporter
is controlled by an inducible promoter. In some embodiments, the at
least one gene encoding the ammonia transporter is controlled by a
promoter that is stronger than its native promoter. In some
embodiments, the at least one gene encoding the ammonia transporter
is controlled by a constitutive promoter.
[0498] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0499] In one embodiment, the at least one gene encoding an ammonia
transporter is located on a plasmid in the bacterial cell. In some
embodiments, the plasmid is a high copy number plasmid. In some
embodiments, the plasmid is a low copy number plasmid. In another
embodiment, the at least one gene encoding an ammonia transporter
is located in the chromosome of the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding an
ammonia transporter is located in the chromosome of the bacterial
cell, and a copy of at least one gene encoding an ammonia
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding an ammonia transporter is
located on a plasmid in the bacterial cell, and a copy of at least
one gene encoding an ammonia transporter from a different species
of bacteria is located on a plasmid in the bacterial cell. In yet
another embodiment, a native copy of the at least one gene encoding
an ammonia transporter is located in the chromosome of the
bacterial cell, and a copy of the at least one gene encoding an
ammonia transporter from a different species of bacteria is located
in the chromosome of the bacterial cell.
[0500] In some embodiments, the at least one native gene encoding
the ammonia transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the ammonia transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0501] In some embodiments, at least one native gene encoding the
ammonia transporter in the bacterial cell is not modified, and one
or more additional copies of at least one native gene encoding the
transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the ammonia transporter in the bacterial
cell is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0502] In one embodiment, the ammonia transporter is the ammonia
transporter AmtB, for example the Escherichia coli AmtB. In one
embodiment the ammonia transporter is encoded by a amtB gene. In
one embodiment, the amtB gene has at least about 80% identity with
the sequence of SEQ ID NO:133. Accordingly, in one embodiment, the
amtB gene has at least about 90% identity with the sequence of SEQ
ID NO:133. Accordingly, in one embodiment, the amtB gene has at
least about 95% identity with the sequence of SEQ ID NO:133.
Accordingly, in one embodiment, the amtB gene has at least about
70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID
NO:133. In another embodiment, the amtB gene comprises the sequence
of SEQ ID NO:133. In yet another embodiment the amtB gene consists
of the sequence of SEQ ID NO:133.
[0503] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0504] In one embodiment, when the ammonia transporter is expressed
in the recombinant bacterial cells, the bacterial cells import 10%
more ammonia into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In another embodiment, when the ammonia
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% more ammonia, into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
ammonia transporter is expressed in the recombinant bacterial
cells, the bacterial cells import two-fold more ammonia into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In yet
another embodiment, when the ammonia transporter is expressed in
the recombinant bacterial cells, the bacterial cells import
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold,
fourty-fold, or fifty-fold more ammonia into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions.
[0505] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous ammonia transporter and a
second heterologous ammonia transporter. In one embodiment, said
first ammonia transporter is derived from a different organism than
said second ammonia transporter. In some embodiments, said first
ammonia transporter is derived from the same organism as said
second ammonia transporter. In some embodiments, said first ammonia
transporter is a wild-type ammonia transporter and said second
ammonia transporter is a mutagenized version of said first ammonia
transporter. In some embodiments, the recombinant bacterial cells
described herein comprise at least a third heterologous ammonia
transporter. In some embodiments, the recombinant bacterial cells
described herein comprise at least four heterologous ammonia
transporters. In some embodiments, the recombinant bacterial cells
described herein comprise at least five heterologous ammonia
transporters or more.
[0506] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an ammonia transporter may
be used to treat a disease, condition, and/or symptom associated
with hyperammonenia. In some embodiment, the recombinant bacterial
cells described herein can be used to treat hepatic encephalopathy.
In some embodiment, the recombinant bacterial cells described
herein can be used to treat Huntington's disease. In some
embodiments, the invention provides methods for reducing,
ameliorating, or eliminating one or more symptom(s) associated with
hepatic encephalopathy and Huntington's disease. In some
embodiments, the symptom(s) associated thereof include, but are not
limited to, seizures, ataxia, stroke-like lesions, coma, psychosis,
vision loss, acute encephalopathy, cerebral edema, as well as
vomiting, respiratory alkalosis, and hypothermia.
I. .gamma.-Aminobutyric Acid (GABA) Transporters
[0507] .gamma.-aminobutyric acid (GABA) is the predominant
inhibitory neurotransmitter (C.sub.4H.sub.9NO.sub.2) in the
mammalian central nervous system. In humans, GABA is also directly
responsible for regulating muscle tone. GABA is capable of
activating the GABAA receptor, which is part of a ligand-gated ion
channel complex, as well as the GABAs metabotropic G
protein-coupled receptor. Neurons that produce GABA are known as
"GABAergic" neurons, and activation of GABA receptors is described
as GABAergic tone (i.e., increased activation of GABA receptors
refers to increased GABAergic tone).
[0508] .gamma.-Aminobutyric acid (GABA) is the predominant
inhibitory neurotransmitter in the mammalian central nervous
system. In humans, GABA activates the postsynaptic GABAA receptor,
which is part of a ligand-gated chloride-specific ion channel
complex. Activation of this complex on a post-synaptic neuron
allows chloride ions to enter the neuron and exert an inhibitory
effect. Alterations of such GABAergic neurotransmission have been
implicated in the pathophysiology of several neurological
disorders, including epilepsy (Jones-Davis and MacDonald (2003)
Curr. Opin. Pharmacol. 3(1): 12-8), Huntington's disease
(Krogsgaard-Larsen (1992) Pharmacol Toxicol. 70(2):95-104), and
hepatic encephalopathy (Jones and Basile (1997) Adv. Exp. Med.
Biol. 420: 75-83). Neurons in the brain that are modulated by GABA
are said to be under inhibitory GABAergic tone. This inhibitory
tone prevents neuronal firing until a sufficiently potent
stimulatory stimulus is received, or until the inhibitory tone is
otherwise released. Increased GABAergic tone in hepatic
encephalopathy (HE) was initially described in the early 1980s,
based on a report of similar visual response patterns in rabbits
with galactosamine-induced liver failure and rabbits treated with
allosteric modulators of the GABAA receptor (e.g., pentobarbital,
diazepam) (Jones and Basile, 1997). Clinical improvements in
hepatic encephalopathy patients treated with a highly selective
benzodiazapene antagonist at the GABAA receptor, flumazenil,
further confirmed these observations (Banksy et al. (1985) Lancet
1: 1324-5 ; Scollo-Lavizzari and Steinmann (1985) Lancet 1: 1324.
Increased GABAergic tone in HE has since been proposed as a
consequence of one or more of the following: (1) increased GABA
concentrations in the brain, (2) altered integrity of the GABAA
receptor, and/or (3) increased concentrations of endogenous
modulators of the GABAA receptor (Ahboucha and Butterworth (2004)
Metab. Brain Dis.1 9(3-4):331-343).
[0509] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a GABA
transporter. In one embodiment, the GABA transporter transports
GABA into the cell.
[0510] The uptake of GABA into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, GABA
uptake in E. coli is driven by membrane potential and facilitated
by the membrane transport protein, GabP (Li et al. (2001) FEBS
Lett. 494(3): 165-169. GabP is a member of the amino
acid/polymaine/organocation (APC) transporter superfamily, one of
the two largest families of secondary active transporters (Jack et
al. (2000) Microbiology 146: 1797-1814). GabP protein, encoded by
the gabP gene, consists of 466 amino acids and 12 transmembrane
alpha helices, wherein both N- and C-termini face the cytosol (Hu
and King, (1998) Biochem J. 336(Pt 1): 69-76. The GabP residue
sequence also includes a consensus amphipathic region (CAR), which
is conserved between members of the APC family from bacteria to
mammals (Hu and King, 1998). Upon entry into the cell, GABA is
converted to succinyl semialdehyde (SSA) by GABA a-ketoglutarate
transaminase (GSST). Succinate-semialdehyde dehydrogenase (SSDH)
then catalyzes the second and only other specific step in GABA
catabolism, the oxidation of succinyl semialdehyde to succinate
(Dover and Halpern (1972) J. Bacteriol. 109(2):835-43). Ultimately,
succinate becomes a substrate for the citric acid (TCA) cycle. In
one embodiment, the at least one gene encoding a GABA transporter
is encoded by an gabP gene. In one embodiment, the bacterial cell
described herein has been genetically engineered to comprise at
least one heterologous an gabP gene.
[0511] In some embodiments, the GABA transporter is encoded by a
GABA transporter gene derived from a bacterial genus or species,
including but not limited to, Bacillus, e.g., Bacillus subtilis, or
Escherichia, e.g., Escherichia coli. In some embodiments, the
bacterial species is Escherichia coli. In some embodiments, the
bacterial species is Escherichia coli strain Nissle.
[0512] Assays for testing the activity of a GABA transporter, a
functional variant of a GABA transporter, or a functional fragment
of transporter of GABA are well known to one of ordinary skill in
the art.
[0513] GABA transporters may be expressed or modified in the
bacteria in order to enhance GABA transport into the cell.
Specifically, when the GABA transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more GABA
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In one embodiment, the bacterial cell comprises a heterologous gene
encoding a GABA transporter. In one embodiment, the bacterial cell
comprises a heterologous gene encoding a GABA transporter and a
genetic modification that reduces export of a GABA, e.g., a genetic
mutation in an exporter gene or promoter. In one embodiment, the
bacterial cell comprises at least one gene encoding a GABA
transporter from a different organism, e.g., a different species of
bacteria. In one embodiment, the bacterial cell comprises at least
one native gene encoding a GABA transporter. In some embodiments,
the at least one native gene encoding a GABA transporter is not
modified. In another embodiment, the bacterial cell comprises more
than one copy of at least one native gene encoding a GABA
transporter. In yet another embodiment, the bacterial cell
comprises a copy of at least one gene encoding a native GABA
transporter, as well as at least one copy of at least one
heterologous gene encoding a GABA transporter from a different
bacterial species. In one embodiment, the bacterial cell comprises
at least one, two, three, four, five, or six copies of the at least
one heterologous gene encoding a GABA transporter. In one
embodiment, the bacterial cell comprises multiple copies of the at
least one heterologous gene encoding a GABA transporter.
[0514] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a GABA transporter, wherein said GABA
transporter comprises a GABA sequence that has at least 70%, 75%,
80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the GABA
sequence of a polypeptide encoded by a GABA transporter gene
disclosed herein.
[0515] The present disclosure further comprises genes encoding
functional fragments of a GABA transporter or functional variants
of a GABA transporter. As used herein, the term "functional
fragment thereof" or "functional variant thereof" of a GABA
transporter relates to an element having qualitative biological
activity in common with the wild-type GABA transporter from which
the fragment or variant was derived. For example, a functional
fragment or a functional variant of a mutated GABA transporter is
one which retains essentially the same ability to import GABA into
the bacterial cell as does the GABA transporter protein from which
the functional fragment or functional variant was derived. In one
embodiment, the recombinant bacterial cell comprises at least one
heterologous gene encoding a functional fragment of a GABA
transporter. In another embodiment, the recombinant bacterial cell
comprises a heterologous gene encoding a functional variant of a
GABA transporter.
[0516] In one embodiment, the genes encoding the GABA transporter
have been codon-optimized for use in the host organism, e.g., a
bacterial cell disclosed herein. In one embodiment, the genes
encoding the GABA transporter have been codon-optimized for use in
Escherichia coli.
[0517] The present disclosure also encompasses genes encoding a
GABA transporter comprising amino acids in its sequence that are
substantially the same as an amino acid sequence described herein
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions. In some embodiments, the at least one
gene encoding a GABA transporter is mutagenized; mutants exhibiting
increased GABA import are selected; and the mutagenized at least
one gene encoding a GABA transporter is isolated and inserted into
the bacterial cell. In some embodiments, the at least one gene
encoding a GABA transporter is mutagenized; mutants exhibiting
decreased GABA import are selected; and the mutagenized at least
one gene encoding a GABA transporter is isolated and inserted into
the bacterial cell. The transporter modifications described herein
may be present on a plasmid or chromosome. In some embodiments, the
bacterial cell comprises a heterologous gene encoding a GABA
transporter operably linked to a promoter. In one embodiment, the
at least one gene encoding a GABA transporter is directly operably
linked to the promoter. In another embodiment, the at least one
gene encoding a GABA transporter is indirectly operably linked to
the promoter.
[0518] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a GABA transporter in nature. In
some embodiments, the at least one gene encoding the GABA
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the GABA transporter is
controlled by an inducible promoter. In some embodiments, the at
least one gene encoding the GABA transporter is controlled by a
promoter that is stronger than its native promoter. In some
embodiments, the at least one gene encoding the GABA transporter is
controlled by a constitutive promoter.
[0519] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0520] In one embodiment, the at least one gene encoding a GABA
transporter is located on a plasmid in the bacterial cell. In some
embodiments, the plasmid is a high copy number plasmid. In some
embodiments, the plasmid is a low copy number plasmid. In another
embodiment, the at least one gene encoding a GABA transporter is
located in the chromosome of the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a GABA
transporter is located in the chromosome of the bacterial cell, and
a copy of at least one gene encoding a GABA transporter from a
different species of bacteria is located on a plasmid in the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a GABA transporter is located on a plasmid
in the bacterial cell, and a copy of at least one gene encoding a
GABA transporter from a different species of bacteria is located on
a plasmid in the bacterial cell. In yet another embodiment, a
native copy of the at least one gene encoding a GABA transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a GABA transporter from a different
species of bacteria is located in the chromosome of the bacterial
cell.
[0521] In some embodiments, the at least one native gene encoding
the GABA transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the GABA transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0522] In some embodiments, at least one native gene encoding the
GABA transporter in the bacterial cell is not modified, and one or
more additional copies of at least one native gene encoding the
transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the GABA transporter in the bacterial cell
is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0523] In one embodiment, the GABA transporter is the GABA
transporter GabP, for example the Escherichia coli GabP. In one
embodiment the GABA transporter is encoded by a amtB gene. In one
embodiment, the gabP gene has at least about 80% identity with the
sequence of SEQ ID NO:134. Accordingly, in one embodiment, the gabP
gene has at least about 90% identity with the sequence of SEQ ID
NO:134. Accordingly, in one embodiment, the gabP gene has at least
about 95% identity with the sequence of SEQ ID NO:134. Accordingly,
in one embodiment, the gabP gene has at least about 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity with the sequence of SEQ ID NO:134. In another
embodiment, the gabP gene comprises the sequence of SEQ ID NO:134.
In yet another embodiment the gabP gene consists of the sequence of
SEQ ID NO:134.
[0524] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0525] In one embodiment, when the GABA transporter is expressed in
the recombinant bacterial cells, the bacterial cells import 10%
more GABA into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In another embodiment, when the GABA transporter
is expressed in the recombinant bacterial cells, the bacterial
cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more
GABA, into the bacterial cell when the transporter is expressed
than unmodified bacteria of the same bacterial subtype under the
same conditions. In yet another embodiment, when the GABA
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import two-fold more GABA into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the GABA transporter is expressed in the
recombinant bacterial cells, the bacterial cells import three-fold,
four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or
fifty-fold more GABA into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions.
[0526] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous GABA transporter and a second
heterologous GABA transporter. In one embodiment, said first GABA
transporter is derived from a different organism than said second
GABA transporter. In some embodiments, said first GABA transporter
is derived from the same organism as said second GABA transporter.
In some embodiments, said first GABA transporter is a wild-type
GABA transporter and said second GABA transporter is a mutagenized
version of said first GABA transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least a
third heterologous GABA transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least four
heterologous GABA transporters. In some embodiments, the
recombinant bacterial cells described herein comprise at least five
heterologous GABA transporters or more.
[0527] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an GABA transporter may be
used to treat a disease, condition, and/or symptom associated with
hyperammonenia. In some embodiment, the recombinant bacterial cells
described herein can be used to treat hepatic encephalopathy. In
some embodiment, the recombinant bacterial cells described herein
can be used to treat Huntington's disease. In some embodiments, the
invention provides methods for reducing, ameliorating, or
eliminating one or more symptom(s) associated with hepatic
encephalopathy and Huntington's disease. In some embodiments, the
symptom(s) associated thereof include, but are not limited to,
seizures, ataxia, stroke-like lesions, coma, psychosis, vision
loss, acute encephalopathy, cerebral edema, as well as vomiting,
respiratory alkalosis, and hypothermia.
J. Manganese Transporters
[0528] In biological systems, manganese (Mn.sup.2+) is an essential
trace metal and plays an important role in enzyme-mediated
catalysis, but can also have deleterious effects. Manganese is a
biologically important trace metal and is required for the survival
of most living organisms. Cells maintain manganese under tight
homeostatic control in order to avoid toxicity. In mammals,
manganese is excreted in the bile, but its disposal is affected by
the impaired flow of bile from the liver to the duodenum (i.e.,
cholestasis) that accompanies liver failure Similar to ammonia,
elevated concentrations of manganese play a role in the development
of hepatic encephalopathy (Rivera-Manda et al. (2012) Neurochem.
Res. 37(5): 1074-1084). Astrocytes in the brain which detoxify
ammonia in a reaction catalyzed by glutamine synthetase, require
manganese as a cofactor and thus have a tendency to accumulate this
metal (Aschner et al. (1999) Neurotoxicology 20(2-3): 173-180). In
vitro studies have demonstrated that manganese can result in the
inhibition of glutamate transport (Hazell and Norenberg, 1997),
abnormalities in astrocyte morphology (Hazell et al. (2006)
Neurosci. Lett. 396(3): 167-71), and increased cell volume (Rama
Rao et al., 2007). Some disorders associated with hyperammonemia
may also be characterized by elevated levels of manganese;
manganese may contribute to disease pathogenesis (e.g., hepatic
encephalopathy) (Rivera-Manda et al., 2012). Manganese and ammonia
have also been shown to act synergistically in the pathogenesis of
hepatic encephalopathy (Jayakumar et al. (2004) Neurochem. Res.
29(11): 2051-6).
[0529] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a manganese
transporter. In one embodiment, the manganese transporter
transports manganese into the cell.
[0530] The uptake of manganese into bacterial cells is mediated by
proteins well known to those of skill in the art. For example, the
manganese transporter MntH is a membrane transport protein capable
of transporting manganese into bacterial cells (see, e.g., Jensen
and Jensen (2014) Chapter 1: Manganese transport, trafficking and
function in invertebrates. In: Manganese in Health and Disease, pp.
1-33). In Escherichia coli, the mntH gene encodes a
proton-stimulated, divalent metal cation uptake system involved in
manganese transport (Porcheron et al. (2013) Front. Cell. Infect.
Microbiol. 3: 90). In one embodiment, the manganese transporter is
selected from the group consisting of mntH, MntABCD, SitABCD,
PsaABCD, YfeABCD. In one embodiment, the at least one gene encoding
a manganese transporter is encoded by an mntH gene. In one
embodiment, the at least one gene encoding a manganese transporter
is encoded by an MntABCD operon. In one embodiment, the at least
one gene encoding a manganese transporter is encoded by an sitABCD
operon. In one embodiment, the at least one gene encoding a
manganese transporter is encoded by an PsaABCD operon. In one
embodiment, the at least one gene encoding a manganese transporter
is encoded by an YfeABCD operon. In one embodiment, the bacterial
cell described herein has been genetically engineered to comprise
at least one heterologous mntH gene.
[0531] Metal ion homeostasis in prokaryotic cells, which lack
internal compartmentalization, is maintained by the tight
regulation of metal ion flux across in cytoplasmic membrane (Jensen
and Jensen, 2014). Manganese uptake in bacteria predominantly
involves two major types of transporters: proton-dependent
Nramprelated transporters, and/or ATP-dependent ABC transporters.
The Nramp (Natural resistance-.sctn..ssociated macrophage Qrotein)
transporter family was first described in plants, animals, and
yeasts (Cellier et al. (1996) Trends Genet. 12(6): 201-4), but MntH
has since been characterized in several bacterial species
(Porcheron et al., 2013). Selectivity of the Nramp1 transporter for
manganese has been shown in metal accumulation studies, wherein
overexpression of Staphylococcus aureus mntH resulted in increased
levels of cell-associated manganese, but no accumulation of
calcium, copper, iron, magnesium, or zinc (Horsburgh et al. (2002)
Mol. Microbiol. 44(5): 1269-86). Additionally, Bacillus subtilis
strains comprising a mutation in the mntH gene exhibited impaired
growth in metal-free medium that was rescued by the addition of
manganese (Que and Heimann (2000) Mol. Microbiol. 35(6):
1454-68).
[0532] High-affinity manganese uptake may also be mediated by ABC
(ATP-binding cassette) transporters. Members of this transporter
superfamily utilize the hydrolysis of ATP to fuel the import or
export of diverse substrates, ranging from ions to macromolecules,
and are well characterized for their role in multi-drug resistance
in both prokaryotic and eukaryotic cells. Non-limiting examples of
bacterial ABC transporters involved in manganese import include
MntABCD (Bacilis subtilis, Staphylococcus aureus), SitABCD
(Salmonella typhimurium, Shigella flexneri), PsaABCD (Streptococcus
pneumoniae), and YfeABCD (Yersinia pestis) (Bearden and Perry
(1999) Mol. Microbiol. 32(2):403-14; Kehres et al. (2002) J.
Bacteriol. 184(12): 3159-66; McAllister et al. (2004) Mol.
Microbiol. 53(3): 889-901; Zhou et al. (1999) Infect. Immun. 67(4):
1974-81). The MntABCD transporter complex consists of three
subunits, wherein MntC and MntD are integral membrane proteins that
comprise the permease subunit mediate cation transport, MntB is the
ATPase, and MntA binds and delivers manganese to the permease
submit. Other ABC transporter operons, such as sitABCD, psaABCD,
and yfeABCD, exhibit similar subunit organization and function
(Higgins, 1992; Rees et al. (2009) Nat. Rev. Mol. Cell Biol. 10(3):
218-227).
[0533] In some embodiments, the manganese transporter is encoded by
a manganese transporter gene derived from a bacterial genus or
species, including but not limited to, Bacillus, e.g., Bacillus
subtilis, Staphylococcus, e.g., Staphylococcus aureus, Salmonella,
e.g., Salmonella typhimurium, Shigella, e.g., Shigella flexneri,
Yersinia, e.g., Yersinia pestis, or Escherichia, e.g., Escherichia
coli. In some embodiments, the bacterial species is Escherichia
coli. In some embodiments, the bacterial species is Escherichia
coli strain Nissle.
[0534] Assays for testing the activity of a manganese transporter,
a functional variant of a manganese transporter, or a functional
fragment of transporter of manganese are well known to one of
ordinary skill in the art.
[0535] Manganese transporters may be expressed or modified in the
bacteria in order to enhance manganese transport into the cell.
Specifically, when the manganese transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
manganese into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a manganese transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a manganese transporter and a genetic modification that
reduces export of a manganese, e.g., a genetic mutation in an
exporter gene or promoter.
[0536] In one embodiment, the bacterial cell comprises at least one
gene encoding a manganese transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a
manganese transporter. In some embodiments, the at least one native
gene encoding a manganese transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding a manganese transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native manganese transporter, as well as at
least one copy of at least one heterologous gene encoding a
manganese transporter from a different bacterial species. In one
embodiment, the bacterial cell comprises at least one, two, three,
four, five, or six copies of the at least one heterologous gene
encoding a manganese transporter. In one embodiment, the bacterial
cell comprises multiple copies of the at least one heterologous
gene encoding a manganese transporter.
[0537] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a manganese transporter, wherein said
manganese transporter comprises a manganese sequence that has at
least 70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity
to the manganese sequence of a polypeptide encoded by a manganese
transporter gene disclosed herein.
[0538] The present disclosure further comprises genes encoding
functional fragments of a manganese transporter or functional
variants of a manganese transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
manganese transporter relates to an element having qualitative
biological activity in common with the wild-type manganese
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
manganese transporter is one which retains essentially the same
ability to import manganese into the bacterial cell as does the
manganese transporter protein from which the functional fragment or
functional variant was derived. In one embodiment, the recombinant
bacterial cell comprises at least one heterologous gene encoding a
functional fragment of a manganese transporter. In another
embodiment, the recombinant bacterial cell comprises a heterologous
gene encoding a functional variant of a manganese transporter.
[0539] In one embodiment, the genes encoding the manganese
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the manganese transporter have been codon-optimized
for use in Escherichia coli.
[0540] The present disclosure also encompasses genes encoding a
manganese transporter comprising amino acids in its sequence that
are substantially the same as an amino acid sequence described
herein Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0541] In some embodiments, the at least one gene encoding a
manganese transporter is mutagenized; mutants exhibiting increased
manganese import are selected; and the mutagenized at least one
gene encoding a manganese transporter is isolated and inserted into
the bacterial cell. In some embodiments, the at least one gene
encoding a manganese transporter is mutagenized; mutants exhibiting
decreased manganese import are selected; and the mutagenized at
least one gene encoding a manganese transporter is isolated and
inserted into the bacterial cell. The transporter modifications
described herein may be present on a plasmid or chromosome.
[0542] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a manganese transporter operably linked
to a promoter. In one embodiment, the at least one gene encoding a
manganese transporter is directly operably linked to the promoter.
In another embodiment, the at least one gene encoding a manganese
transporter is indirectly operably linked to the promoter.
[0543] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a manganese transporter in nature.
In some embodiments, the at least one gene encoding the manganese
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the manganese
transporter is controlled by an inducible promoter. In some
embodiments, the at least one gene encoding the manganese
transporter is controlled by a promoter that is stronger than its
native promoter. In some embodiments, the at least one gene
encoding the manganese transporter is controlled by a constitutive
promoter.
[0544] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0545] In one embodiment, the at least one gene encoding a
manganese transporter is located on a plasmid in the bacterial
cell. In some embodiments, the plasmid is a high copy number
plasmid. In some embodiments, the plasmid is a low copy number
plasmid. In another embodiment, the at least one gene encoding a
manganese transporter is located in the chromosome of the bacterial
cell. In yet another embodiment, a native copy of the at least one
gene encoding a manganese transporter is located in the chromosome
of the bacterial cell, and a copy of at least one gene encoding a
manganese transporter from a different species of bacteria is
located on a plasmid in the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a
manganese transporter is located on a plasmid in the bacterial
cell, and a copy of at least one gene encoding a manganese
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a manganese transporter is
located in the chromosome of the bacterial cell, and a copy of the
at least one gene encoding a manganese transporter from a different
species of bacteria is located in the chromosome of the bacterial
cell.
[0546] In some embodiments, the at least one native gene encoding
the manganese transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the manganese transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0547] In some embodiments, at least one native gene encoding the
manganese transporter in the bacterial cell is not modified, and
one or more additional copies of at least one native gene encoding
the transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the manganese transporter in the bacterial
cell is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0548] In one embodiment, the manganese transporter is the
manganese transporter GabP, for example the Escherichia coli mntH
gene. In one embodiment the manganese transporter is encoded by a
mntH gene. In one embodiment, the mntH gene has at least about 80%
identity with the sequence of SEQ ID NO:135. Accordingly, in one
embodiment, the mntH gene has at least about 90% identity with the
sequence of SEQ ID NO:135. Accordingly, in one embodiment, the mntH
gene has at least about 95% identity with the sequence of SEQ ID
NO:135. Accordingly, in one embodiment, the mntH gene has at least
about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ
ID NO:135. In another embodiment, the mntH gene comprises the
sequence of SEQ ID NO:135. In yet another embodiment the mntH gene
consists of the sequence of SEQ ID NO:135.
[0549] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0550] In one embodiment, when the manganese transporter is
expressed in the recombinant bacterial cells, the bacterial cells
import 10% more manganese into the bacterial cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In another embodiment,
when the manganese transporter is expressed in the recombinant
bacterial cells, the bacterial cells import 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100% more manganese, into the bacterial cell
when the transporter is expressed than unmodified bacteria of the
same bacterial subtype under the same conditions. In yet another
embodiment, when the manganese transporter is expressed in the
recombinant bacterial cells, the bacterial cells import two-fold
more manganese into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In yet another embodiment, when the manganese
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import three-fold, four-fold, five-fold, six-fold,
seven-fold, eight-fold, nine-fold, ten-fold, fifteen-fold,
twenty-fold, thirty-fold, fourty-fold, or fifty-fold more manganese
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same
conditions.
[0551] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous manganese transporter and a
second heterologous manganese transporter. In one embodiment, said
first manganese transporter is derived from a different organism
than said second manganese transporter. In some embodiments, said
first manganese transporter is derived from the same organism as
said second manganese transporter. In some embodiments, said first
manganese transporter is a wild-type manganese transporter and said
second manganese transporter is a mutagenized version of said first
manganese transporter. In some embodiments, the recombinant
bacterial cells described herein comprise at least a third
heterologous manganese transporter. In some embodiments, the
recombinant bacterial cells described herein comprise at least four
heterologous manganese transporters. In some embodiments, the
recombinant bacterial cells described herein comprise at least five
heterologous manganese transporters or more.
[0552] In some embodiment, the recombinant bacterial cell
comprising a heterologous gene encoding an manganese transporter
may be used to treat a disease, condition, and/or symptom
associated with hyperammonenia. In some embodiment, the recombinant
bacterial cells described herein can be used to treat hepatic
encephalopathy. In some embodiment, the recombinant bacterial cells
described herein can be used to treat Huntington's disease. In some
embodiments, the invention provides methods for reducing,
ameliorating, or eliminating one or more symptom(s) associated with
hepatic encephalopathy and Huntington's disease. In some
embodiments, the symptom(s) associated thereof include, but are not
limited to, seizures, ataxia, stroke-like lesions, coma, psychosis,
vision loss, acute encephalopathy, cerebral edema, as well as
vomiting, respiratory alkalosis, and hypothermia.
K. Toxin Transporters
[0553] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a toxin
transporter. In one embodiment, the toxin transporter transports
toxin into the cell.
[0554] In some embodiments, the toxin transporter is encoded by a
toxin transporter gene derived from a bacterial genus or species,
including but not limited to, Escherichia, Corynebacterium,
Escherichia coli, Saccharomyces cerevisiae or Corynebacterium
glutamicum. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0555] Assays for testing the activity of a toxin transporter, a
functional variant of a toxin transporter, or a functional fragment
of transporter of toxin are well known to one of ordinary skill in
the art.
[0556] Toxin transporters may be expressed or modified in the
bacteria in order to enhance toxin transport into the cell.
Specifically, when the toxin transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more toxin
into the cell when the transporter is expressed than unmodified
bacteria of the same bacterial subtype under the same conditions.
In one embodiment, the bacterial cell comprises a heterologous gene
encoding a toxin transporter. In one embodiment, the bacterial cell
comprises a heterologous gene encoding a toxin transporter and a
genetic modification that reduces export of a toxin, e.g., a
genetic mutation in an exporter gene or promoter.
[0557] In one embodiment, the bacterial cell comprises at least one
gene encoding a toxin transporter from a different organism, e.g.,
a different species of bacteria. In one embodiment, the bacterial
cell comprises at least one native gene encoding a toxin
transporter. In some embodiments, the at least one native gene
encoding a toxin transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding a toxin transporter. In yet another
embodiment, the bacterial cell comprises a copy of at least one
gene encoding a native toxin transporter, as well as at least one
copy of at least one heterologous gene encoding a toxin transporter
from a different bacterial species. In one embodiment, the
bacterial cell comprises at least one, two, three, four, five, or
six copies of the at least one heterologous gene encoding a toxin
transporter. In one embodiment, the bacterial cell comprises
multiple copies of the at least one heterologous gene encoding a
toxin transporter.
[0558] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a toxin transporter, wherein said
toxin transporter comprises a toxin sequence that has at least 70%,
75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the
toxin sequence of a polypeptide encoded by a toxin transporter gene
disclosed herein.
[0559] The present disclosure further comprises genes encoding
functional fragments of a toxin transporter or functional variants
of a toxin transporter. As used herein, the term "functional
fragment thereof" or "functional variant thereof" of a toxin
transporter relates to an element having qualitative biological
activity in common with the wild-type toxin transporter from which
the fragment or variant was derived. For example, a functional
fragment or a functional variant of a mutated toxin transporter is
one which retains essentially the same ability to import toxin into
the bacterial cell as does the toxin transporter protein from which
the functional fragment or functional variant was derived. In one
embodiment, the recombinant bacterial cell comprises at least one
heterologous gene encoding a functional fragment of a toxin
transporter. In another embodiment, the recombinant bacterial cell
comprises a heterologous gene encoding a functional variant of a
toxin transporter.
[0560] In one embodiment, the genes encoding the toxin transporter
have been codon-optimized for use in the host organism, e.g., a
bacterial cell disclosed herein. In one embodiment, the genes
encoding the toxin transporter have been codon-optimized for use in
Escherichia coli.
[0561] The present disclosure also encompasses genes encoding a
toxin transporter comprising amino acids in its sequence that are
substantially the same as an amino acid sequence described herein
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0562] In some embodiments, the at least one gene encoding a toxin
transporter is mutagenized; mutants exhibiting increased toxin
import are selected; and the mutagenized at least one gene encoding
a toxin transporter is isolated and inserted into the bacterial
cell. In some embodiments, the at least one gene encoding a toxin
transporter is mutagenized; mutants exhibiting decreased toxin
import are selected; and the mutagenized at least one gene encoding
a toxin transporter is isolated and inserted into the bacterial
cell. The transporter modifications described herein may be present
on a plasmid or chromosome.
[0563] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a toxin transporter operably linked to a
promoter. In one embodiment, the at least one gene encoding a toxin
transporter is directly operably linked to the promoter. In another
embodiment, the at least one gene encoding a toxin transporter is
indirectly operably linked to the promoter.
[0564] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a toxin transporter in nature. In
some embodiments, the at least one gene encoding the toxin
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the toxin transporter
is controlled by an inducible promoter. In some embodiments, the at
least one gene encoding the toxin transporter is controlled by a
promoter that is stronger than its native promoter. In some
embodiments, the at least one gene encoding the toxin transporter
is controlled by a constitutive promoter.
[0565] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0566] In one embodiment, the at least one gene encoding a toxin
transporter is located on a plasmid in the bacterial cell. In some
embodiments, the plasmid is a high copy number plasmid. In some
embodiments, the plasmid is a low copy number plasmid. In another
embodiment, the at least one gene encoding a toxin transporter is
located in the chromosome of the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a toxin
transporter is located in the chromosome of the bacterial cell, and
a copy of at least one gene encoding a toxin transporter from a
different species of bacteria is located on a plasmid in the
bacterial cell. In yet another embodiment, a native copy of the at
least one gene encoding a toxin transporter is located on a plasmid
in the bacterial cell, and a copy of at least one gene encoding a
toxin transporter from a different species of bacteria is located
on a plasmid in the bacterial cell. In yet another embodiment, a
native copy of the at least one gene encoding a toxin transporter
is located in the chromosome of the bacterial cell, and a copy of
the at least one gene encoding a toxin transporter from a different
species of bacteria is located in the chromosome of the bacterial
cell.
[0567] In some embodiments, the at least one native gene encoding
the toxin transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the toxin transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0568] In some embodiments, at least one native gene encoding the
toxin transporter in the bacterial cell is not modified, and one or
more additional copies of at least one native gene encoding the
transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the toxin transporter in the bacterial
cell is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0569] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0570] In one embodiment, when the toxin transporter is expressed
in the recombinant bacterial cells, the bacterial cells import 10%
more toxin into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In another embodiment, when the toxin
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% more PGE2, into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
toxin transporter is expressed in the recombinant bacterial cells,
the bacterial cells import two-fold more toxin into the cell when
the transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions. In yet another
embodiment, when the toxin transporter is expressed in the
recombinant bacterial cells, the bacterial cells import three-fold,
four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold,
ten-fold, fifteen-fold, twenty-fold, thirty-fold, fourty-fold, or
fifty-fold more toxin into the cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions.
[0571] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous toxin transporter and a second
heterologous toxin transporter. In one embodiment, said first toxin
transporter is derived from a different organism than said second
toxin transporter. In some embodiments, said first toxin
transporter is derived from the same organism as said second toxin
transporter. In some embodiments, said first toxin transporter
imports the same toxin as said second toxin transporter. In other
embodiment, said first toxin transporter imports a different toxin
from said second toxin transporter. In some embodiments, said first
toxin transporter is a wild-type toxin transporter and said second
toxin transporter is a mutagenized version of said first toxin
transporter. In some embodiments, the recombinant bacterial cells
described herein comprise at least a third heterologous toxin
transporter. In some embodiments, the recombinant bacterial cells
described herein comprise at least four heterologous toxin
transporters. In some embodiments, the recombinant bacterial cells
described herein comprise at least five heterologous toxin
transporters or more.
L. Peptide Transporters
[0572] In one embodiment, the recombinant bacterial cell of the
invention comprises a heterologous gene encoding a substrate
transporter, wherein the substrate transporter is a peptide
transporter.
[0573] In some embodiments, the peptide transporter is encoded by a
peptide transporter gene derived from a bacterial genus or species,
including but not limited to, Escherichia, Corynebacterium,
Escherichia coli, Saccharomyces cerevisiae or Corynebacterium
glutamicum. In some embodiments, the bacterial species is
Escherichia coli. In some embodiments, the bacterial species is
Escherichia coli strain Nissle.
[0574] Assays for testing the activity of a peptide transporter, a
functional variant of a peptide transporter, or a functional
fragment of transporter of peptide are well known to one of
ordinary skill in the art.
[0575] Peptide transporters may be expressed or modified in the
bacteria in order to enhance peptide transport into the cell.
Specifically, when the peptide transporter is expressed in the
recombinant bacterial cells, the bacterial cells import more
peptide into the cell when the transporter is expressed than
unmodified bacteria of the same bacterial subtype under the same
conditions. In one embodiment, the bacterial cell comprises a
heterologous gene encoding a peptide transporter. In one
embodiment, the bacterial cell comprises a heterologous gene
encoding a peptide transporter and a genetic modification that
reduces export of a peptide, e.g., a genetic mutation in an
exporter gene or promoter.
[0576] In one embodiment, the bacterial cell comprises at least one
gene encoding a peptide transporter from a different organism,
e.g., a different species of bacteria. In one embodiment, the
bacterial cell comprises at least one native gene encoding a
peptide transporter. In some embodiments, the at least one native
gene encoding a peptide transporter is not modified. In another
embodiment, the bacterial cell comprises more than one copy of at
least one native gene encoding a peptide transporter. In yet
another embodiment, the bacterial cell comprises a copy of at least
one gene encoding a native peptide transporter, as well as at least
one copy of at least one heterologous gene encoding a peptide
transporter from a different bacterial species. In one embodiment,
the bacterial cell comprises at least one, two, three, four, five,
or six copies of the at least one heterologous gene encoding a
peptide transporter. In one embodiment, the bacterial cell
comprises multiple copies of the at least one heterologous gene
encoding a peptide transporter.
[0577] In one embodiment, the recombinant bacterial cell comprises
a heterologous gene encoding a peptide transporter, wherein said
peptide transporter comprises a peptide sequence that has at least
70%, 75%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to
the peptide sequence of a polypeptide encoded by a peptide
transporter gene disclosed herein.
[0578] The present disclosure further comprises genes encoding
functional fragments of a peptide transporter or functional
variants of a peptide transporter. As used herein, the term
"functional fragment thereof" or "functional variant thereof" of a
peptide transporter relates to an element having qualitative
biological activity in common with the wild-type peptide
transporter from which the fragment or variant was derived. For
example, a functional fragment or a functional variant of a mutated
peptide transporter is one which retains essentially the same
ability to import peptide into the bacterial cell as does the
peptide transporter protein from which the functional fragment or
functional variant was derived. In one embodiment, the recombinant
bacterial cell comprises at least one heterologous gene encoding a
functional fragment of a peptide transporter. In another
embodiment, the recombinant bacterial cell comprises a heterologous
gene encoding a functional variant of a peptide transporter.
[0579] In one embodiment, the genes encoding the peptide
transporter have been codon-optimized for use in the host organism,
e.g., a bacterial cell disclosed herein. In one embodiment, the
genes encoding the peptide transporter have been codon-optimized
for use in Escherichia coli.
[0580] The present disclosure also encompasses genes encoding a
peptide transporter comprising amino acids in its sequence that are
substantially the same as an amino acid sequence described herein
Amino acid sequences that are substantially the same as the
sequences described herein include sequences comprising
conservative amino acid substitutions, as well as amino acid
deletions and/or insertions.
[0581] In some embodiments, the at least one gene encoding a
peptide transporter is mutagenized; mutants exhibiting increased
peptide import are selected; and the mutagenized at least one gene
encoding a peptide transporter is isolated and inserted into the
bacterial cell. In some embodiments, the at least one gene encoding
a peptide transporter is mutagenized; mutants exhibiting decreased
peptide import are selected; and the mutagenized at least one gene
encoding a peptide transporter is isolated and inserted into the
bacterial cell. The transporter modifications described herein may
be present on a plasmid or chromosome.
[0582] In some embodiments, the bacterial cell comprises a
heterologous gene encoding a peptide transporter operably linked to
a promoter. In one embodiment, the at least one gene encoding a
peptide transporter is directly operably linked to the promoter. In
another embodiment, the at least one gene encoding a peptide
transporter is indirectly operably linked to the promoter.
[0583] In one embodiment, the promoter is not operably linked with
the at least one gene encoding a peptide transporter in nature. In
some embodiments, the at least one gene encoding the peptide
transporter is controlled by its native promoter. In some
embodiments, the at least one gene encoding the peptide transporter
is controlled by an inducible promoter. In some embodiments, the at
least one gene encoding the peptide transporter is controlled by a
promoter that is stronger than its native promoter. In some
embodiments, the at least one gene encoding the peptide transporter
is controlled by a constitutive promoter.
[0584] In another embodiment, the promoter is an inducible
promoter. Inducible promoters are described in more detail
infra.
[0585] In one embodiment, the at least one gene encoding a peptide
transporter is located on a plasmid in the bacterial cell. In some
embodiments, the plasmid is a high copy number plasmid. In some
embodiments, the plasmid is a low copy number plasmid. In another
embodiment, the at least one gene encoding a peptide transporter is
located in the chromosome of the bacterial cell. In yet another
embodiment, a native copy of the at least one gene encoding a
peptide transporter is located in the chromosome of the bacterial
cell, and a copy of at least one gene encoding a peptide
transporter from a different species of bacteria is located on a
plasmid in the bacterial cell. In yet another embodiment, a native
copy of the at least one gene encoding a peptide transporter is
located on a plasmid in the bacterial cell, and a copy of at least
one gene encoding a peptide transporter from a different species of
bacteria is located on a plasmid in the bacterial cell. In yet
another embodiment, a native copy of the at least one gene encoding
a peptide transporter is located in the chromosome of the bacterial
cell, and a copy of the at least one gene encoding a peptide
transporter from a different species of bacteria is located in the
chromosome of the bacterial cell.
[0586] In some embodiments, the at least one native gene encoding
the peptide transporter in the recombinant bacterial cell is not
modified, and one or more additional copies of the native
transporter are inserted into the genome. In alternate embodiments,
the at least one native gene encoding the transporter is not
modified, and one or more additional copies of the transporter from
a different bacterial species is inserted into the genome of the
recombinant bacterial cell. In some embodiments, the at least one
native gene encoding the peptide transporter in the recombinant
bacterial cell is modified, and one or more additional copies of
the native transporter are inserted into the genome. In alternate
embodiments, the at least one native gene encoding the transporter
is modified, and one or more additional copies of the transporter
from a different bacterial species is inserted into the genome of
the recombinant bacterial cell.
[0587] In some embodiments, at least one native gene encoding the
peptide transporter in the bacterial cell is not modified, and one
or more additional copies of at least one native gene encoding the
transporter are present in the bacterial cell on a plasmid. In
alternate embodiments, the at least one native gene encoding the
transporter is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
present in the bacteria on a plasmid. In some embodiments, at least
one native gene encoding the peptide transporter in the bacterial
cell is modified, and one or more additional copies of at least one
native gene encoding the transporter are present in the bacterial
cell on a plasmid. In alternate embodiments, the at least one
native gene encoding the transporter is modified, and a copy of at
least one gene encoding the transporter from a different bacterial
species is present in the bacteria on a plasmid.
[0588] In some embodiments, the bacterium is E. coli Nissle, and
the at least one native gene encoding the transporter in E. coli
Nissle is not modified; one or more additional copies at least one
native gene encoding the transporter from E. coli Nissle is
inserted into the E. coli Nissle genome. In an alternate
embodiment, the at least one native gene encoding the transporter
in E. coli Nissle is not modified, and a copy of at least one gene
encoding the transporter from a different bacterial species is
inserted into the E. coli Nissle genome.
[0589] In one embodiment, when the peptide transporter is expressed
in the recombinant bacterial cells, the bacterial cells import 10%
more peptide into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In another embodiment, when the peptide
transporter is expressed in the recombinant bacterial cells, the
bacterial cells import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% more PGE2, into the bacterial cell when the transporter is
expressed than unmodified bacteria of the same bacterial subtype
under the same conditions. In yet another embodiment, when the
peptide transporter is expressed in the recombinant bacterial
cells, the bacterial cells import two-fold more peptide into the
cell when the transporter is expressed than unmodified bacteria of
the same bacterial subtype under the same conditions. In yet
another embodiment, when the peptide transporter is expressed in
the recombinant bacterial cells, the bacterial cells import
three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold,
nine-fold, ten-fold, fifteen-fold, twenty-fold, thirty-fold,
fourty-fold, or fifty-fold more peptide into the cell when the
transporter is expressed than unmodified bacteria of the same
bacterial subtype under the same conditions.
[0590] In one embodiment, the recombinant bacterial cells described
herein comprise a first heterologous peptide transporter and a
second heterologous peptide transporter. In one embodiment, said
first peptide transporter is derived from a different organism than
said second peptide transporter. In some embodiments, said first
peptide transporter is derived from the same organism as said
second peptide transporter. In some embodiments, said first peptide
transporter imports the same peptide as said second peptide
transporter. In other embodiment, said first peptide transporter
imports a different peptide from said second peptide transporter.
In some embodiments, said first peptide transporter is a wild-type
peptide transporter and said second peptide transporter is a
mutagenized version of said first peptide transporter. In some
embodiments, the recombinant bacterial cells described herein
comprise at least a third heterologous peptide transporter. In some
embodiments, the recombinant bacterial cells described herein
comprise at least four heterologous peptide transporters. In some
embodiments, the recombinant bacterial cells described herein
comprise at least five heterologous peptide transporters or
more.
[0591] Inducible Promoters
[0592] In some embodiments, the bacterial cell comprises a stably
maintained plasmid or chromosome carrying the gene(s) encoding the
tranporter(s), such that the tranporter(s) can be expressed in the
host cell, and the host cell is capable of survival and/or growth
in vitro, e.g., in medium, and/or in vivo, e.g., in the gut. In
some embodiments, bacterial cell comprises two or more distinct
tranporters or operons, e.g., two or more tranporter genes. In some
embodiments, bacterial cell comprises three or more distinct
transporters or operons, e.g., three or more tranporter genes. In
some embodiments, bacterial cell comprises 4, 5, 6, 7, 8, 9, 10, or
more distinct tranporters or operons, e.g., 4, 5, 6, 7, 8, 9, 10,
or more tranporter genes.
[0593] In some embodiments, the genetically engineered bacteria
comprise multiple copies of the same tranporter gene(s). In some
embodiments, the gene encoding the tranporter is present on a
plasmid and operably linked to a directly or indirectly inducible
promoter. In some embodiments, the gene encoding the tranporter is
present on a plasmid and operably linked to a promoter that is
induced under low-oxygen or anaerobic conditions. In some
embodiments, the gene encoding the tranporter is present on a
chromosome and operably linked to a directly or indirectly
inducible promoter. In some embodiments, the gene encoding the
tranporter is present in the chromosome and operably linked to a
promoter that is induced under low-oxygen or anaerobic conditions.
In some embodiments, the gene encoding the tranporter is present on
a plasmid and operably linked to a promoter that is induced by
exposure to tetracycline or arabinose.
[0594] In some embodiments, the promoter that is operably linked to
the gene encoding the tranporter is directly induced by exogenous
environmental conditions. In some embodiments, the promoter that is
operably linked to the gene encoding the tranporter is indirectly
induced by exogenous environmental conditions. In some embodiments,
the promoter is directly or indirectly induced by exogenous
environmental conditions specific to the gut of a mammal. In some
embodiments, the promoter is directly or indirectly induced by
exogenous environmental conditions specific to the small intestine
of a mammal In some embodiments, the promoter is directly or
indirectly induced by low-oxygen or anaerobic conditions such as
the environment of the mammalian gut. In some embodiments, the
promoter is directly or indirectly induced by molecules or
metabolites that are specific to the gut of a mammal In some
embodiments, the promoter is directly or indirectly induced by a
molecule that is co-administered with the bacterial cell.
[0595] In certain embodiments, the bacterial cell comprises a gene
encoding an tranporter expressed under the control of a fumarate
and nitrate reductase regulator (FNR) responsive promoter. In E.
coli, FNR is a major transcriptional activator that controls the
switch from aerobic to anaerobic metabolism (Unden et al., 1997).
In the anaerobic state, FNR dimerizes into an active DNA binding
protein that activates hundreds of genes responsible for adapting
to anaerobic growth. In the aerobic state, FNR is prevented from
dimerizing by oxygen and is inactive. FNR responsive promoters
include, but are not limited to, the FNR responsive promoters
listed in the chart, below. Underlined sequences are predicted
ribosome binding sites, and bolded sequences are restriction sites
used for cloning.
TABLE-US-00005 FNR Responsive Promoter Sequence SEQ ID NO:
GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT
CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAA
TCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCCGT
GACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAAA
GGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGTAGGC
GGTAATAGAAAAGAAATCGAGGCAAAA SEQ ID NO:
ATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCTC
ATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCACT
CGACAGGAGTATTTATATTGCGCCCGTTACGTGGGCTTCGACTGTAAATCAG
AAAGGAGAAAACACCT SEQ ID NO:
GTCAGCATAACACCCTGACCTCTCATTAATTGTTCATGCCGGGCGGCACTAT
CGTCGTCCGGCCTTTTCCTCTCTTACTCTGCTACGTACATCTATTTCTATAAA
TCCGTTCAATTTGTCTGTTTTTTGCACAAACATGAAATATCAGACAATTCCGT
GACTTAAGAAAATTTATACAAATCAGCAATATACCCCTTAAGGAGTATATAAA
GGTGAATTTGATTTACATCAATAAGCGGGGTTGCTGAATCGTTAAGGATCCC
TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT SEQ ID NO:
CATTTCCTCTCATCCCATCCGGGGTGAGAGTCTTTTCCCCCGACTTATGGCT
CATGCATGCATCAAAAAAGATGTGAGCTTGATCAAAAACAAAAAATATTTCAC
TCGACAGGAGTATTTATATTGCGCCCGGATCCCTCTAGAAATAATTTTGTTTA
ACTTTAAGAAGGAGATATACAT SEQ ID NO:
AGTTGTTCTTATTGGTGGTGTTGCTTTATGGTTGCATCGTAGTAAATGGTTGT
AACAAAAGCAATTTTTCCGGCTGTCTGTATACAAAAACGCCGTAAAGTTTGAG
CGAAGTCAATAAACTCTCTACCCATTCAGGGCAATATCTCTCTTGGATCCCT
CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT
[0596] In one embodiment, the FNR responsive promoter comprises SEQ
ID NO:1. In another embodiment, the FNR responsive promoter
comprises SEQ ID NO:2. In another embodiment, the FNR responsive
promoter comprises SEQ ID NO:3. In another embodiment, the FNR
responsive promoter comprises SEQ ID NO:4. In yet another
embodiment, the FNR responsive promoter comprises SEQ ID NO:5.
[0597] In some embodiments, multiple distinct FNR nucleic acid
sequences are inserted in the genetically engineered bacteria. In
alternate embodiments, the genetically engineered bacteria comprise
a gene encoding an tranporter expressed under the control of an
alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al.,
2010) or ANR (Ray et al., 1997). In these embodiments, expression
of the tranporter gene is particularly activated in a low-oxygen or
anaerobic environment, such as in the gut. In some embodiments,
gene expression is further optimized by methods known in the art,
e.g., by optimizing ribosomal binding sites and/or increasing mRNA
stability. In one embodiment, the mammalian gut is a human
mammalian gut.
[0598] In some embodiments, the bacterial cell comprises an
oxygen-level dependent transcriptional regulator, e.g., FNR, ANR,
or DNR, and corresponding promoter from a different bacterial
species. The heterologous oxygen-level dependent transcriptional
regulator and promoter increase the transcription of genes operably
linked to said promoter, e.g., the gene encoding the tranporter, in
a low-oxygen or anaerobic environment, as compared to the native
gene(s) and promoter in the bacteria under the same conditions. In
certain embodiments, the non-native oxygen-level dependent
transcriptional regulator is an FNR protein from N. gonorrhoeae
(see, e.g., Isabella et al., 2011). In some embodiments, the
corresponding wild-type transcriptional regulator is left intact
and retains wild-type activity. In alternate embodiments, the
corresponding wild-type transcriptional regulator is deleted or
mutated to reduce or eliminate wild-type activity.
[0599] In some embodiments, the genetically engineered bacteria
comprise a wild-type oxygen-level dependent transcriptional
regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that
is mutated relative to the wild-type promoter from bacteria of the
same subtype. The mutated promoter enhances binding to the
wild-type transcriptional regulator and increases the transcription
of genes operably linked to said promoter, e.g., the gene encoding
the tranporter, in a low-oxygen or anaerobic environment, as
compared to the wild-type promoter under the same conditions. In
some embodiments, the genetically engineered bacteria comprise a
wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR
promoter, and corresponding transcriptional regulator that is
mutated relative to the wild-type transcriptional regulator from
bacteria of the same subtype. The mutated transcriptional regulator
enhances binding to the wild-type promoter and increases the
transcription of genes operably linked to said promoter, e.g., the
gene encoding the tranporter, in a low-oxygen or anaerobic
environment, as compared to the wild-type transcriptional regulator
under the same conditions. In certain embodiments, the mutant
oxygen-level dependent transcriptional regulator is an FNR protein
comprising amino acid substitutions that enhance dimerization and
FNR activity (see, e.g., Moore et al., (2006).
[0600] In some embodiments, the bacterial cells comprise multiple
copies of the endogenous gene encoding the oxygen level-sensing
transcriptional regulator, e.g., the FNR gene. In some embodiments,
the gene encoding the oxygen level-sensing transcriptional
regulator is present on a plasmid. In some embodiments, the gene
encoding the oxygen level-sensing transcriptional regulator and the
gene encoding the tranporter are present on different plasmids. In
some embodiments, the gene encoding the oxygen level-sensing
transcriptional regulator and the gene encoding the tranporter are
present on the same plasmid. In some embodiments, the gene encoding
the oxygen level-sensing transcriptional regulator is present on a
chromosome. In some embodiments, the gene encoding the oxygen
level-sensing transcriptional regulator and the gene encoding the
tranporter are present on different chromosomes. In some
embodiments, the gene encoding the oxygen level-sensing
transcriptional regulator and the gene encoding the tranporter are
present on the same chromosome. In some instances, it may be
advantageous to express the oxygen level-sensing transcriptional
regulator under the control of an inducible promoter in order to
enhance expression stability. In some embodiments, expression of
the transcriptional regulator is controlled by a different promoter
than the promoter that controls expression of the gene encoding the
tranporter. In some embodiments, expression of the transcriptional
regulator is controlled by the same promoter that controls
expression of the tranporter. In some embodiments, the
transcriptional regulator and the tranporter are divergently
transcribed from a promoter region.
[0601] RNS-Dependent Regulation
[0602] In some embodiments, the genetically engineered bacteria or
genetically engineered virus comprise a gene encoding an tranporter
that is expressed under the control of an inducible promoter. In
some embodiments, the genetically engineered bacterium or
genetically engineered virus that expresses an tranporter under the
control of a promoter that is activated by inflammatory conditions.
In one embodiment, the gene for producing the tranporter is
expressed under the control of an inflammatory-dependent promoter
that is activated in inflammatory environments, e.g., a reactive
nitrogen species or RNS promoter.
[0603] As used herein, "reactive nitrogen species" and "RNS" are
used interchangeably to refer to highly active molecules, ions,
and/or radicals derived from molecular nitrogen. RNS can cause
deleterious cellular effects such as nitrosative stress. RNS
includes, but is not limited to, nitric oxide (NO.), peroxynitrite
or peroxynitrite anion (ONOO--), nitrogen dioxide (.cndot.NO2),
dinitrogen trioxide (N2O3), peroxynitrous acid (ONOOH--), and
nitroperoxycarbonate (.cndot.ONOOCO2--) (unpaired electrons denoted
by .cndot.). Bacteria have evolved transcription factors that are
capable of sensing RNS levels. Different RNS signaling pathways are
triggered by different RNS levels and occur with different
kinetics.
[0604] As used herein, "RNS-inducible regulatory region" refers to
a nucleic acid sequence to which one or more RNS-sensing
transcription factors is capable of binding, wherein the binding
and/or activation of the corresponding transcription factor
activates downstream gene expression; in the presence of RNS, the
transcription factor binds to and/or activates the regulatory
region. In some embodiments, the RNS-inducible regulatory region
comprises a promoter sequence. In some embodiments, the
transcription factor senses RNS and subsequently binds to the
RNS-inducible regulatory region, thereby activating downstream gene
expression. In alternate embodiments, the transcription factor is
bound to the RNS-inducible regulatory region in the absence of RNS;
in the presence of RNS, the transcription factor undergoes a
conformational change, thereby activating downstream gene
expression. The RNS-inducible regulatory region may be operatively
linked to a gene or genes, e.g., an tranporter gene sequence(s),
e.g., any of the tranporters described herein. For example, in the
presence of RNS, a transcription factor senses RNS and activates a
corresponding RNS-inducible regulatory region, thereby driving
expression of an operatively linked gene sequence. Thus, RNS
induces expression of the gene or gene sequences.
[0605] As used herein, "RNS-derepressible regulatory region" refers
to a nucleic acid sequence to which one or more RNS-sensing
transcription factors is capable of binding, wherein the binding of
the corresponding transcription factor represses downstream gene
expression; in the presence of RNS, the transcription factor does
not bind to and does not repress the regulatory region. In some
embodiments, the RNS-derepressible regulatory region comprises a
promoter sequence. The RNS-derepressible regulatory region may be
operatively linked to a gene or genes, e.g., an tranporter gene
sequence(s). For example, in the presence of RNS, a transcription
factor senses RNS and no longer binds to and/or represses the
regulatory region, thereby derepressing an operatively linked gene
sequence or gene cassette. Thus, RNS derepresses expression of the
gene or genes.
[0606] As used herein, "RNS-repressible regulatory region" refers
to a nucleic acid sequence to which one or more RNS-sensing
transcription factors is capable of binding, wherein the binding of
the corresponding transcription factor represses downstream gene
expression; in the presence of RNS, the transcription factor binds
to and represses the regulatory region. In some embodiments, the
RNS-repressible regulatory region comprises a promoter sequence. In
some embodiments, the transcription factor that senses RNS is
capable of binding to a regulatory region that overlaps with part
of the promoter sequence. In alternate embodiments, the
transcription factor that senses RNS is capable of binding to a
regulatory region that is upstream or downstream of the promoter
sequence. The RNS-repressible regulatory region may be operatively
linked to a gene sequence or gene cassette. For example, in the
presence of RNS, a transcription factor senses RNS and binds to a
corresponding RNS-repressible regulatory region, thereby blocking
expression of an operatively linked gene sequence or gene
sequences. Thus, RNS represses expression of the gene or gene
sequences.
[0607] As used herein, a "RNS-responsive regulatory region" refers
to a RNS-inducible regulatory region, a RNS-repressible regulatory
region, and/or a RNS-derepressible regulatory region. In some
embodiments, the RNS-responsive regulatory region comprises a
promoter sequence. Each regulatory region is capable of binding at
least one corresponding RNS-sensing transcription factor. Examples
of transcription factors that sense RNS and their corresponding
RNS-responsive genes, promoters, and/or regulatory regions include,
but are not limited to, those shown in Table 3.
[0608] Examples of RNS-Sensing Transcription Factors and
RNS-Responsive Genes
TABLE-US-00006 Primarily RNS-sensing capable of Examples of
responsive genes, transcription factor: sensing: promoters, and/or
regulatory regions: NsrR NO norB, aniA, nsrR, hmpA, ytfE, ygbA,
hcp, hcr, nrfA, aox NorR NO norVW, norR DNR NO norCB, nir, nor,
nos
[0609] In some embodiments, the genetically engineered bacteria of
the invention comprise a tunable regulatory region that is directly
or indirectly controlled by a transcription factor that is capable
of sensing at least one reactive nitrogen species. The tunable
regulatory region is operatively linked to a gene or genes capable
of directly or indirectly driving the expression of an tranporter,
thus controlling expression of the tranporter relative to RNS
levels. For example, the tunable regulatory region is a
RNS-inducible regulatory region, and the payload is an tranporter,
such as any of the tranporters provided herein; when RNS is
present, e.g., in an inflamed tissue, a RNS-sensing transcription
factor binds to and/or activates the regulatory region and drives
expression of the tranporter gene or genes. Subsequently, when
inflammation is ameliorated, RNS levels are reduced, and production
of the tranporter is decreased or eliminated.
[0610] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region; in the presence of RNS, a
transcription factor senses RNS and activates the RNS-inducible
regulatory region, thereby driving expression of an operatively
linked gene or genes. In some embodiments, the transcription factor
senses RNS and subsequently binds to the RNS-inducible regulatory
region, thereby activating downstream gene expression. In alternate
embodiments, the transcription factor is bound to the RNS-inducible
regulatory region in the absence of RNS; when the transcription
factor senses RNS, it undergoes a conformational change, thereby
inducing downstream gene expression.
[0611] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region, and the transcription factor that
senses RNS is NorR. NorR "is an NO-responsive transcriptional
activator that regulates expression of the norVW genes encoding
flavorubredoxin and an associated flavoprotein, which reduce NO to
nitrous oxide" (Spiro 2006). The genetically engineered bacteria of
the invention may comprise any suitable RNS-responsive regulatory
region from a gene that is activated by NorR. Genes that are
capable of being activated by NorR are known in the art (see, e.g.,
Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012; Table 1). In
certain embodiments, the genetically engineered bacteria of the
invention comprise a RNS-inducible regulatory region from norVW
that is operatively linked to a gene or genes, e.g., one or more
tranporter gene sequence(s). In the presence of RNS, a NorR
transcription factor senses RNS and activates to the norVW
regulatory region, thereby driving expression of the operatively
linked gene(s) and producing the tranporter.
[0612] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region, and the transcription factor that
senses RNS is DNR. DNR (dissimilatory nitrate respiration
regulator) "promotes the expression of the nir, the nor and the nos
genes" in the presence of nitric oxide (Castiglione et al., 2009).
The genetically engineered bacteria of the invention may comprise
any suitable RNS-responsive regulatory region from a gene that is
activated by DNR. Genes that are capable of being activated by DNR
are known in the art (see, e.g., Castiglione et al., 2009; Giardina
et al., 2008; Table 1). In certain embodiments, the genetically
engineered bacteria of the invention comprise a RNS-inducible
regulatory region from norCB that is operatively linked to a gene
or gene cassette, e.g., a butyrogenic gene cassette. In the
presence of RNS, a DNR transcription factor senses RNS and
activates to the norCB regulatory region, thereby driving
expression of the operatively linked gene or genes and producing
one or more tranporters. In some embodiments, the DNR is
Pseudomonas aeruginosa DNR.
[0613] In some embodiments, the tunable regulatory region is a
RNS-derepressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of RNS, the transcription factor no longer binds to the
regulatory region, thereby derepressing the operatively linked gene
or gene cassette.
[0614] In some embodiments, the tunable regulatory region is a
RNS-derepressible regulatory region, and the transcription factor
that senses RNS is NsrR. NsrR is "an Rrf2-type transcriptional
repressor [that] can sense NO and control the expression of genes
responsible for NO metabolism" (Isabella et al., 2009). The
genetically engineered bacteria of the invention may comprise any
suitable RNS-responsive regulatory region from a gene that is
repressed by NsrR. In some embodiments, the NsrR is Neisseria
gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR
are known in the art (see, e.g., Isabella et al., 2009; Dunn et
al., 2010; Table 1). In certain embodiments, the genetically
engineered bacteria of the invention comprise a RNS-derepressible
regulatory region from norB that is operatively linked to a gene or
genes, e.g., an tranporter gene or genes. In the presence of RNS,
an NsrR transcription factor senses RNS and no longer binds to the
norB regulatory region, thereby derepressing the operatively linked
an tranporter gene or genes and producing the encoding an
tranporter(s).
[0615] In some embodiments, it is advantageous for the genetically
engineered bacteria to express a RNS-sensing transcription factor
that does not regulate the expression of a significant number of
native genes in the bacteria. In some embodiments, the genetically
engineered bacterium of the invention expresses a RNS-sensing
transcription factor from a different species, strain, or substrain
of bacteria, wherein the transcription factor does not bind to
regulatory sequences in the genetically engineered bacterium of the
invention. In some embodiments, the genetically engineered
bacterium of the invention is Escherichia coli, and the RNS-sensing
transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae,
wherein the Escherichia coli does not comprise binding sites for
said NsrR. In some embodiments, the heterologous transcription
factor minimizes or eliminates off-target effects on endogenous
regulatory regions and genes in the genetically engineered
bacteria.
[0616] In some embodiments, the tunable regulatory region is a
RNS-repressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of RNS, the transcription factor senses RNS and binds to
the RNS-repressible regulatory region, thereby repressing
expression of the operatively linked gene or gene cassette. In some
embodiments, the RNS-sensing transcription factor is capable of
binding to a regulatory region that overlaps with part of the
promoter sequence. In alternate embodiments, the RNS-sensing
transcription factor is capable of binding to a regulatory region
that is upstream or downstream of the promoter sequence.
[0617] In these embodiments, the genetically engineered bacteria
may comprise a two repressor activation regulatory circuit, which
is used to express an tranporter. The two repressor activation
regulatory circuit comprises a first RNS-sensing repressor and a
second repressor, which is operatively linked to a gene or gene
cassette, e.g., encoding an tranporter. In one aspect of these
embodiments, the RNS-sensing repressor inhibits transcription of
the second repressor, which inhibits the transcription of the gene
or gene cassette. Examples of second repressors useful in these
embodiments include, but are not limited to, TetR, Cl, and LexA. In
the absence of binding by the first repressor (which occurs in the
absence of RNS), the second repressor is transcribed, which
represses expression of the gene or genes. In the presence of
binding by the first repressor (which occurs in the presence of
RNS), expression of the second repressor is repressed, and the gene
or genes, e.g., an tranporter gene or genes is expressed.
[0618] RNS-responsive transcription factor may induce, derepress,
or repress gene expression depending upon the regulatory region
sequence used in the genetically engineered bacteria. One or more
types of RNS-sensing transcription factors and corresponding
regulatory region sequences may be present in genetically
engineered bacteria. In some embodiments, the genetically
engineered bacteria comprise one type of RNS-sensing transcription
factor, e.g., NsrR, and one corresponding regulatory region
sequence, e.g., from norB. In some embodiments, the genetically
engineered bacteria comprise one type of RNS-sensing transcription
factor, e.g., NsrR, and two or more different corresponding
regulatory region sequences, e.g., from norB and aniA. In some
embodiments, the genetically engineered bacteria comprise two or
more types of RNS-sensing transcription factors, e.g., NsrR and
NorR, and two or more corresponding regulatory region sequences,
e.g., from norB and norR, respectively. One RNS-responsive
regulatory region may be capable of binding more than one
transcription factor. In some embodiments, the genetically
engineered bacteria comprise two or more types of RNS-sensing
transcription factors and one corresponding regulatory region
sequence. Nucleic acid sequences of several RNS-regulated
regulatory regions are known in the art (see, e.g., Spiro 2006;
Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011;
Karlinsey et al., 2012).
[0619] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene encoding a RNS-sensing transcription
factor, e.g., the nsrR gene, that is controlled by its native
promoter, an inducible promoter, a promoter that is stronger than
the native promoter, e.g., the GlnRS promoter or the P(Bla)
promoter, or a constitutive promoter. In some instances, it may be
advantageous to express the RNS-sensing transcription factor under
the control of an inducible promoter in order to enhance expression
stability. In some embodiments, expression of the RNS-sensing
transcription factor is controlled by a different promoter than the
promoter that controls expression of the therapeutic molecule. In
some embodiments, expression of the RNS-sensing transcription
factor is controlled by the same promoter that controls expression
of the therapeutic molecule. In some embodiments, the RNS-sensing
transcription factor and therapeutic molecule are divergently
transcribed from a promoter region.
[0620] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene for a RNS-sensing transcription
factor from a different species, strain, or substrain of bacteria.
In some embodiments, the genetically engineered bacteria comprise a
RNS-responsive regulatory region from a different species, strain,
or substrain of bacteria. In some embodiments, the genetically
engineered bacteria comprise a RNS-sensing transcription factor and
corresponding RNS-responsive regulatory region from a different
species, strain, or substrain of bacteria. The heterologous
RNS-sensing transcription factor and regulatory region may increase
the transcription of genes operatively linked to said regulatory
region in the presence of RNS, as compared to the native
transcription factor and regulatory region from bacteria of the
same subtype under the same conditions.
[0621] In some embodiments, the genetically engineered bacteria
comprise a RNS-sensing transcription factor, NsrR, and
corresponding regulatory region, nsrR, from Neisseria gonorrhoeae.
In some embodiments, the native RNS-sensing transcription factor,
e.g., NsrR, is left intact and retains wild-type activity. In
alternate embodiments, the native RNS-sensing transcription factor,
e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type
activity.
[0622] In some embodiments, the genetically engineered bacteria of
the invention comprise multiple copies of the endogenous gene
encoding the RNS-sensing transcription factor, e.g., the nsrR gene.
In some embodiments, the gene encoding the RNS-sensing
transcription factor is present on a plasmid. In some embodiments,
the gene encoding the RNS-sensing transcription factor and the gene
or gene cassette for producing the therapeutic molecule are present
on different plasmids. In some embodiments, the gene encoding the
RNS-sensing transcription factor and the gene or gene cassette for
producing the therapeutic molecule are present on the same plasmid.
In some embodiments, the gene encoding the RNS-sensing
transcription factor is present on a chromosome. In some
embodiments, the gene encoding the RNS-sensing transcription factor
and the gene or gene cassette for producing the therapeutic
molecule are present on different chromosomes. In some embodiments,
the gene encoding the RNS-sensing transcription factor and the gene
or gene cassette for producing the therapeutic molecule are present
on the same chromosome.
[0623] In some embodiments, the genetically engineered bacteria
comprise a wild-type gene encoding a RNS-sensing transcription
factor, e.g., the NsrR gene, and a corresponding regulatory region,
e.g., a norB regulatory region, that is mutated relative to the
wild-type regulatory region from bacteria of the same subtype. The
mutated regulatory region increases the expression of the
tranporter in the presence of RNS, as compared to the wild-type
regulatory region under the same conditions. In some embodiments,
the genetically engineered bacteria comprise a wild-type
RNS-responsive regulatory region, e.g., the norB regulatory region,
and a corresponding transcription factor, e.g., NsrR, that is
mutated relative to the wild-type transcription factor from
bacteria of the same subtype. The mutant transcription factor
increases the expression of the tranporter in the presence of RNS,
as compared to the wild-type transcription factor under the same
conditions. In some embodiments, both the RNS-sensing transcription
factor and corresponding regulatory region are mutated relative to
the wild-type sequences from bacteria of the same subtype in order
to increase expression of the tranporter in the presence of
RNS.
[0624] In some embodiments, the gene or gene cassette for producing
the anti-inflammation and/or gut barrier function enhancer molecule
is present on a plasmid and operably linked to a promoter that is
induced by RNS. In some embodiments, expression is further
optimized by methods known in the art, e.g., by optimizing
ribosomal binding sites, manipulating transcriptional regulators,
and/or increasing mRNA stability.
[0625] In some embodiments, any of the gene(s) of the present
disclosure may be integrated into the bacterial chromosome at one
or more integration sites. For example, one or more copies of one
or more encoding an tranporter gene(s) may be integrated into the
bacterial chromosome. Having multiple copies of the gene or gen(s)
integrated into the chromosome allows for greater production of the
tranporter(s) and also permits fine-tuning of the level of
expression. Alternatively, different circuits described herein,
such as any of the secretion or exporter circuits, in addition to
the therapeutic gene(s) or gene cassette(s) could be integrated
into the bacterial chromosome at one or more different integration
sites to perform multiple different functions.
[0626] ROS-Dependent Regulation
[0627] In some embodiments, the genetically engineered bacteria or
genetically engineered virus comprise a gene for producing an
tranporter that is expressed under the control of an inducible
promoter. In some embodiments, the genetically engineered bacterium
or genetically engineered virus that expresses an tranporter under
the control of a promoter that is activated by conditions of
cellular damage. In one embodiment, the gene for producing the
tranporter is expressed under the control of an cellular
damaged-dependent promoter that is activated in environments in
which there is cellular or tissue damage, e.g., a reactive oxygen
species or ROS promoter.
[0628] As used herein, "reactive oxygen species" and "ROS" are used
interchangeably to refer to highly active molecules, ions, and/or
radicals derived from molecular oxygen. ROS can be produced as
byproducts of aerobic respiration or metal-catalyzed oxidation and
may cause deleterious cellular effects such as oxidative damage.
ROS includes, but is not limited to, hydrogen peroxide (H2O2),
organic peroxide (ROOH), hydroxyl ion (OH--), hydroxyl radical
(.cndot.OH), superoxide or superoxide anion (.cndot.O2--), singlet
oxygen (1O2), ozone (O3), carbonate radical, peroxide or peroxyl
radical (.cndot.O2-2), hypochlorous acid (HOCl), hypochlorite ion
(OCl--), sodium hypochlorite (NaOCl), nitric oxide (NO.cndot.), and
peroxynitrite or peroxynitrite anion (ONOO--) (unpaired electrons
denoted by .cndot.). Bacteria have evolved transcription factors
that are capable of sensing ROS levels. Different ROS signaling
pathways are triggered by different ROS levels and occur with
different kinetics (Marinho et al., 2014).
[0629] As used herein, "ROS-inducible regulatory region" refers to
a nucleic acid sequence to which one or more ROS-sensing
transcription factors is capable of binding, wherein the binding
and/or activation of the corresponding transcription factor
activates downstream gene expression; in the presence of ROS, the
transcription factor binds to and/or activates the regulatory
region. In some embodiments, the ROS-inducible regulatory region
comprises a promoter sequence. In some embodiments, the
transcription factor senses ROS and subsequently binds to the
ROS-inducible regulatory region, thereby activating downstream gene
expression. In alternate embodiments, the transcription factor is
bound to the ROS-inducible regulatory region in the absence of ROS;
in the presence of ROS, the transcription factor undergoes a
conformational change, thereby activating downstream gene
expression. The ROS-inducible regulatory region may be operatively
linked to a gene sequence or gene sequence, e.g., a sequence or
sequences encoding one or more tranporter(s). For example, in the
presence of ROS, a transcription factor, e.g., OxyR, senses ROS and
activates a corresponding ROS-inducible regulatory region, thereby
driving expression of an operatively linked gene sequence or gene
sequences. Thus, ROS induces expression of the gene or genes.
[0630] As used herein, "ROS-derepressible regulatory region" refers
to a nucleic acid sequence to which one or more ROS-sensing
transcription factors is capable of binding, wherein the binding of
the corresponding transcription factor represses downstream gene
expression; in the presence of ROS, the transcription factor does
not bind to and does not repress the regulatory region. In some
embodiments, the ROS-derepressible regulatory region comprises a
promoter sequence. The ROS-derepressible regulatory region may be
operatively linked to a gene or genes, e.g., one or more genes
encoding one or more tranporter(s). For example, in the presence of
ROS, a transcription factor, e.g., OhrR, senses ROS and no longer
binds to and/or represses the regulatory region, thereby
derepressing an operatively linked gene sequence or gene cassette.
Thus, ROS derepresses expression of the gene or gene cassette.
[0631] As used herein, "ROS-repressible regulatory region" refers
to a nucleic acid sequence to which one or more ROS-sensing
transcription factors is capable of binding, wherein the binding of
the corresponding transcription factor represses downstream gene
expression; in the presence of ROS, the transcription factor binds
to and represses the regulatory region. In some embodiments, the
ROS-repressible regulatory region comprises a promoter sequence. In
some embodiments, the transcription factor that senses ROS is
capable of binding to a regulatory region that overlaps with part
of the promoter sequence. In alternate embodiments, the
transcription factor that senses ROS is capable of binding to a
regulatory region that is upstream or downstream of the promoter
sequence. The ROS-repressible regulatory region may be operatively
linked to a gene sequence or gene sequences. For example, in the
presence of ROS, a transcription factor, e.g., PerR, senses ROS and
binds to a corresponding ROS-repressible regulatory region, thereby
blocking expression of an operatively linked gene sequence or gene
sequences. Thus, ROS represses expression of the gene or genes.
[0632] As used herein, a "ROS-responsive regulatory region" refers
to a ROS-inducible regulatory region, a ROS-repressible regulatory
region, and/or a ROS-derepressible regulatory region. In some
embodiments, the ROS-responsive regulatory region comprises a
promoter sequence. Each regulatory region is capable of binding at
least one corresponding ROS-sensing transcription factor. Examples
of transcription factors that sense ROS and their corresponding
ROS-responsive genes, promoters, and/or regulatory regions include,
but are not limited to, those shown in Table 4.
[0633] Examples of ROS-Sensing Transcription Factors and
ROS-Responsive Genes
TABLE-US-00007 ROS-sensing Primarily transcription capable of
Examples of responsive genes, factor: sensing: promoters, and/or
regulatory regions: OxyR H.sub.2O.sub.2 ahpC; ahpF; dps; dsbG;
fhuF; flu; fur; gor; grxA; hemH; katG; oxyS; sufA; sufB; sufC;
sufD; sufE; sufS; trxC; uxuA; yaaA; yaeH; yaiA; ybjM; ydcH; ydeN;
ygaQ; yljA; ytfK PerR H.sub.2O.sub.2 katA; ahpCF; mrgA; zoaA; fur;
hemAXCDBL; srfA OhrR Organic ohrA peroxides NaOCl SoxR
.cndot.O.sub.2.sup.- soxS NO.cndot. (also capable of sensing
H.sub.2O.sub.2) RosR H.sub.2O.sub.2 rbtT; tnp16a; rluC1; tnp5a;
mscL; tnp2d; phoD; tnp15b; pstA; tnp5b; xylC; gabD1; rluC2; cgtS9;
azlC; narKGHJI; rosR
[0634] In some embodiments, the genetically engineered bacteria
comprise a tunable regulatory region that is directly or indirectly
controlled by a transcription factor that is capable of sensing at
least one reactive oxygen species. The tunable regulatory region is
operatively linked to a gene or gene cassette capable of directly
or indirectly driving the expression of an tranporter, thus
controlling expression of the tranporter relative to ROS levels.
For example, the tunable regulatory region is a ROS-inducible
regulatory region, and the molecule is an tranporter; when ROS is
present, e.g., in an inflamed tissue, a ROS-sensing transcription
factor binds to and/or activates the regulatory region and drives
expression of the gene sequence for the tranporter, thereby
producing the tranporter. Subsequently, when inflammation is
ameliorated, ROS levels are reduced, and production of the
tranporter is decreased or eliminated.
[0635] In some embodiments, the tunable regulatory region is a
ROS-inducible regulatory region; in the presence of ROS, a
transcription factor senses ROS and activates the ROS-inducible
regulatory region, thereby driving expression of an operatively
linked gene or gene cassette. In some embodiments, the
transcription factor senses ROS and subsequently binds to the
ROS-inducible regulatory region, thereby activating downstream gene
expression. In alternate embodiments, the transcription factor is
bound to the ROS-inducible regulatory region in the absence of ROS;
when the transcription factor senses ROS, it undergoes a
conformational change, thereby inducing downstream gene
expression.
[0636] In some embodiments, the tunable regulatory region is a
ROS-inducible regulatory region, and the transcription factor that
senses ROS is OxyR. OxyR "functions primarily as a global regulator
of the peroxide stress response" and is capable of regulating
dozens of genes, e.g., "genes involved in H2O2 detoxification
(katE, ahpCF), heme biosynthesis (hemH), reductant supply (grxA,
gor, trxC), thiol-disulfide isomerization (dsbG), Fe--S center
repair (sufA-E, sufS), iron binding (yaaA), repression of iron
import systems (fur)" and "OxyS, a small regulatory RNA" (Dubbs et
al., 2012). The genetically engineered bacteria may comprise any
suitable ROS-responsive regulatory region from a gene that is
activated by OxyR. Genes that are capable of being activated by
OxyR are known in the art (see, e.g., Zheng et al., 2001; Dubbs et
al., 2012; Table 1). In certain embodiments, the genetically
engineered bacteria of the invention comprise a ROS-inducible
regulatory region from oxyS that is operatively linked to a gene,
e.g., an tranporter gene. In the presence of ROS, e.g., H2O2, an
OxyR transcription factor senses ROS and activates to the oxyS
regulatory region, thereby driving expression of the operatively
linked tranporter gene and producing the tranporter. In some
embodiments, OxyR is encoded by an E. coli oxyR gene. In some
embodiments, the oxyS regulatory region is an E. coli oxyS
regulatory region. In some embodiments, the ROS-inducible
regulatory region is selected from the regulatory region of katG,
dps, and ahpC.
[0637] In alternate embodiments, the tunable regulatory region is a
ROS-inducible regulatory region, and the corresponding
transcription factor that senses ROS is SoxR. When SoxR is
"activated by oxidation of its [2Fe--2S ] cluster, it increases the
synthesis of SoxS, which then activates its target gene expression"
(Koo et al., 2003). "SoxR is known to respond primarily to
superoxide and nitric oxide" (Koo et al., 2003), and is also
capable of responding to H2O2. The genetically engineered bacteria
of the invention may comprise any suitable ROS-responsive
regulatory region from a gene that is activated by SoxR. Genes that
are capable of being activated by SoxR are known in the art (see,
e.g., Koo et al., 2003; Table 1). In certain embodiments, the
genetically engineered bacteria of the invention comprise a
ROS-inducible regulatory region from soxS that is operatively
linked to a gene, e.g., an tranporter. In the presence of ROS, the
SoxR transcription factor senses ROS and activates the soxS
regulatory region, thereby driving expression of the operatively
linked an tranporter gene and producing the an tranporter.
[0638] In some embodiments, the tunable regulatory region is a
ROS-derepressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of ROS, the transcription factor no longer binds to the
regulatory region, thereby derepressing the operatively linked gene
or gene cassette.
[0639] In some embodiments, the tunable regulatory region is a
ROS-derepressible regulatory region, and the transcription factor
that senses ROS is OhrR. OhrR "binds to a pair of inverted repeat
DNA sequences overlapping the ohrA promoter site and thereby
represses the transcription event," but oxidized OhrR is "unable to
bind its DNA target" (Duarte et al., 2010). OhrR is a
"transcriptional repressor [that] . . . senses both organic
peroxides and NaOCl" (Dubbs et al., 2012) and is "weakly activated
by H2O2 but it shows much higher reactivity for organic
hydroperoxides" (Duarte et al., 2010). The genetically engineered
bacteria of the invention may comprise any suitable ROS-responsive
regulatory region from a gene that is repressed by OhrR. Genes that
are capable of being repressed by OhrR are known in the art (see,
e.g., Dubbs et al., 2012; Table 1). In certain embodiments, the
genetically engineered bacteria of the invention comprise a
ROS-derepressible regulatory region from ohrA that is operatively
linked to a gene or gene cassette, e.g., an tranporter gene. In the
presence of ROS, e.g., NaOCl, an OhrR transcription factor senses
ROS and no longer binds to the ohrA regulatory region, thereby
derepressing the operatively linked tranporter gene and producing
the an tranporter.
[0640] OhrR is a member of the MarR family of ROS-responsive
regulators. "Most members of the MarR family are transcriptional
repressors and often bind to the -10 or -35 region in the promoter
causing a steric inhibition of RNA polymerase binding" (Bussmann et
al., 2010). Other members of this family are known in the art and
include, but are not limited to, OspR, MgrA, RosR, and SarZ. In
some embodiments, the transcription factor that senses ROS is OspR,
MgRA, RosR, and/or SarZ, and the genetically engineered bacteria of
the invention comprises one or more corresponding regulatory region
sequences from a gene that is repressed by OspR, MgRA, RosR, and/or
SarZ. Genes that are capable of being repressed by OspR, MgRA,
RosR, and/or SarZ are known in the art (see, e.g., Dubbs et al.,
2012).
[0641] In some embodiments, the tunable regulatory region is a
ROS-derepressible regulatory region, and the corresponding
transcription factor that senses ROS is RosR. RosR is "a MarR-type
transcriptional regulator" that binds to an "18-bp inverted repeat
with the consensus sequence TTGTTGAYRYRTCAACWA" and is "reversibly
inhibited by the oxidant H2O2" (Bussmann et al., 2010). RosR is
capable of repressing numerous genes and putative genes, including
but not limited to "a putative polyisoprenoid-binding protein
(cg1322, gene upstream of and divergent from rosR), a sensory
histidine kinase (cgtS9), a putative transcriptional regulator of
the Crp/FNR family (cg3291), a protein of the glutathione
S-transferase family (cg1426), two putative FMN reductases (cg1150
and cg1850), and four putative monooxygenases (cg0823, cg1848,
cg2329, and cg3084)" (Bussmann et al., 2010). The genetically
engineered bacteria of the invention may comprise any suitable
ROS-responsive regulatory region from a gene that is repressed by
RosR. Genes that are capable of being repressed by RosR are known
in the art (see, e.g., Bussmann et al., 2010; Table 1). In certain
embodiments, the genetically engineered bacteria of the invention
comprise a ROS-derepressible regulatory region from cgtS9 that is
operatively linked to a gene or gene cassette, e.g., an tranporter.
In the presence of ROS, e.g., H2O2, a RosR transcription factor
senses ROS and no longer binds to the cgtS9 regulatory region,
thereby derepressing the operatively linked tranporter gene and
producing the tranporter.
[0642] In some embodiments, it is advantageous for the genetically
engineered bacteria to express a ROS-sensing transcription factor
that does not regulate the expression of a significant number of
native genes in the bacteria. In some embodiments, the genetically
engineered bacterium of the invention expresses a ROS-sensing
transcription factor from a different species, strain, or substrain
of bacteria, wherein the transcription factor does not bind to
regulatory sequences in the genetically engineered bacterium of the
invention. In some embodiments, the genetically engineered
bacterium of the invention is Escherichia coli, and the ROS-sensing
transcription factor is RosR, e.g., from Corynebacterium
glutamicum, wherein the Escherichia coli does not comprise binding
sites for said RosR. In some embodiments, the heterologous
transcription factor minimizes or eliminates off-target effects on
endogenous regulatory regions and genes in the genetically
engineered bacteria.
[0643] In some embodiments, the tunable regulatory region is a
ROS-repressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of ROS, the transcription factor senses ROS and binds to
the ROS-repressible regulatory region, thereby repressing
expression of the operatively linked gene or gene cassette. In some
embodiments, the ROS-sensing transcription factor is capable of
binding to a regulatory region that overlaps with part of the
promoter sequence. In alternate embodiments, the ROS-sensing
transcription factor is capable of binding to a regulatory region
that is upstream or downstream of the promoter sequence.
[0644] In some embodiments, the tunable regulatory region is a
ROS-repressible regulatory region, and the transcription factor
that senses ROS is PerR. In Bacillus subtilis, PerR "when bound to
DNA, represses the genes coding for proteins involved in the
oxidative stress response (katA, ahpC, and mrgA), metal homeostasis
(hemAXCDBL, fur, and zoaA) and its own synthesis (perR)" (Marinho
et al., 2014). PerR is a "global regulator that responds primarily
to H2O2" (Dubbs et al., 2012) and "interacts with DNA at the per
box, a specific palindromic consensus sequence (TTATAATNATTATAA)
residing within and near the promoter sequences of PerR-controlled
genes" (Marinho et al., 2014). PerR is capable of binding a
regulatory region that "overlaps part of the promoter or is
immediately downstream from it" (Dubbs et al., 2012). The
genetically engineered bacteria of the invention may comprise any
suitable ROS-responsive regulatory region from a gene that is
repressed by PerR. Genes that are capable of being repressed by
PerR are known in the art (see, e.g., Dubbs et al., 2012; Table
1).
[0645] In these embodiments, the genetically engineered bacteria
may comprise a two repressor activation regulatory circuit, which
is used to express an tranporter. The two repressor activation
regulatory circuit comprises a first ROS-sensing repressor, e.g.,
PerR, and a second repressor, e.g., TetR, which is operatively
linked to a gene or gene cassette, e.g., an tranporter. In one
aspect of these embodiments, the ROS-sensing repressor inhibits
transcription of the second repressor, which inhibits the
transcription of the gene or gene cassette. Examples of second
repressors useful in these embodiments include, but are not limited
to, TetR, Cl, and LexA. In some embodiments, the ROS-sensing
repressor is PerR. In some embodiments, the second repressor is
TetR. In this embodiment, a PerR-repressible regulatory region
drives expression of TetR, and a TetR-repressible regulatory region
drives expression of the gene or gene cassette, e.g., an
tranporter. In the absence of PerR binding (which occurs in the
absence of ROS), tetR is transcribed, and TetR represses expression
of the gene or gene cassette, e.g., an tranporter. In the presence
of PerR binding (which occurs in the presence of ROS), tetR
expression is repressed, and the gene or gene cassette, e.g., an
tranporter, is expressed.
[0646] A ROS-responsive transcription factor may induce, derepress,
or repress gene expression depending upon the regulatory region
sequence used in the genetically engineered bacteria. For example,
although "OxyR is primarily thought of as a transcriptional
activator under oxidizing conditions . . . OxyR can function as
either a repressor or activator under both oxidizing and reducing
conditions" (Dubbs et al., 2012), and OxyR "has been shown to be a
repressor of its own expression as well as that of fhuF (encoding a
ferric ion reductase) and flu (encoding the antigen 43 outer
membrane protein)" (Zheng et al., 2001). The genetically engineered
bacteria of the invention may comprise any suitable ROS-responsive
regulatory region from a gene that is repressed by OxyR. In some
embodiments, OxyR is used in a two repressor activation regulatory
circuit, as described above. Genes that are capable of being
repressed by OxyR are known in the art (see, e.g., Zheng et al.,
2001; Table 1). Or, for example, although RosR is capable of
repressing a number of genes, it is also capable of activating
certain genes, e.g., the narKGHJI operon. In some embodiments, the
genetically engineered bacteria comprise any suitable
ROS-responsive regulatory region from a gene that is activated by
RosR. In addition, "PerR-mediated positive regulation has also been
observed . . . and appears to involve PerR binding to distant
upstream sites" (Dubbs et al., 2012). In some embodiments, the
genetically engineered bacteria comprise any suitable
ROS-responsive regulatory region from a gene that is activated by
PerR.
[0647] One or more types of ROS-sensing transcription factors and
corresponding regulatory region sequences may be present in
genetically engineered bacteria. For example, "OhrR is found in
both Gram-positive and Gram-negative bacteria and can coreside with
either OxyR or PerR or both" (Dubbs et al., 2012). In some
embodiments, the genetically engineered bacteria comprise one type
of ROS-sensing transcription factor, e.g., OxyR, and one
corresponding regulatory region sequence, e.g., from oxyS. In some
embodiments, the genetically engineered bacteria comprise one type
of ROS-sensing transcription factor, e.g., OxyR, and two or more
different corresponding regulatory region sequences, e.g., from
oxyS and katG. In some embodiments, the genetically engineered
bacteria comprise two or more types of ROS-sensing transcription
factors, e.g., OxyR and PerR, and two or more corresponding
regulatory region sequences, e.g., from oxyS and katA,
respectively. One ROS-responsive regulatory region may be capable
of binding more than one transcription factor. In some embodiments,
the genetically engineered bacteria comprise two or more types of
ROS-sensing transcription factors and one corresponding regulatory
region sequence.
[0648] Nucleic acid sequences of several exemplary OxyR-regulated
regulatory regions are shown in Table 5. OxyR binding sites are
underlined and bolded. In some embodiments, genetically engineered
bacteria comprise a nucleic acid sequence that is at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 99% homologous to the DNA sequence of SEQ ID NO: 46,
47, 48, or 49, or a functional fragment thereof.
[0649] Nucleotide Sequences of Exemplary OxyR-Regulated Regulatory
Regions
TABLE-US-00008 Regulatory sequence
01234567890123456789012345678901234567890123456789 katG
TGTGGCTTTTATGAAAATCACACAGTGATCACAAATTTTAAACA (SEQ ID NO:)
GAGCACAAAATGCTGCCTCGAAATGAGGGCGGGAAAATAAGGT
TATCAGCCTTGTTTTCTCCCTCATTACTTGAAGGATATGAAGCTA
AAACCCTTTTTTATAAAGCATTTGTCCGAATTCGGACATAATCA
AAAAAGCTTAATTAAGATCAATTTGATCTACATCTCTTTAACCA
ACAATATGTAAGATCTCAACTATCGCATCCGTGGATTAATTC
AATTATAACTTCTCTCTAACGCTGTGTATCGTAACGGTAACACT
GTAGAGGGGAGCACATTGATGCGAATTCATTAAAGAGGAGAAA GGTACC dps
TTCCGAAAATTCCTGGCGAGCAGATAAATAAGAATTGTTCTTAT (SEQ ID NO:)
CAATATATCTAACTCATTGAATCTTTATTAGTTTTGTTTTTCACG
CTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCG
GAACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGA
CATAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGA GAAAGGTACC ahpC
GCTTAGATCAGGTGATTGCCCTTTGTTTATGAGGGTGTTGTAATC (SEQ ID NO:)
CATGTCGTTGTTGCATTTGTAAGGGCAACACCTCAGCCTGCAGG
CAGGCACTGAAGATACCAAAGGGTAGTTCAGATTACACGGTCA
CCTGGAAAGGGGGCCATTTTACTTTTTATCGCCGCTGGCGGTGC
AAAGTTCACAAAGTTGTCTTACGAAGGTTGTAAGGTAAAACTT
ATCGATTTGATAATGGAAACGCATTAGCCGAATCGGCAAAAAT
TGGTTACCTTACATCTCATCGAAAACACGGAGGAAGTATAGATG
CGAATTCATTAAAGAGGAGAAAGGTACC oxyS
CTCGAGTTCATTATCCATCCTCCATCGCCACGATAGTTCATGGC (SEQ ID NO:)
GATAGGTAGAATAGCAATGAACGATTATCCCTATCAAGCATTC
TGACTGATAATTGCTCACACGAATTCATTAAAGAGGAGAAAGGT ACC
[0650] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene encoding a ROS-sensing transcription
factor, e.g., the oxyR gene, that is controlled by its native
promoter, an inducible promoter, a promoter that is stronger than
the native promoter, e.g., the GlnRS promoter or the P(Bla)
promoter, or a constitutive promoter. In some instances, it may be
advantageous to express the ROS-sensing transcription factor under
the control of an inducible promoter in order to enhance expression
stability. In some embodiments, expression of the ROS-sensing
transcription factor is controlled by a different promoter than the
promoter that controls expression of the therapeutic molecule. In
some embodiments, expression of the ROS-sensing transcription
factor is controlled by the same promoter that controls expression
of the therapeutic molecule. In some embodiments, the ROS-sensing
transcription factor and therapeutic molecule are divergently
transcribed from a promoter region.
[0651] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene for a ROS-sensing transcription
factor from a different species, strain, or substrain of bacteria.
In some embodiments, the genetically engineered bacteria comprise a
ROS-responsive regulatory region from a different species, strain,
or substrain of bacteria. In some embodiments, the genetically
engineered bacteria comprise a ROS-sensing transcription factor and
corresponding ROS-responsive regulatory region from a different
species, strain, or substrain of bacteria. The heterologous
ROS-sensing transcription factor and regulatory region may increase
the transcription of genes operatively linked to said regulatory
region in the presence of ROS, as compared to the native
transcription factor and regulatory region from bacteria of the
same subtype under the same conditions.
[0652] In some embodiments, the genetically engineered bacteria
comprise a ROS-sensing transcription factor, OxyR, and
corresponding regulatory region, oxyS, from Escherichia coli. In
some embodiments, the native ROS-sensing transcription factor,
e.g., OxyR, is left intact and retains wild-type activity. In
alternate embodiments, the native ROS-sensing transcription factor,
e.g., OxyR, is deleted or mutated to reduce or eliminate wild-type
activity.
[0653] In some embodiments, the genetically engineered bacteria of
the invention comprise multiple copies of the endogenous gene
encoding the ROS-sensing transcription factor, e.g., the oxyR gene.
In some embodiments, the gene encoding the ROS-sensing
transcription factor is present on a plasmid. In some embodiments,
the gene encoding the ROS-sensing transcription factor and the gene
or gene cassette for producing the therapeutic molecule are present
on different plasmids. In some embodiments, the gene encoding the
ROS-sensing transcription factor and the gene or gene cassette for
producing the therapeutic molecule are present on the same. In some
embodiments, the gene encoding the ROS-sensing transcription factor
is present on a chromosome. In some embodiments, the gene encoding
the ROS-sensing transcription factor and the gene or gene cassette
for producing the therapeutic molecule are present on different
chromosomes. In some embodiments, the gene encoding the ROS-sensing
transcription factor and the gene or gene cassette for producing
the therapeutic molecule are present on the same chromosome.
[0654] In some embodiments, the genetically engineered bacteria
comprise a wild-type gene encoding a ROS-sensing transcription
factor, e.g., the soxR gene, and a corresponding regulatory region,
e.g., a soxS regulatory region, that is mutated relative to the
wild-type regulatory region from bacteria of the same subtype. The
mutated regulatory region increases the expression of the
tranporter in the presence of ROS, as compared to the wild-type
regulatory region under the same conditions. In some embodiments,
the genetically engineered bacteria comprise a wild-type
ROS-responsive regulatory region, e.g., the oxyS regulatory region,
and a corresponding transcription factor, e.g., OxyR, that is
mutated relative to the wild-type transcription factor from
bacteria of the same subtype. The mutant transcription factor
increases the expression of the tranporter in the presence of ROS,
as compared to the wild-type transcription factor under the same
conditions. In some embodiments, both the ROS-sensing transcription
factor and corresponding regulatory region are mutated relative to
the wild-type sequences from bacteria of the same subtype in order
to increase expression of the tranporter in the presence of
ROS.
[0655] In some embodiments, the gene or gene cassette for producing
the tranporter is present on a plasmid and operably linked to a
promoter that is induced by ROS. In some embodiments, the gene or
gene cassette for producing the tranporter is present in the
chromosome and operably linked to a promoter that is induced by
ROS. In some embodiments, the gene or gene cassette for producing
the tranporter is present on a chromosome and operably linked to a
promoter that is induced by exposure to tetracycline. In some
embodiments, the gene or gene cassette for producing the tranporter
is present on a plasmid and operably linked to a promoter that is
induced by exposure to tetracycline. In some embodiments,
expression is further optimized by methods known in the art, e.g.,
by optimizing ribosomal binding sites, manipulating transcriptional
regulators, and/or increasing mRNA stability.
[0656] In some embodiments, the genetically engineered bacteria may
comprise multiple copies of the gene(s) capable of producing an
tranporter(s). In some embodiments, the gene(s) capable of
producing an tranporter(s) is present on a plasmid and operatively
linked to a ROS-responsive regulatory region. In some embodiments,
the gene(s) capable of producing an tranporter is present in a
chromosome and operatively linked to a ROS-responsive regulatory
region.
[0657] Thus, in some embodiments, the genetically engineered
bacteria or genetically engineered virus produce one or more
tranporters under the control of an oxygen level-dependent
promoter, a reactive oxygen species (ROS)-dependent promoter, or a
reactive nitrogen species (RNS)-dependent promoter, and a
corresponding transcription factor.
[0658] In some embodiments, the genetically engineered bacteria
comprise a stably maintained plasmid or chromosome carrying a gene
for producing an tranporter, such that the tranporter can be
expressed in the host cell, and the host cell is capable of
survival and/or growth in vitro, e.g., in medium, and/or in vivo.
In some embodiments, a bacterium may comprise multiple copies of
the gene encoding the tranporter. In some embodiments, the gene
encoding the tranporter is expressed on a low-copy plasmid. In some
embodiments, the low-copy plasmid may be useful for increasing
stability of expression. In some embodiments, the low-copy plasmid
may be useful for decreasing leaky expression under non-inducing
conditions. In some embodiments, the gene encoding the tranporter
is expressed on a high-copy plasmid. In some embodiments, the
high-copy plasmid may be useful for increasing expression of the
tranporter. In some embodiments, the gene encoding the tranporter
is expressed on a chromosome.
[0659] In some embodiments, the bacteria are genetically engineered
to include multiple mechanisms of action (MOAs), e.g., circuits
producing multiple copies of the same product (e.g., to enhance
copy number) or circuits performing multiple different functions.
For example, the genetically engineered bacteria may include four
copies of the gene encoding a particular tranporter inserted at
four different insertion sites. Alternatively, the genetically
engineered bacteria may include three copies of the gene encoding a
particular tranporter inserted at three different insertion sites
and three copies of the gene encoding a different tranporter
inserted at three different insertion sites.
[0660] In some embodiments, under conditions where the tranporter
is expressed, the genetically engineered bacteria of the disclosure
produce at least about 1.5-fold, at least about 2-fold, at least
about 10-fold, at least about 15-fold, at least about 20-fold, at
least about 30-fold, at least about 50-fold, at least about
100-fold, at least about 200-fold, at least about 300-fold, at
least about 400-fold, at least about 500-fold, at least about
600-fold, at least about 700-fold, at least about 800-fold, at
least about 900-fold, at least about 1,000-fold, or at least about
1,500-fold more of the tranporter, and/or transcript of the gene(s)
in the operon as compared to unmodified bacteria of the same
subtype under the same conditions.
[0661] In some embodiments, quantitative PCR (qPCR) is used to
amplify, detect, and/or quantify mRNA expression levels of the
tranporter gene(s). Primers specific for tranporter the gene(s) may
be designed and used to detect mRNA in a sample according to
methods known in the art. In some embodiments, a fluorophore is
added to a sample reaction mixture that may contain tranporter
mRNA, and a thermal cycler is used to illuminate the sample
reaction mixture with a specific wavelength of light and detect the
subsequent emission by the fluorophore. The reaction mixture is
heated and cooled to predetermined temperatures for predetermined
time periods. In certain embodiments, the heating and cooling is
repeated for a predetermined number of cycles. In some embodiments,
the reaction mixture is heated and cooled to 90-100.degree. C.,
60-70.degree. C., and 30-50.degree. C. for a predetermined number
of cycles. In a certain embodiment, the reaction mixture is heated
and cooled to 93-97.degree. C., 55-65.degree. C., and 35-45.degree.
C. for a predetermined number of cycles. In some embodiments, the
accumulating amplicon is quantified after each cycle of the qPCR.
The number of cycles at which fluorescence exceeds the threshold is
the threshold cycle (CT). At least one CT result for each sample is
generated, and the CT result(s) may be used to determine mRNA
expression levels of the tranporter gene(s).
[0662] In some embodiments, quantitative PCR (qPCR) is used to
amplify, detect, and/or quantify mRNA expression levels of the
tranporter gene(s). Primers specific for tranporter the gene(s) may
be designed and used to detect mRNA in a sample according to
methods known in the art. In some embodiments, a fluorophore is
added to a sample reaction mixture that may contain tranporter
mRNA, and a thermal cycler is used to illuminate the sample
reaction mixture with a specific wavelength of light and detect the
subsequent emission by the fluorophore. The reaction mixture is
heated and cooled to predetermined temperatures for predetermined
time periods. In certain embodiments, the heating and cooling is
repeated for a predetermined number of cycles. In some embodiments,
the reaction mixture is heated and cooled to 90-100.degree. C.,
60-70.degree. C., and 30-50.degree. C. for a predetermined number
of cycles. In a certain embodiment, the reaction mixture is heated
and cooled to 93-97.degree. C., 55-65.degree. C., and 35-45.degree.
C. for a predetermined number of cycles. In some embodiments, the
accumulating amplicon is quantified after each cycle of the qPCR.
The number of cycles at which fluorescence exceeds the threshold is
the threshold cycle (CT). At least one CT result for each sample is
generated, and the CT result(s) may be used to determine mRNA
expression levels of the tranporter gene(s).
[0663] Essential Genes and Auxotrophs
[0664] As used herein, the term "essential gene" refers to a gene
that is necessary to for cell growth and/or survival. Bacterial
essential genes are well known to one of ordinary skill in the art,
and can be identified by directed deletion of genes and/or random
mutagenesis and screening (see, for example, Zhang and Lin, 2009,
DEG 5.0, a database of essential genes in both prokaryotes and
eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al.,
Essential genes on metabolic maps, Curr. Opin. Biotechnol.,
17(5):448-456, the entire contents of each of which are expressly
incorporated herein by reference).
[0665] An "essential gene" may be dependent on the circumstances
and environment in which an organism lives. For example, a mutation
of, modification of, or excision of an essential gene may result in
the recombinant bacteria of the disclosure becoming an auxotroph.
An auxotrophic modification is intended to cause bacteria to die in
the absence of an exogenously added nutrient essential for survival
or growth because they lack the gene(s) necessary to produce that
essential nutrient.
[0666] An auxotrophic modification is intended to cause bacteria to
die in the absence of an exogenously added nutrient essential for
survival or growth because they lack the gene(s) necessary to
produce that essential nutrient. In some embodiments, any of the
genetically engineered bacteria described herein also comprise a
deletion or mutation in a gene required for cell survival and/or
growth. In one embodiment, the essential gene is a DNA synthesis
gene, for example, thyA. In another embodiment, the essential gene
is a cell wall synthesis gene, for example, dapA. In yet another
embodiment, the essential gene is an amino acid gene, for example,
serA or MetA. Any gene required for cell survival and/or growth may
be targeted, including but not limited to, cysE, glnA, ilvD, leuB,
lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA,
thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB,
and thiI, as long as the corresponding wild-type gene product is
not produced in the bacteria. For example, thymine is a nucleic
acid that is required for bacterial cell growth; in its absence,
bacteria undergo cell death. The thyA gene encodes thimidylate
synthetase, an enzyme that catalyzes the first step in thymine
synthesis by converting dUMP to dTMP (Sat et al. (2003) J
Bacteriol. (2003) 185(6):1803-7). In some embodiments, the
bacterial cell of the disclosure is a thyA auxotroph in which the
thyA gene is deleted and/or replaced with an unrelated gene. A thyA
auxotroph can grow only when sufficient amounts of thymine are
present, e.g., by adding thymine to growth media in vitro. Without
sufficient amounts of thymine, the thyA auxotroph dies. In some
embodiments, the auxotrophic modification is used to ensure that
the bacterial cell does not survive in the absence of the
auxotrophic gene product, e.g., outside of the hypoxic tumor
environment.
[0667] Diaminopimelic acid (DAP) is an amino acid synthetized
within the lysine biosynthetic pathway and is required for
bacterial cell wall growth (Meadow et al., 1959; Clarkson et al.,
1971). In some embodiments, any of the genetically engineered
bacteria described herein is a dapD auxotroph in which the dapD
gene is deleted and/or replaced with an unrelated gene. A dapD
auxotroph can grow only when sufficient amounts of DAP are present,
e.g., by adding DAP to growth media in vitro. Without sufficient
amounts of DAP, the dapD auxotroph dies. In some embodiments, the
auxotrophic modification is used to ensure that the bacterial cell
does not survive in the absence of the auxotrophic gene
product.
[0668] In other embodiments, the genetically engineered bacterium
of the present disclosure is a uraA auxotroph in which the uraA
gene is deleted and/or replaced with an unrelated gene. The uraA
gene codes for UraA, a membrane-bound transporter that facilitates
the uptake and subsequent metabolism of the pyrimidine uracil
(Andersen et al., 1995). A uraA auxotroph can grow only when
sufficient amounts of uracil are present, e.g., by adding uracil to
growth media in vitro. Without sufficient amounts of uracil, the
uraA auxotroph dies. In some embodiments, auxotrophic modifications
are used to ensure that the bacteria do not survive in the absence
of the auxotrophic gene product.
[0669] In complex communities, it is possible for bacteria to share
DNA. In very rare circumstances, an auxotrophic bacterial strain
may receive DNA from a non-auxotrophic strain, which repairs the
genomic deletion and permanently rescues the auxotroph. Therefore,
engineering a bacterial strain with more than one auxotroph may
greatly decrease the probability that DNA transfer will occur
enough times to rescue the auxotrophy. In some embodiments, the
genetically engineered bacteria of the invention comprise a
deletion or mutation in two or more genes required for cell
survival and/or growth.
[0670] Other examples of essential genes include, but are not
limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs,
ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE,
gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA,
nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS,
ispG, suhB, tadA, acpS, era, mc, ftsB, eno, pyrG, chpR, lgt, fbaA,
pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF,
yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB,
rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE,
rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, lspA, ispH, dapB,
folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG,
murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR,
dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA,
yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC,
yrdC, def fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA,
nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB,
csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS,
yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA,
coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA,
yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG,
secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN,
rpsQ, rpmC, rplP, rpsL, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD,
fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA,
rlpB, leuS, int, glnS, fldA, cydA, infA, cydC, ftsK, A, serS, rpsA,
msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, yceQ,
fabD, fabG, acpP, tmk, holB, C, D, E, purB, ymfK, minE, mind, pth,
rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, racR,
dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ,
and gpsA. Other essential genes are known to those of ordinary
skill in the art.
[0671] In some embodiments, the genetically engineered bacterium of
the present disclosure is a synthetic ligand-dependent essential
gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic
auxotrophs with a mutation in one or more essential genes that only
grow in the presence of a particular ligand (see Lopez and Anderson
"Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a
BL21 (DE3 Biosafety Strain, "ACS Synthetic Biology (2015) DOI:
10.1021/acssynbio.5b00085, the entire contents of which are
expressly incorporated herein by reference).
[0672] In some embodiments, the SLiDE bacterial cell comprises a
mutation in an essential gene. In some embodiments, the essential
gene is selected from the group consisting of pheS, dnaN, tyrS,
metG, and adk. In some embodiments, the essential gene is dnaN
comprising one or more of the following mutations: H191N, R240C,
1317S, F319V, L340T, V347I, and S345C. In some embodiments, the
essential gene is dnaN comprising the mutations H191N, R240C,
I317S, F319V, L340T, V347I, and S345C. In some embodiments, the
essential gene is pheS comprising one or more of the following
mutations: F125G, P183T, P184A, R186A, and I188L. In some
embodiments, the essential gene is pheS comprising the mutations
F125G, P183T, P184A, R186A, and I188L. In some embodiments, the
essential gene is tyrS comprising one or more of the following
mutations: L36V, C38A, and F40G. In some embodiments, the essential
gene is tyrS comprising the mutations L36V, C38A, and F40G. In some
embodiments, the essential gene is metG comprising one or more of
the following mutations: E45Q, N47R, I49G, and A51C. In some
embodiments, the essential gene is metG comprising the mutations
E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene
is adk comprising one or more of the following mutations: I4L, L5I,
and L6G. In some embodiments, the essential gene is adk comprising
the mutations I4L, L5I, and L6G.
[0673] In some embodiments, the genetically engineered bacterium is
complemented by a ligand. In some embodiments, the ligand is
selected from the group consisting of benzothiazole, indole,
2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid,
and L-histidine methyl ester. For example, bacterial cells
comprising mutations in metG (E45Q, N47R, I49G, and A51C) are
complemented by benzothiazole, indole, 2-aminobenzothiazole,
indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl
ester. Bacterial cells comprising mutations in dnaN (H191N, R240C,
I317S, F319V, L340T, V347I, and S345C) are complemented by
benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells
comprising mutations in pheS (F125G, P183T, P184A, R186A, and
I188L) are complemented by benzothiazole or 2-aminobenzothiazole.
Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G)
are complemented by benzothiazole or 2-aminobenzothiazole.
Bacterial cells comprising mutations in adk (I4L, L5I, and L6G) are
complemented by benzothiazole or indole.
[0674] In some embodiments, the genetically engineered bacterium
comprises more than one mutant essential gene that renders it
auxotrophic to a ligand. In some embodiments, the bacterial cell
comprises mutations in two essential genes. For example, in some
embodiments, the bacterial cell comprises mutations in tyrS (L36V,
C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other
embodiments, the bacterial cell comprises mutations in three
essential genes. For example, in some embodiments, the bacterial
cell comprises mutations in tyrS (L36V, C38A, and F40G), metG
(E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A,
and I188L).
[0675] In some embodiments, the genetically engineered bacterium is
a conditional auxotroph whose essential gene(s) is replaced using
an arabinose system.
[0676] In some embodiments, the genetically engineered bacterium of
the disclosure is an auxotroph and also comprises kill switch
circuitry, such as any of the kill switch components and systems
described herein. For example, the recombinant bacteria may
comprise a deletion or mutation in an essential gene required for
cell survival and/or growth, for example, in a DNA synthesis gene,
for example, thyA, cell wall synthesis gene, for example, dapA
and/or an amino acid gene, for example, serA or MetA and may also
comprise a toxin gene that is regulated by one or more
transcriptional activators that are expressed in response to an
environmental condition(s) and/or signal(s) (such as low oxygen
levels) or regulated by one or more recombinases that are expressed
upon sensing an exogenous environmental condition(s) and/or
signal(s) (such as the recombinase systems described herein). Other
embodiments are described in Wright et al., "GeneGuard: A Modular
Plasmid System Designed for Biosafety," ACS Synthetic Biology
(2015) 4: 307-16, the entire contents of which are expressly
incorporated herein by reference). In some embodiments, the
genetically engineered bacterium of the disclosure is an auxotroph
and also comprises kill switch circuitry, such as any of the kill
switch components and systems described herein, as well as another
biosecurity system, such a conditional origin of replication (see
Wright et al., supra). In other embodiments, auxotrophic
modifications may also be used to screen for mutant bacteria that
produce the substrate transporter.
[0677] Genetic Regulatory Circuits
[0678] In some embodiments, the genetically engineered bacteria
comprise multi-layered genetic regulatory circuits for expressing
the constructs described herein (see, e.g., U.S. Provisional
Application No. 62/184,811, incorporated herein by reference in its
entirety). The genetic regulatory circuits are useful to screen for
mutant bacteria that produce a substrate transporter or rescue an
auxotroph. In certain embodiments, the invention provides methods
for selecting genetically engineered bacteria that produce one or
more genes of interest.
[0679] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload and a T7 polymerase-regulated genetic
regulatory circuit. For example, the genetically engineered
bacteria comprise a first gene encoding a T7 polymerase, wherein
the first gene is operably linked to a fumarate and nitrate
reductase regulator (FNR)-responsive promoter; a second gene or
gene cassette for producing a payload, wherein the second gene or
gene cassette is operably linked to a T7 promoter that is induced
by the T7 polymerase; and a third gene encoding an inhibitory
factor, lysY, that is capable of inhibiting the T7 polymerase. In
the presence of oxygen, FNR does not bind the FNR-responsive
promoter, and the payload is not expressed. LysY is expressed
constitutively (P-lac constitutive) and further inhibits T7
polymerase. In the absence of oxygen, FNR dimerizes and binds to
the FNR-responsive promoter, T7 polymerase is expressed at a level
sufficient to overcome lysY inhibition, and the payload is
expressed. In some embodiments, the lysY gene is operably linked to
an additional FNR binding site. In the absence of oxygen, FNR
dimerizes to activate T7 polymerase expression as described above,
and also inhibits lysY expression.
[0680] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload, and a protease-regulated genetic regulatory
circuit. For example, the genetically engineered bacteria comprise
a first gene encoding an mf-lon protease, wherein the first gene is
operably linked to a FNR-responsive promoter; a second gene or gene
cassette for producing a payload operably linked to a tet
regulatory region (tetO); and a third gene encoding an mf-lon
degradation signal linked to a tet repressor (tetR), wherein the
tetR is capable of binding to the tet regulatory region and
repressing expression of the second gene or gene cassette. The
mf-lon protease is capable of recognizing the mf-lon degradation
signal and degrading the tetR. In the presence of oxygen, FNR does
not bind the FNR-responsive promoter, the repressor is not
degraded, and the payload is not expressed. In the absence of
oxygen, FNR dimerizes and binds the FNR-responsive promoter,
thereby inducing expression of mf-lon protease. The mf-lon protease
recognizes the mf-lon degradation signal and degrades the tetR, and
the payload is expressed.
[0681] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload, and a repressor-regulated genetic regulatory
circuit. For example, the genetically engineered bacteria comprise
a first gene encoding a first repressor, wherein the first gene is
operably linked to a FNR-responsive promoter; a second gene or gene
cassette for producing a payload operably linked to a first
regulatory region comprising a constitutive promoter; and a third
gene encoding a second repressor, wherein the second repressor is
capable of binding to the first regulatory region and repressing
expression of the second gene or gene cassette. The third gene is
operably linked to a second regulatory region comprising a
constitutive promoter, wherein the first repressor is capable of
binding to the second regulatory region and inhibiting expression
of the second repressor. In the presence of oxygen, FNR does not
bind the FNR-responsive promoter, the first repressor is not
expressed, the second repressor is expressed, and the payload is
not expressed. In the absence of oxygen, FNR dimerizes and binds
the FNR-responsive promoter, the first repressor is expressed, the
second repressor is not expressed, and the payload is
expressed.
[0682] Examples of repressors useful in these embodiments include,
but are not limited to, ArgR, TetR, ArsR, AscG, LacI, CscR, DeoR,
DgoR, FruR, GalR, GatR, CI, LexA, RafR, QacR, and PtxS
(US20030166191).
[0683] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload and a regulatory RNA-regulated genetic
regulatory circuit. For example, the genetically engineered
bacteria comprise a first gene encoding a regulatory RNA, wherein
the first gene is operably linked to a FNR-responsive promoter, and
a second gene or gene cassette for producing a payload. The second
gene or gene cassette is operably linked to a constitutive promoter
and further linked to a nucleotide sequence capable of producing an
mRNA hairpin that inhibits translation of the payload. The
regulatory RNA is capable of eliminating the mRNA hairpin and
inducing payload translation via the ribosomal binding site. In the
presence of oxygen, FNR does not bind the FNR-responsive promoter,
the regulatory RNA is not expressed, and the mRNA hairpin prevents
the payload from being translated. In the absence of oxygen, FNR
dimerizes and binds the FNR-responsive promoter, the regulatory RNA
is expressed, the mRNA hairpin is eliminated, and the payload is
expressed.
[0684] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload, and a CRISPR-regulated genetic regulatory
circuit. For example, the genetically engineered bacteria comprise
a Cas9 protein; a first gene encoding a CRISPR guide RNA, wherein
the first gene is operably linked to a FNR-responsive promoter; a
second gene or gene cassette for producing a payload, wherein the
second gene or gene cassette is operably linked to a regulatory
region comprising a constitutive promoter; and a third gene
encoding a repressor operably linked to a constitutive promoter,
wherein the repressor is capable of binding to the regulatory
region and repressing expression of the second gene or gene
cassette. The third gene is further linked to a CRISPR target
sequence that is capable of binding to the CRISPR guide RNA,
wherein said binding to the CRISPR guide RNA induces cleavage by
the Cas9 protein and inhibits expression of the repressor. In the
presence of oxygen, FNR does not bind the FNR-responsive promoter,
the guide RNA is not expressed, the repressor is expressed, and the
payload is not expressed. In the absence of oxygen, FNR dimerizes
and binds the FNR-responsive promoter, the guide RNA is expressed,
the repressor is not expressed, and the payload is expressed.
[0685] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload and a recombinase-regulated genetic regulatory
circuit. For example, the genetically engineered bacteria comprise
a first gene encoding a recombinase, wherein the first gene is
operably linked to a FNR-responsive promoter, and a second gene or
gene cassette for producing a payload operably linked to a
constitutive promoter. The second gene or gene cassette is inverted
in orientation (3' to 5') and flanked by recombinase binding sites,
and the recombinase is capable of binding to the recombinase
binding sites to induce expression of the second gene or gene
cassette by reverting its orientation (5' to 3'). In the presence
of oxygen, FNR does not bind the FNR-responsive promoter, the
recombinase is not expressed, the payload remains in the 3' to 5'
orientation, and no functional payload is produced. In the absence
of oxygen, FNR dimerizes and binds the FNR-responsive promoter, the
recombinase is expressed, the payload is reverted to the 5' to 3'
orientation, and functional payload is produced.
[0686] In some embodiments, the invention provides genetically
engineered bacteria comprising a gene or gene cassette for
producing a payload and a polymerase- and recombinase-regulated
genetic regulatory circuit. For example, the genetically engineered
bacteria comprise a first gene encoding a recombinase, wherein the
first gene is operably linked to a FNR-responsive promoter; a
second gene or gene cassette for producing a payload operably
linked to a T7 promoter; a third gene encoding a T7 polymerase,
wherein the T7 polymerase is capable of binding to the T7 promoter
and inducing expression of the payload. The third gene encoding the
T7 polymerase is inverted in orientation (3' to 5') and flanked by
recombinase binding sites, and the recombinase is capable of
binding to the recombinase binding sites to induce expression of
the T7 polymerase gene by reverting its orientation (5' to 3'). In
the presence of oxygen, FNR does not bind the FNR-responsive
promoter, the recombinase is not expressed, the T7 polymerase gene
remains in the 3' to 5' orientation, and the payload is not
expressed. In the absence of oxygen, FNR dimerizes and binds the
FNR-responsive promoter, the recombinase is expressed, the T7
polymerase gene is reverted to the 5' to 3' orientation, and the
payload is expressed.
[0687] Host-Plasmid Mutual Dependency
[0688] In some embodiments, the genetically engineered bacteria of
the invention also comprise a plasmid that has been modified to
create a host-plasmid mutual dependency. In certain embodiments,
the mutually dependent host-plasmid platform is GeneGuard (Wright
et al., 2015). In some embodiments, the GeneGuard plasmid comprises
(i) a conditional origin of replication, in which the requisite
replication initiator protein is provided in trans; (ii) an
auxotrophic modification that is rescued by the host via genomic
translocation and is also compatible for use in rich media; and/or
(iii) a nucleic acid sequence which encodes a broad-spectrum toxin.
The toxin gene may be used to select against plasmid spread by
making the plasmid DNA itself disadvantageous for strains not
expressing the anti-toxin (e.g., a wild-type bacterium). In some
embodiments, the GeneGuard plasmid is stable for at least 100
generations without antibiotic selection. In some embodiments, the
GeneGuard plasmid does not disrupt growth of the host. The
GeneGuard plasmid is used to greatly reduce unintentional plasmid
propagation in the genetically engineered bacteria of the
invention.
[0689] The mutually dependent host-plasmid platform may be used
alone or in combination with other biosafety mechanisms, such as
those described herein (e.g., kill switches, auxotrophies). In some
embodiments, the genetically engineered bacteria comprise a
GeneGuard plasmid. In other embodiments, the genetically engineered
bacteria comprise a GeneGuard plasmid and/or one or more kill
switches. In other embodiments, the genetically engineered bacteria
comprise a GeneGuard plasmid and/or one or more auxotrophies. In
still other embodiments, the genetically engineered bacteria
comprise a GeneGuard plasmid, one or more kill switches, and/or one
or more auxotrophies.
[0690] Kill Switch
[0691] In some embodiments, the genetically engineered bacteria of
the invention also comprise a kill switch (see, e.g., U.S.
Provisional Application Nos. 183,935 and 62/263,329 incorporated
herein by reference in their entireties). The kill switch is
intended to actively kill engineered microbes in response to
external stimuli. As opposed to an auxotrophic mutation where
bacteria die because they lack an essential nutrient for survival,
the kill switch is triggered by a particular factor in the
environment that induces the production of toxic molecules within
the microbe that cause cell death.
[0692] Bacteria engineered with kill switches have been engineered
for in vitro research purposes, e.g., to limit the spread of a
biofuel-producing microorganism outside of a laboratory
environment. Bacteria engineered for in vivo administration to
treat a disease or disorder may also be programmed to die at a
specific time after the expression and delivery of a heterologous
gene or genes, for example, a therapeutic gene(s) or after the
subject has experienced the therapeutic effect. For example, in
some embodiments, the kill switch is activated to kill the bacteria
after a period of time following oxygen level-dependent expression
of the substrate transporter. In some embodiments, the kill switch
is activated in a delayed fashion following oxygen level-dependent
expression of the substrate transporter. Alternatively, the
bacteria may be engineered to die if the bacteria have spread
outside of a target site (e.g., a tumor site). Specifically, it may
be useful to prevent the spread of the microorganism outside the
area of interest (for example, outside of the tumor site) within
the subject, or spread of the microorganism outside of the subject
into the environment (for example, spread to the environment
through the blood or stool of the subject). Examples of such toxins
that can be used in kill switches include, but are not limited to,
bacteriocins, lysins, and other molecules that cause cell death by
lysing cell membranes, degrading cellular DNA, or other mechanisms.
Such toxins can be used individually or in combination. The
switches that control their production can be based on, for
example, transcriptional activation (toggle switches; see, e.g.,
Gardner et al. (2000) Nature 403: 339-42), translation
(riboregulators), or DNA recombination (recombinase-based
switches), and can sense environmental stimuli such as anaerobiosis
or reactive oxygen species. These switches can be activated by a
single environmental factor or may require several activators in
AND, OR, NAND and NOR logic configurations to induce cell death.
For example, an AND riboregulator switch is activated by
tetracycline, isopropyl .beta.-D-1-thiogalactopyranoside (IPTG),
and arabinose to induce the expression of lysins, which
permeabilize the cell membrane and kill the cell. IPTG induces the
expression of the endolysin and holin mRNAs, which are then
derepressed by the addition of arabinose and tetracycline. All
three inducers must be present to cause cell death. Examples of
kill switches are known in the art (Callura et al. (2010) Proc.
Natl. Acad. Sci. USA 107(36): 15898-903.). In some embodiments, the
kill switch is activated to kill the bacteria after a period of
time following oxygen level-dependent expression of the substrate
transporter. In some embodiments, the kill switch is activated in a
delayed fashion following oxygen level-dependent expression of the
substrate transporter.
[0693] Kill switches can be designed such that a toxin is produced
in response to an environmental condition or external signal (e.g.,
the bacteria is killed in response to an external cue) or,
alternatively designed such that a toxin is produced once an
environmental condition no longer exists or an external signal is
ceased.
[0694] Thus, in some embodiments, the genetically engineered
bacteria of the disclosure are further programmed to die after
sensing an exogenous environmental signal, for example, in a low
oxygen environment. In some embodiments, the genetically engineered
bacteria of the present disclosure comprise one or more genes
encoding one or more recombinase(s), whose expression is induced in
response to an environmental condition or signal and causes one or
more recombination events that ultimately leads to the expression
of a toxin which kills the cell. In some embodiments, the at least
one recombination event is the flipping of an inverted heterologous
gene encoding a bacterial toxin which is then constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, constitutive expression of the bacterial toxin kills
the genetically engineered bacterium. In these types of kill switch
systems, once the engineered bacterial cell senses the exogenous
environmental condition and expresses the heterologous gene of
interest, the recombinant bacterial cell is no longer viable.
[0695] In another embodiment in which the genetically engineered
bacteria of the present disclosure express one or more
recombinase(s) in response to an environmental condition or signal
causing at least one recombination event, the genetically
engineered bacterium further expresses a heterologous gene encoding
an anti-toxin in response to an exogenous environmental condition
or signal. In one embodiment, the at least one recombination event
is flipping of an inverted heterologous gene encoding a bacterial
toxin by a first recombinase. In one embodiment, the inverted
heterologous gene encoding the bacterial toxin is located between a
first forward recombinase recognition sequence and a first reverse
recombinase recognition sequence. In one embodiment, the
heterologous gene encoding the bacterial toxin is constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, the anti-toxin inhibits the activity of the toxin,
thereby delaying death of the genetically engineered bacterium. In
one embodiment, the genetically engineered bacterium is killed by
the bacterial toxin when the heterologous gene encoding the
anti-toxin is no longer expressed when the exogenous environmental
condition is no longer present.
[0696] In another embodiment, the at least one recombination event
is flipping of an inverted heterologous gene encoding a second
recombinase by a first recombinase, followed by the flipping of an
inverted heterologous gene encoding a bacterial toxin by the second
recombinase. In one embodiment, the inverted heterologous gene
encoding the second recombinase is located between a first forward
recombinase recognition sequence and a first reverse recombinase
recognition sequence. In one embodiment, the inverted heterologous
gene encoding the bacterial toxin is located between a second
forward recombinase recognition sequence and a second reverse
recombinase recognition sequence. In one embodiment, the
heterologous gene encoding the second recombinase is constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, the heterologous gene encoding the bacterial toxin is
constitutively expressed after it is flipped by the second
recombinase. In one embodiment, the genetically engineered
bacterium is killed by the bacterial toxin. In one embodiment, the
genetically engineered bacterium further expresses a heterologous
gene encoding an anti-toxin in response to the exogenous
environmental condition. In one embodiment, the anti-toxin inhibits
the activity of the toxin when the exogenous environmental
condition is present, thereby delaying death of the genetically
engineered bacterium. In one embodiment, the genetically engineered
bacterium is killed by the bacterial toxin when the heterologous
gene encoding the anti-toxin is no longer expressed when the
exogenous environmental condition is no longer present.
[0697] In one embodiment, the at least one recombination event is
flipping of an inverted heterologous gene encoding a second
recombinase by a first recombinase, followed by flipping of an
inverted heterologous gene encoding a third recombinase by the
second recombinase, followed by flipping of an inverted
heterologous gene encoding a bacterial toxin by the third
recombinase.
[0698] In one embodiment, the at least one recombination event is
flipping of an inverted heterologous gene encoding a first excision
enzyme by a first recombinase. In one embodiment, the inverted
heterologous gene encoding the first excision enzyme is located
between a first forward recombinase recognition sequence and a
first reverse recombinase recognition sequence. In one embodiment,
the heterologous gene encoding the first excision enzyme is
constitutively expressed after it is flipped by the first
recombinase. In one embodiment, the first excision enzyme excises a
first essential gene. In one embodiment, the programmed recombinant
bacterial cell is not viable after the first essential gene is
excised.
[0699] In one embodiment, the first recombinase further flips an
inverted heterologous gene encoding a second excision enzyme. In
one embodiment, the wherein the inverted heterologous gene encoding
the second excision enzyme is located between a second forward
recombinase recognition sequence and a second reverse recombinase
recognition sequence. In one embodiment, the heterologous gene
encoding the second excision enzyme is constitutively expressed
after it is flipped by the first recombinase. In one embodiment,
the genetically engineered bacterium dies or is no longer viable
when the first essential gene and the second essential gene are
both excised. In one embodiment, the genetically engineered
bacterium dies or is no longer viable when either the first
essential gene is excised or the second essential gene is excised
by the first recombinase.
[0700] In one embodiment, the genetically engineered bacterium dies
after the at least one recombination event occurs. In another
embodiment, the genetically engineered bacterium is no longer
viable after the at least one recombination event occurs.
[0701] In any of these embodiment, the recombinase can be a
recombinase selected from the group consisting of: BxbI, PhiC31,
TP901, BxbI, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Inti, Int4,
Int5, Int6, Int1, Int8, Int9, Int10, Int11, Int12, Int13, Int14,
Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23,
Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32,
Int33, and Int34, or a biologically active fragment thereof.
[0702] In the above-described kill switch circuits, a toxin is
produced in the presence of an environmental factor or signal. In
another aspect of kill switch circuitry, a toxin may be repressed
in the presence of an environmental factor (not produced) and then
produced once the environmental condition or external signal is no
longer present. Such kill switches are called repression-based kill
switches and represent systems in which the bacterial cells are
viable only in the presence of an external factor or signal, such
as arabinose or other sugar. Exemplary kill switch designs in which
the toxin is repressed in the presence of an external factor or
signal (and activated once the external signal is removed) are
shown in FIGS. 12-16. The disclosure provides recombinant bacterial
cells which express one or more heterologous gene(s) upon sensing
arabinose or other sugar in the exogenous environment. In this
aspect, the recombinant bacterial cells contain the araC gene,
which encodes the AraC transcription factor, as well as one or more
genes under the control of the araBAD promoter. In the absence of
arabinose, the AraC transcription factor adopts a conformation that
represses transcription of genes under the control of the araBAD
promoter. In the presence of arabinose, the AraC transcription
factor undergoes a conformational change that allows it to bind to
and activate the AraBAD promoter, which induces expression of the
desired gene, for example tetR, which represses expression of a
toxin gene. In this embodiment, the toxing gene is repressed in the
presence of arabinose or other sugar. In an environment where
arabinose is not present, the tetR gene is not activated and the
toxin is expressed, thereby killing the bacteria. The arbinoase
system can also be used to express an essential gene, in which the
essential gene is only expressed in the presence of arabinose or
other sugar and is not expressed when arabinose or other sugar is
absent from the environment.
[0703] Thus, in some embodiments in which one or more heterologous
gene(s) are expressed upon sensing arabinose in the exogenous
environment, the one or more heterologous genes are directly or
indirectly under the control of the araBAD promoter. In some
embodiments, the expressed heterologous gene is selected from one
or more of the following: a heterologous therapeutic gene, a
heterologous gene encoding an antitoxin, a heterologous gene
encoding a repressor protein or polypeptide, for example, a TetR
repressor, a heterologous gene encoding an essential protein not
found in the bacterial cell, and/or a heterologous encoding a
regulatory protein or polypeptide.
[0704] Arabinose inducible promoters are known in the art,
including P.sub.ara, P.sub.araB, P.sub.araC, and P.sub.araBAD. In
one embodiment, the arabinose inducible promoter is from E. coli.
In some embodiments, the P.sub.araC promoter and the P.sub.araBAD
promoter operate as a bidirectional promoter, with the P.sub.araBAD
promoter controlling expression of a heterologous gene(s) in one
direction, and the P.sub.araC (in close proximity to, and on the
opposite strand from the P.sub.araBAD promoter), controlling
expression of a heterologous gene(s) in the other direction. In the
presence of arabinose, transcription of both heterologous genes
from both promoters is induced. However, in the absence of
arabinose, transcription of both heterologous genes from both
promoters is not induced.
[0705] In one exemplary embodiment of the disclosure, the
genetically engineered bacteria of the present disclosure contains
a kill-switch having at least the following sequences: a
P.sub.araBAD promoter operably linked to a heterologous gene
encoding a Tetracycline Repressor Protein (TetR), a P.sub.araC
promoter operably linked to a heterologous gene encoding AraC
transcription factor, and a heterologous gene encoding a bacterial
toxin operably linked to a promoter which is repressed by the
Tetracycline Repressor Protein (P.sub.TetR). In the presence of
arabinose, the AraC transcription factor activates the P.sub.araBAD
promoter, which activates transcription of the TetR protein, which,
in turn, represses transcription of the toxin. In the absence of
arabinose, however, AraC suppresses transcription from the the
P.sub.araBAD promoter and no TetR protein is expressed. In this
case, expression of the heterologous toxin gene is activated, and
the toxin is expressed. The toxin builds up in the recombinant
bacterial cell, and the recombinant bacterial cell is killed. In
one embodiment, the AraC gene encoding the AraC transcription
factor is under the control of a constitutive promoter and is
therefore constitutively expressed.
[0706] In one embodiment of the disclosure, the genetically
engineered bacterium further comprises an antitoxin under the
control of a constitutive promoter. In this situation, in the
presence of arabinose, the toxin is not expressed due to repression
by TetR protein, and the antitoxin protein builds-up in the cell.
However, in the absence of arabinose, TetR protein is not
expressed, and expression of the toxin is induced. The toxin begins
to build-up within the recombinant bacterial cell. The recombinant
bacterial cell is no longer viable once the toxin protein is
present at either equal or greater amounts than that of the
anti-toxin protein in the cell, and the recombinant bacterial cell
will be killed by the toxin.
[0707] In another embodiment of the disclosure, the genetically
engineered bacterium further comprises an antitoxin under the
control of the P.sub.araBAD promoter. In this situation, in the
presence of arabinose, TetR and the anti-toxin are expressed, the
anti-toxin builds up in the cell, and the toxin is not expressed
due to repression by TetR protein. However, in the absence of
arabinose, both the TetR protein and the anti-toxin are not
expressed, and expression of the toxin is induced. The toxin begins
to build-up within the recombinant bacterial cell. The recombinant
bacterial cell is no longer viable once the toxin protein is
expressed, and the recombinant bacterial cell will be killed by the
toxin.
[0708] In another exemplary embodiment of the disclosure, the
genetically engineered bacteria of the present disclosure contain a
kill-switch having at least the following sequences: a P.sub.araBAD
promoter operably linked to a heterologous gene encoding an
essential polypeptide not found in the recombinant bacterial cell
(and required for survival), and a P.sub.araC promoter operably
linked to a heterologous gene encoding AraC transcription factor.
In the presence of arabinose, the AraC transcription factor
activates the P.sub.araBAD promoter, which activates transcription
of the heterologous gene encoding the essential polypeptide,
allowing the recombinant bacterial cell to survive. In the absence
of arabinose, however, AraC suppresses transcription from the
P.sub.araBAD promoter and the essential protein required for
survival is not expressed. In this case, the recombinant bacterial
cell dies in the absence of arabinose. In some embodiments, the
sequence of P.sub.araBAD promoter operably linked to a heterologous
gene encoding an essential polypeptide not found in the recombinant
bacterial cell can be present in the bacterial cell in conjunction
with the TetR/toxin kill-switch system described directly above. In
some embodiments, the sequence of P.sub.araBAD promoter operably
linked to a heterologous gene encoding an essential polypeptide not
found in the recombinant bacterial cell can be present in the
bacterial cell in conjunction with the TetR/toxin/anto-toxin
kill-switch system described directly above.
[0709] In yet other embodiments, the bacteria may comprise a
plasmid stability system with a plasmid that produces both a
short-lived anti-toxin and a long-lived toxin. In this system, the
bacterial cell produces equal amounts of toxin and anti-toxin to
neutralize the toxin. Howevere, if/when the cell loses the plasmid,
the short-lived anti-toxin begins to decay. When the anti-toxin
decays completely the cell dies as a result of the longer-lived
toxin killing it.
[0710] In some embodiments, the engineered bacteria of the present
disclosure that are capable of producing a substrate transporter
further comprise the gene(s) encoding the components of any of the
above-described kill switch circuits.
[0711] In any of the above-described embodiments, the bacterial
toxin is selected from the group consisting of a lysin, Hok, Fst,
TisB, LdrD, Kid, SymE, MazF, FlmA, Ibs, XCV2162, dinJ, CcdB, MazF,
ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B,
microcin B17, microcin C, microcin C7-C51, microcin J25, microcin
ColV, microcin 24, microcin L, microcin D93, microcin L, microcin
E492, microcin H47, microcin 147, microcin M, colicin A, colicin
E1, colicin K, colicin N, colicin U, colicin B, colicin Ia, colicin
Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2,
colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin
E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a
biologically active fragment thereof.
[0712] In any of the above-described embodiments, the anti-toxin is
selected from the group consisting of an anti-lysin, Sok, RNAII,
IstR, Rd1D, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE,
ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE,
MccE.sup.CTD, MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi,
Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin
immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically
active fragment thereof.
[0713] In one embodiment, the bacterial toxin is bactericidal to
the genetically engineered bacterium. In one embodiment, the
bacterial toxin is bacteriostatic to the genetically engineered
bacterium.
[0714] In some embodiments, provided herein are genetically
engineered bacteria comprising a heterologous gene encoding a
substrate transporter, wherein the gene or gene cassette for
producing the substrate transporter is controlled by a promoter
that is induced under low-oxygen or anaerobic conditions. In some
embodiments, the promoter is selected from the fumarate and nitrate
reductase regulator (FNR) promoter, arginine deiminiase and nitrate
reduction (ANR) promoter, and dissimilatory nitrate respiration
regulator (DNR) promoter.
[0715] In some embodiments, the genetically engineered bacteria
comprising a heterologous gene encoding a substrate transporter is
an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA,
metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA,
dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thil1
auxotroph. In some embodiments, the engineered bacteria have more
than one auxotrophy, for example, they may be a .DELTA.thyA and
.DELTA.dapA auxotroph.
[0716] In some embodiments, the genetically engineered bacteria
comprising a heterologous gene encoding a substrate transporter
further comprises a kill switch circuit, such as any of the kill
switch circuits provided herein. For example, in some embodiments,
the genetically engineered bacteria further comprise one or more
genes encoding one or more recombinase(s) under the control of an
inducible promoter and an inverted toxin sequence. In some
embodiments, the genetically engineered bacteria further comprise
one or more genes encoding an anti-toxin. In some embodiments, the
engineered bacteria further comprise one or more genes encoding one
or more recombinase(s) under the control of an inducible promoter
and one or more inverted excision genes, wherein the excision
gene(s) encode an enzyme that deletes an essential gene. In some
embodiments, the genetically engineered bacteria further comprise
one or more genes encoding an anti-toxin.
[0717] In some instances, basal or leaky expression from an
inducible promoter may result in the activation of the kill switch,
thereby creating strong selective pressure for one or more
mutations that disable the switch and thus the ability to kill the
cell. In some embodiments, an environmental factor, e.g. arabinose,
is present during manufacturing, and activates the production of a
repressor that shuts down toxin production. Mutations in this
circuit, with the exception of the toxin gene itself, will result
in death with reduced chance for negative selection. When the
environmental factor is absent, the repressor stops being made, and
the toxin is produced. When the toxin concentration overcomes that
of the antitoxin, the cell dies. In some embodiments, variations in
the promoter and ribosome binding sequences of the antitoxin and
the toxin allow for tuning of the circuit to produce variations in
the timing of cell death. In alternate embodiments, the circuit
comprises recombinases that are repressed by tetR and produced in
the absence of tetR. These recombinases are capable of flipping the
toxin gene or its promoter into the active configuration, thereby
resulting in toxin production.
[0718] Synthetic gene circuits express on plasmids may function
well in the short term but lose ability and/or function in the long
term, e.g., in the stringent conditions found in a tumor
microenvironment (Danino et al. (2015) Sci. Transl. Med.
7(289):289ra84). In some embodiments, the genetically engineered
bacteria comprise stable circuits for expressing genes of interest,
e.g., a substrate transporter, over prolonged periods. In some
embodiments, the genetically engineered bacteria are capable of
targeting cancerous cells and producing a substrate transporter and
further comprise a toxin-antitoxin system that simultaneously
produces a toxin (hok) and a short-lived antitoxin (sok), wherein
loss of the plasmid causes the cell to be killed by the long-lived
toxin (Danino et al., 2015; FIG. 21). In some embodiments, the
genetically engineered bacteria further comprise alp7 from B.
subtilis plasmid pL20 and produces filaments that are capable of
pushing plasmids to the poles of the cells in order to ensure equal
segregation during cell division (Danino et al., 2015).
[0719] In some embodiments, the genetically engineered bacteria
comprising a heterologous gene encoding a substrate transporter is
an auxotroph and further comprises a kill switch circuit, such as
any of the kill switch circuits described herein.
[0720] In some embodiments of the above described genetically
engineered bacteria, the gene encoding the substrate transporter is
present on a plasmid in the bacterium and operatively linked on the
plasmid to the promoter that is induced under low-oxygen or
anaerobic conditions. The genetically engineered bacteria are
capable of local and tumor-specific delivery of the substrate
transporter, e.g., an amino acid transporter. In other embodiments,
the gene encoding the substrate transporter is present in the
bacterial chromosome and is operatively linked in the chromosome to
the promoter that is induced under low-oxygen or anaerobic
conditions. The genetically engineered bacteria are capable of
local and tumor-specific delivery of the substrate transporter.
[0721] Pharmaceutical Compositions and Formulations
[0722] Pharmaceutical compositions comprising the genetically
engineered microrganisms of the invention may be used to treat,
manage, ameliorate, and/or prevent a disease or condition disclosed
herein. Pharmaceutical compositions of the invention comprising one
or more genetically engineered bacteria with prophylactic agents,
therapeutic agents, and/or pharmaceutically acceptable carriers are
provided.
[0723] In certain embodiments, the pharmaceutical composition
comprises one species, strain, or subtype of bacteria that are
engineered to comprise the genetic modifications described herein,
e.g., one or more genes encoding one or more substrate
transporters. In alternate embodiments, the pharmaceutical
composition comprises two or more species, strains, and/or subtypes
of bacteria that are each engineered to comprise the genetic
modifications described herein, e.g., one or more genes encoding
one or more substrate transporters.
[0724] In some embodiments, the genetically engineered bacteria are
administered systemically or intratumorally as spores. As a
non-limiting example, the genetically engineered bacteria are
Clostridia, and administration results in a selective colonization
of hypoxic/necrotic areas within a tumor. In some embodiments, the
spores germinate exclusively in the hypoxic/necrotic regions
present in solid tumours and nowhere else in the body.
[0725] The pharmaceutical compositions of the invention may be
formulated in a conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients
into compositions for pharmaceutical use. Methods of formulating
pharmaceutical compositions are known in the art (see, e.g.,
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa.). In some embodiments, the pharmaceutical compositions are
subjected to tabletting, lyophilizing, direct compression,
conventional mixing, dissolving, granulating, levigating,
emulsifying, encapsulating, entrapping, or spray drying to form
tablets, granulates, nanoparticles, nanocapsules, microcapsules,
microtablets, pellets, or powders, which may be enterically coated
or uncoated. Appropriate formulation depends on the route of
administration.
[0726] The genetically engineered microorganisms may be formulated
into pharmaceutical compositions in any suitable dosage form (e.g.,
liquids, capsules, sachet, hard capsules, soft capsules, tablets,
enteric coated tablets, suspension powders, granules, or matrix
sustained release formations for oral administration) and for any
suitable type of administration (e.g., oral, topical, injectable,
intravenous, sub-cutaneous, intratumoral, peritumor,
immediate-release, pulsatile-release, delayed-release, or sustained
release). Suitable dosage amounts for the genetically engineered
bacteria may range from about 10.sup.4 to 10.sup.12 bacteria. The
composition may be administered once or more daily, weekly, or
monthly. The composition may be administered before, during, or
following a meal. In one embodiment, the pharmaceutical composition
is administered before the subject eats a meal. In one embodiment,
the pharmaceutical composition is administered currently with a
meal. In on embodiment, the pharmaceutical composition is
administered after the subject eats a meal.
[0727] The genetically engineered bacteria or genetically
engineered virus may be formulated into pharmaceutical compositions
comprising one or more pharmaceutically acceptable carriers,
thickeners, diluents, buffers, buffering agents, surface active
agents, neutral or cationic lipids, lipid complexes, liposomes,
penetration enhancers, carrier compounds, and other
pharmaceutically acceptable carriers or agents. For example, the
pharmaceutical composition may include, but is not limited to, the
addition of calcium bicarbonate, sodium bicarbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils, polyethylene glycols, and
surfactants, including, for example, polysorbate 20. In some
embodiments, the genetically engineered bacteria of the invention
may be formulated in a solution of sodium bicarbonate, e.g., 1
molar solution of sodium bicarbonate (to buffer an acidic cellular
environment, such as the stomach, for example). The genetically
engineered bacteria may be administered and formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0728] The genetically engineered microorganisms may be
administered intravenously, e.g., by infusion or injection.
Alternatively, the genetically engineered microorganisms may be
administered intratumorally and/or peritumorally. In other
embodiments, the genetically engineered microorganisms may be
administered intra-arterially, intramuscularly, or
intraperitoneally. In some embodiments, the genetically engineered
bacteria colonize about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
more of the target site (e.g., a tumor). In some embodiments, the
genetically engineered bacteria are co-administered with a
PEGylated form of rHuPH20 (PEGPH20) or other agent in order to
destroy the tumor septae in order to enhance penetration of the
tumor capsule, collagen, and/or stroma. In some embodiments, the
genetically engineered bacteria are capable of producing a
substrate transporter as well as one or more enzymes that degrade
fibrous tissue.
[0729] The genetically engineered microroganisms of the disclosure
may be administered via intratumoral injection, resulting in
bacterial cells that are directly deposited within the target
tissue (e.g., a tumor). Intratumoral injection of the engineered
bacteria may elicit a potent localized inflammatory response as
well as an adaptive immune response against tumor cells. Bacteria
are suspended in solution before being withdrawn into a 1-ml
syringe. In some embodiments, the tumor is injected with an
18-gauge multipronged needle (Quadra-Fuse, Rex Medical). The
injection site is aseptically prepared. If available, ultrasound or
CT may be used to identify a necrotic region of the tumor for
injection. If a necrotic region is not identified, the injection
can be directed to the center of the tumor. The needle is inserted
once into a predefined region, and dispensed with even pressure.
The injection needle is removed slowly, and the injection site is
sterilized.
[0730] Direct intratumoral injection of the genetically engineered
bacteria of the invention into a target tissue (e.g., a solid
tumor) may be advantageous as compared to intravenous
administration. Using an intravenous injection method, only a small
proporation of the bacteria may reach the target tumor. For
example, following E. coli Nissle injection into the tail vein of
4T1 tumor-bearing mice, most bacteria (>99%) are quickly cleared
from the animals and only a small percentage of the administered
bacteria colonize the tumor (Stritzker et al., 2007). In
particular, in large animals and human patients, which have
relatively large blood volumes and relatively small tumors compared
to mice, intratumoral injection may be especially beneficial.
Injection directly into the tumor allows the delivery of a higher
concentration of therapeutic agent and avoids the toxicity, which
can result from systemic administration. In addition, intratumoral
injection of bacteria induces robust and localized immune responses
within the tumor.
[0731] Depending on the location, tumor type, and tumor size,
different administration techniques may be used, including but not
limited to, cutaneous, subcutaneous, and percutaneous injection,
therapeutic endoscopic ultrasonography, or endobronchial intratumor
delivery. Prior to the intratumor administration procedures,
sedation in combination with a local anesthetic and standard
cardiac, pressure, and oxygen monitoring, or full anesthesia of the
patient is performed.
[0732] For some tumors, percutaneous injection can be employed,
which is the least invasive administration method. Ultrasound,
computed tomography (CT) or fluoroscopy can be used as guidance to
introduce and position the needle. Percutaneous intratumoral
injection is for example described for hepatocellular carcinoma in
Lencioni et al. (2010) J. Vasc Interv Radiol. 21(10): 1533-8).
Intratumoral injection of cutaneous, subcutaneous, and nodal tumors
is for example described in WO/2014/036412 (Amgen) for late stage
melanoma.
[0733] Single insertion points or multiple insertion points can be
used in percutaneous injection protocols. Using a single insertion
point, the solution may be injected percutaneously along multiple
tracks, as far as the radial reach of the needle allows. In other
embodiments, multiple injection points may be used if the tumor is
larger than the radial reach of the needle. The needle can be
pulled back without exiting, and redirected as often as necessary
until the full dose is injected and dispersed. To maintain
sterility, a separate needle is used for each injection. Needle
size and length varies depending on the tumor type and size.
[0734] In some embodiments, the tumor is injected percutaneously
with an 18-gauge multipronged needle (Quadra-Fuse, Rex Medical).
The device consists of an 18 gauge puncture needle 20 cm in length.
The needle has three retractable prongs, each with four terminal
side holes and a connector with extension tubing clamp. The prongs
are deployed from the lateral wall of the needle. The needle can be
introduced percutaneously into the center of the tumor and can be
positioned at the deepest margin of the tumor. The prongs are
deployed to the margins of the tumor. The prongs are deployed at
maximum length and then are retracted at defined intervals.
Optionally, one or more rotation-injection-rotation maneuvers can
be performed, in which the prongs are retracted, the needle is
rotated by a 60 degrees, which is followed by repeat deployment of
the prongs and additional injection.
[0735] Therapeutic endoscopic ultrasonography (EUS) is employed to
overcome the anatomical constraints inherent in gaining access to
certain other tumors (Shirley et al. (2013) Gastroenterol Res.
Pract. 2013: 207129). EUS-guided fine needle injection (EUS-FNI)
has been successfully used for antitumor therapies for the
treatment of head and neck, esophageal, pancreatic, hepatic, and
adrenal masses (Verna et al. (2008) Therap. Adv Gastroenterol.
1(2): 103-9). EUS-FNI has been extensively used for pancreatic
cancer injections. Fine-needle injection requires the use of the
curvilinear echoendoscope. The esophagus is carefully intubated and
the echoendoscope is passed into the stomach and duodenum where the
pancreatic examination occurs and the target tumor is identified.
The largest plane is measured to estimate the tumor volume and to
calculate the injection volume. The appropriate volume is drawn
into a syringe. A primed 22-gauge fine needle aspiration (FNA)
needle is passed into the working channel of the echoendoscope.
Under ultrasound guidance, the needle is passed into the tumor.
Depending on the size of the tumor, administration can be performed
by dividing the tumor into sections and then injecting the
corresponding fractions of the volume into each section. Use of an
installed endoscopic ultrasound processor with Doppler technology
assures there are no arterial or venous structures that may
interfere with the needle passage into the tumor (Shirley et al.,
2013). In some embodiments, `multiple injectable needle` (MIN) for
EUS-FNI can be used to improvement the injection distribution to
the tumor in comparison with straight-type needles (Ohara et al.
(2013) Mol. Clin. Oncol. 1(2): 231-4).
[0736] Intratumoral administration for lung cancer, such as
non-small cell lung cancer, can be achieved through endobronchial
intratumor delivery methods, as described in Celikoglu et al.,
2008. Bronchoscopy (trans-nasal or oral) is conducted to visualize
the lesion to be treated. The tumor volume can be estimated
visually from visible length-width height measurements over the
bronchial surface. The needle device is then introduced through the
working channel of the bronchoscope. The needle catheter, which
consists of a metallic needle attached to a plastic catheter, is
placed within a sheath to prevent damage by the needle to the
working channel during advancement. The needle size and length
varies and is determined according to tumor type and size of the
tumor. Needles made from plastic are less rigid than metal needles
and are ideal, since they can be passed around sharper bends in the
working channel The needle is inserted into the lesion and the
genetically engineered bacteria of the invention are in injected.
Needles are inserted repeatedly at several insertion points until
the tumor mass is completely perfused. After each injection, the
needle is withdrawn entirely from the tumor and is then embedded at
another location. At the end of the bronchoscopic injection
session, removal of any necrotic debris caused by the treatment may
be removed using mechanical dissection, or other ablation
techniques accompanied by irrigation and aspiration.
[0737] In some embodiments, the genetically engineered bacteria are
administrated directly into the tumor using methods, including but
not limited to, percutaneous injection, EUS-FNI, or endobronchial
intratumor delivery methods. In some cases other techniques, such
as laproscopic or open surgical techniques are used to access the
target tumor, however, these techniques are much more invasive and
bring with them much greater morbidity and longer hospital
stays.
[0738] In some embodiments, bacteria, e.g., E. coli Nissle, or
spores, e.g., Clostridium novyi NT, are disolved in sterile
phosphate buffered saline (PBS) for systemic or intratumor
injection.
[0739] The dose to be injected is derived from the type and size of
the tumor. The dose of a drug or the genetically engineered
bacteria or virus of the invention is typically lower, e.g., orders
of magniture lower, than a dose for systemic intravenous
administration.
[0740] The volume injected into each lesion is based on the size of
the tumor. To obtain the tumor volume, a measurement of the largest
plane can be conducted. The estimated tumor volume can then inform
the determination of the injection volume as a percentage of the
total volume. For example, an injection volume of approximately
20-40% of the total tumor volume can be used.
[0741] For example, as is described, for example, in WO
2014/036412, for tumors larger than 5 cm in their largest
dimension, up to 4 ml can be injected. For tumors between 2.5 and 5
cm in their largest dimension, up to 2 ml can be injected. For
tumors between 2.5 and 5 cm in their largest dimension, up to 2 ml
can be injected. For tumors between 1.5 and 2.5 cm in their largest
dimension, up to 1 ml can be injected. For tumors between 0.5 and
1.5 cm in their largest dimension, up to 0.5 ml can be injected.
For tumors equal or small than 0.5 in their largest dimension, up
to 0.1 ml can be injected. Alternatively, ultrasound scan can be
used to determine the injection volume that can be taken up by the
tumor without leakage into surrounding tissue.
[0742] In some embodiments, the treatment regimen will include one
or more intratumoral administrations. In some embodiments, a
treatment regimen will include an initial dose, which followed by
at least one subsequent dose. One or more doses can be administered
sequentially in two or more cycles.
[0743] For example a first dose may be administered at day 1, and a
second dose may be administered after 1, 2, 3, 4, 5, 6, days or 1,
2, 3, or 4 weeks or after a longer interval. Additional doses may
be administered after 1, 2, 3, 4, 5, 6, days or after 1, 2, 3, or 4
weeks or longer intervals. In some embodiments, the first and
subsequent administrations have the same dosage. In other
embodiments, different doses are administered. In some embodiments,
more than one dose is administered per day, for example, two, three
or more doses can be administered per day.
[0744] The routes of administration and dosages described are
intended only as a guide. The optimum route of administration and
dosage can be readily determined by a skilled practitioner. The
dosage may be determined according to various parameters,
especially according to the location of the tumor, the size of the
tumor, the age, weight and condition of the patient to be treated
and the route and method of administration.
[0745] In one embodiment, Clostridium spores are delivered
systemically. In another embodiment, Clostridium spores are
delivered via intratumor injection. In one embodiment, E. coli
Nissle are delivered via intratumor injection In other embodiments,
E. coli Nissle, which is known to hone to tumors, is administered
via intravenous injection or orally, as described in a mouse model
in for example in Danino et al. 2015, or Stritzker et al., 2007,
the contents of which is herein incorporated by reference in its
entirety. E. coli Nissle mutations to reduce toxicity include but
are not limited to msbB mutants resulting in non-myristoylated LPS
and reduced endotoxin activity, as described in Stritzker et al.,
2010 (Stritzker et al, Bioengineered Bugs 1:2, 139-145;
Myroystoation negative msbB-mutants of probiotic E. coli Nissle
1917 retain tumor specific colonization properties but show less
side effects in immunocompetent mice.
[0746] For intravenous injection a preferred dose of bacteria is
the dose in which the greatest number of bacteria is found in the
tumor and the lowest amount found in other tissues. In mice,
Stritzker et al. (2007) Int. J. Med. Microbiol. 297 (2007) 151-162)
found that the lowest number of bacteria needed for successful
tumor colonization was 2.times.10.sup.4 CFU, in which half of the
mice showed tumor colonization. Injection of 2.times.10.sup.5 and
2.times.10.sup.6 CFU resulted in colonization of all tumors, and
numbers of bacteria in the tumors increased. However, at higher
concentrations, bacterial counts became detectable in the liver and
the spleen.
[0747] In some embodiments, the genetically engineered
microorganisms of the invention may be administered orally. In some
embodiments the genetically engineered bacteria may be useful in
the prevention, treatment or management of liver cancer or liver
metastases. For example, Danino et al. showed that orally
administered E. coli Nissle is able to colonize liver metastases by
crossing the gastrointestinal tract in a mouse model of liver
metastases (Danino et al., Science Translational Medicine 7 (289):
1-10, the contents of which is herein incorporated by reference in
its entirety).
[0748] Tumor types into which the engineered bacteria of the
current invention are intratumorally delivered include locally
advanced and metastatic tumors, including but not limited to, B, T,
and NK cell lymphomas, colon and rectal cancers, melanoma,
including metastatic melanoma, mycosis fungoides, Merkel carcinoma,
liver cancer, including hepatocellular carcinoma and liver
metastasis secondary to colorectal cancer, pancreatic cancer,
breast cancer, follicular lymphoma, prostate cancer, refractory
liver cancer, and Merkel cell carcinoma.
[0749] The genetically engineered microorganisms disclosed herein
may be administered topically and formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form well known to one of
skill in the art. See, e.g., "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa. In an embodiment, for
non-sprayable topical dosage forms, viscous to semi-solid or solid
forms comprising a carrier or one or more excipients compatible
with topical application and having a dynamic viscosity greater
than water are employed. Suitable formulations include, but are not
limited to, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, etc., which may be sterilized or mixed
with auxiliary agents (e.g., preservatives, stabilizers, wetting
agents, buffers, or salts) for influencing various properties,
e.g., osmotic pressure. Other suitable topical dosage forms include
sprayable aerosol preparations wherein the active ingredient in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon) or in a squeeze bottle. Moisturizers or humectants
can also be added to pharmaceutical compositions and dosage forms.
Examples of such additional ingredients are well known in the art.
In one embodiment, the pharmaceutical composition comprising the
recombinant bacteria of the invention may be formulated as a
hygiene product. For example, the hygiene product may be an
antibacterial formulation, or a fermentation product such as a
fermentation broth. Hygiene products may be, for example, shampoos,
conditioners, creams, pastes, lotions, and lip balms.
[0750] The genetically engineered microorganisms disclosed herein
may be administered orally and formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
etc. Pharmacological compositions for oral use can be made using a
solid excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients include, but are not limited to, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
compositions such as maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP) or polyethylene glycol (PEG). Disintegrating agents may also
be added, such as cross-linked polyvinylpyrrolidone, agar, alginic
acid or a salt thereof such as sodium alginate.
[0751] Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone,
hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene
glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and
tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or
calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum,
zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate,
starch, sodium benzoate, L-leucine, magnesium stearate, talc, or
silica); disintegrants (e.g., starch, potato starch, sodium starch
glycolate, sugars, cellulose derivatives, silica powders); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. A coating shell may be
present, and common membranes include, but are not limited to,
polylactide, polyglycolic acid, polyanhydride, other biodegradable
polymers, alginate-polylysine-alginate (APA),
alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),
hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered
HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC),
acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene
glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane
(PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous
encapsulates, cellulose sulphate/sodium
alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate
phthalate, calcium alginate, k-carrageenan-locust bean gum gel
beads, gellan-xanthan beads, poly(lactide-co-glycolides),
carrageenan, starch poly-anhydrides, starch polymethacrylates,
polyamino acids, and enteric coating polymers.
[0752] In some embodiments, the genetically engineered
microorganisms are enterically coated for release into the gut or a
particular region of the gut, for example, the large intestine. The
typical pH profile from the stomach to the colon is about 1-4
(stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon).
In some diseases, the pH profile may be modified. In some
embodiments, the coating is degraded in specific pH environments in
order to specify the site of release. In some embodiments, at least
two coatings are used. In some embodiments, the outside coating and
the inside coating are degraded at different pH levels.
[0753] Liquid preparations for oral administration may take the
form of solutions, syrups, suspensions, or a dry product for
constitution with water or other suitable vehicle before use. Such
liquid preparations may be prepared by conventional means with
pharmaceutically acceptable agents such as suspending agents (e.g.,
sorbitol syrup, cellulose derivatives, or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol, or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated for slow release, controlled release, or
sustained release of the genetically engineered microorganisms
described herein.
[0754] In one embodiment, the genetically engineered microorganisms
of the disclosure may be formulated in a composition suitable for
administration to pediatric subjects. As is well known in the art,
children differ from adults in many aspects, including different
rates of gastric emptying, pH, gastrointestinal permeability, etc.
(Ivanovska et al., Pediatrics, 134(2):361-372, 2014). Moreover,
pediatric formulation acceptability and preferences, such as route
of administration and taste attributes, are critical for achieving
acceptable pediatric compliance. Thus, in one embodiment, the
composition suitable for administration to pediatric subjects may
include easy-to-swallow or dissolvable dosage forms, or more
palatable compositions, such as compositions with added flavors,
sweeteners, or taste blockers. In one embodiment, a composition
suitable for administration to pediatric subjects may also be
suitable for administration to adults.
[0755] In one embodiment, the composition suitable for
administration to pediatric subjects may include a solution, syrup,
suspension, elixir, powder for reconstitution as suspension or
solution, dispersible/effervescent tablet, chewable tablet, gummy
candy, lollipop, freezer pop, troche, chewing gum, oral thin strip,
orally disintegrating tablet, sachet, soft gelatin capsule,
sprinkle oral powder, or granules. In one embodiment, the
composition is a gummy candy, which is made from a gelatin base,
giving the candy elasticity, desired chewy consistency, and longer
shelf-life. In some embodiments, the gummy candy may also comprise
sweeteners or flavors.
[0756] In one embodiment, the composition suitable for
administration to pediatric subjects may include a flavor. As used
herein, "flavor" is a substance (liquid or solid) that provides a
distinct taste and aroma to the formulation. Flavors also help to
improve the palatability of the formulation. Flavors include, but
are not limited to, strawberry, vanilla, lemon, grape, bubble gum,
and cherry.
[0757] In certain embodiments, the genetically engineered
microorganisms may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound may
also be enclosed in a hard or soft shell gelatin capsule,
compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
by other than parenteral administration, it may be necessary to
coat the compound with, or co-administer the compound with, a
material to prevent its inactivation.
[0758] In another embodiment, the pharmaceutical composition
comprising the recombinant bacteria of the invention may be a
comestible product, for example, a food product. In one embodiment,
the food product is milk, concentrated milk, fermented milk
(yogurt, sour milk, frozen yogurt, lactic acid bacteria-fermented
beverages), milk powder, ice cream, cream cheeses, dry cheeses,
soybean milk, fermented soybean milk, vegetable-fruit juices, fruit
juices, sports drinks, confectionery, candies, infant foods (such
as infant cakes), nutritional food products, animal feeds, or
dietary supplements. In one embodiment, the food product is a
fermented food, such as a fermented dairy product. In one
embodiment, the fermented dairy product is yogurt. In another
embodiment, the fermented dairy product is cheese, milk, cream, ice
cream, milk shake, or kefir. In another embodiment, the recombinant
bacteria of the invention are combined in a preparation containing
other live bacterial cells intended to serve as probiotics. In
another embodiment, the food product is a beverage. In one
embodiment, the beverage is a fruit juice-based beverage or a
beverage containing plant or herbal extracts. In another
embodiment, the food product is a jelly or a pudding. Other food
products suitable for administration of the recombinant bacteria of
the invention are well known in the art. For example, see U.S.
2015/0359894 and US 2015/0238545, the entire contents of each of
which are expressly incorporated herein by reference. In yet
another embodiment, the pharmaceutical composition of the invention
is injected into, sprayed onto, or sprinkled onto a food product,
such as bread, yogurt, or cheese.
[0759] In some embodiments, the composition is formulated for
intraintestinal administration, intrajejunal administration,
intraduodenal administration, intraileal administration, gastric
shunt administration, or intracolic administration, via
nanoparticles, nanocapsules, microcapsules, or microtablets, which
are enterically coated or uncoated. The pharmaceutical compositions
may also be formulated in rectal compositions such as suppositories
or retention enemas, using, e.g., conventional suppository bases
such as cocoa butter or other glycerides. The compositions may be
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain suspending, stabilizing and/or dispersing
agents.
[0760] The genetically engineered microorganisms described herein
may be administered intranasally, formulated in an aerosol form,
spray, mist, or in the form of drops, and conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
Pressurized aerosol dosage units may be determined by providing a
valve to deliver a metered amount. Capsules and cartridges (e.g.,
of gelatin) for use in an inhaler or insufflator may be formulated
containing a powder mix of the compound and a suitable powder base
such as lactose or starch.
[0761] The genetically engineered microorganisms may be
administered and formulated as depot preparations. Such long acting
formulations may be administered by implantation or by injection,
including intravenous injection, subcutaneous injection, local
injection, direct injection, or infusion. For example, the
compositions may be formulated with suitable polymeric or
hydrophobic materials (e.g., as an emulsion in an acceptable oil)
or ion exchange resins, or as sparingly soluble derivatives (e.g.,
as a sparingly soluble salt).
[0762] In some embodiments, disclosed herein are pharmaceutically
acceptable compositions in single dosage forms. Single dosage forms
may be in a liquid or a solid form. Single dosage forms may be
administered directly to a patient without modification or may be
diluted or reconstituted prior to administration. In certain
embodiments, a single dosage form may be administered in bolus
form, e.g., single injection, single oral dose, including an oral
dose that comprises multiple tablets, capsule, pills, etc. In
alternate embodiments, a single dosage form may be administered
over a period of time, e.g., by infusion.
[0763] Single dosage forms of the pharmaceutical composition may be
prepared by portioning the pharmaceutical composition into smaller
aliquots, single dose containers, single dose liquid forms, or
single dose solid forms, such as tablets, granulates,
nanoparticles, nanocapsules, microcapsules, microtablets, pellets,
or powders, which may be enterically coated or uncoated. A single
dose in a solid form may be reconstituted by adding liquid,
typically sterile water or saline solution, prior to administration
to a patient.
[0764] In other embodiments, the composition can be delivered in a
controlled release or sustained release system. In one embodiment,
a pump may be used to achieve controlled or sustained release. In
another embodiment, polymeric materials can be used to achieve
controlled or sustained release of the therapies of the present
disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of
polymers used in sustained release formulations include, but are
not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl
methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The
polymer used in a sustained release formulation may be inert, free
of leachable impurities, stable on storage, sterile, and
biodegradable. In some embodiments, a controlled or sustained
release system can be placed in proximity of the prophylactic or
therapeutic target, thus requiring only a fraction of the systemic
dose. Any suitable technique known to one of skill in the art may
be used.
[0765] Dosage regimens may be adjusted to provide a therapeutic
response. Dosing can depend on several factors, including severity
and responsiveness of the disease, route of administration, time
course of treatment (days to months to years), and time to
amelioration of the disease. For example, a single bolus may be
administered at one time, several divided doses may be administered
over a predetermined period of time, or the dose may be reduced or
increased as indicated by the therapeutic situation. The
specification for the dosage is dictated by the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved. Dosage values may vary with the
type and severity of the condition to be alleviated. For any
particular subject, specific dosage regimens may be adjusted over
time according to the individual need and the professional judgment
of the treating clinician. Toxicity and therapeutic efficacy of
compounds provided herein can be determined by standard
pharmaceutical procedures in cell culture or animal models. For
example, LD.sub.50, ED.sub.50, EC.sub.50, and IC.sub.50 may be
determined, and the dose ratio between toxic and therapeutic
effects (LD.sub.50/ED.sub.50) may be calculated as the therapeutic
index. Compositions that exhibit toxic side effects may be used,
with careful modifications to minimize potential damage to reduce
side effects. Dosing may be estimated initially from cell culture
assays and animal models. The data obtained from in vitro and in
vivo assays and animal studies can be used in formulating a range
of dosage for use in humans
[0766] The ingredients are supplied either separately or mixed
together in unit dosage form, for example, as a dry lyophilized
powder or water-free concentrate in a hermetically sealed container
such as an ampoule or sachet indicating the quantity of active
agent. If the mode of administration is by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0767] The pharmaceutical compositions may be packaged in a
hermetically sealed container such as an ampoule or sachet
indicating the quantity of the agent. In one embodiment, one or
more of the pharmaceutical compositions is supplied as a dry
sterilized lyophilized powder or water-free concentrate in a
hermetically sealed container and can be reconstituted (e.g., with
water or saline) to the appropriate concentration for
administration to a subject. In an embodiment, one or more of the
prophylactic or therapeutic agents or pharmaceutical compositions
is supplied as a dry sterile lyophilized powder in a hermetically
sealed container stored between 2.degree. C. and 8.degree. C. and
administered within 1 hour, within 3 hours, within 5 hours, within
6 hours, within 12 hours, within 24 hours, within 48 hours, within
72 hours, or within one week after being reconstituted.
Cryoprotectants can be included for a lyophilized dosage form,
principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable
cryoprotectants include trehalose and lactose. Other suitable
bulking agents include glycine and arginine, either of which can be
included at a concentration of 0-0.05%, and polysorbate-80
(optimally included at a concentration of 0.005-0.01%). Additional
surfactants include but are not limited to polysorbate 20 and BRIJ
surfactants. The pharmaceutical composition may be prepared as an
injectable solution and can further comprise an agent useful as an
adjuvant, such as those used to increase absorption or dispersion,
e.g., hyaluronidase.
[0768] In some embodiments, the genetically engineered
microorganisms and composition thereof is formulated for
intravenous administration, intratumor administration, or peritumor
administration. The genetically engineered microorganisms may be
formulated as depot preparations. Such long acting formulations may
be administered by implantation or by injection. For example, the
compositions may be formulated with suitable polymeric or
hydrophobic materials (e.g., as an emulsion in an acceptable oil)
or ion exchange resins, or as sparingly soluble derivatives (e.g.,
as a sparingly soluble salt).
[0769] In another embodiment, the composition can be delivered in a
controlled release or sustained release system. In one embodiment,
a pump may be used to achieve controlled or sustained release. In
another embodiment, polymeric materials can be used to achieve
controlled or sustained release of the therapies of the present
disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of
polymers used in sustained release formulations include, but are
not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl
methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The
polymer used in a sustained release formulation may be inert, free
of leachable impurities, stable on storage, sterile, and
biodegradable. In some embodiments, a controlled or sustained
release system can be placed in proximity of the prophylactic or
therapeutic target, thus requiring only a fraction of the systemic
dose. Any suitable technique known to one of skill in the art may
be used.
[0770] The genetically engineered bacteria of the invention may be
administered and formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions
such as those derived from hydrochloric, phosphoric, acetic,
oxalic, tartaric acids, etc., and those formed with cations such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0771] Methods of Treatment
[0772] In one aspect of the invention provides methods of treating
a disease, disorder and/or a symptom of a disease or disorder
described herein. In one aspect aspect of the invention provides
methods of treating cancer. In some embodiments, the invention
provides methods for reducing, ameliorating, or eliminating one or
more symptom(s) associated with cancer. In some embodiments, the
cancer is selected from adrenal cancer, adrenocortical carcinoma,
anal cancer, appendix cancer, bile duct cancer, bladder cancer,
bone cancer (e.g., Ewing sarcoma tumors, osteosarcoma, malignant
fibrous histiocytoma), brain cancer (e.g., astrocytomas, brain stem
glioma, craniopharyngioma, ependymoma), bronchial tumors, central
nervous system tumors, breast cancer, Castleman disease, cervical
cancer, colon cancer, rectal cancer, colorectal cancer, endometrial
cancer, esophageal cancer, eye cancer, gallbladder cancer,
gastrointestinal cancer, gastrointestinal carcinoid tumors,
gastrointestinal stromal tumors, gestational trophoblastic disease,
heart cancer, Kaposi sarcoma, kidney cancer, largyngeal cancer,
hypopharyngeal cancer, leukemia (e.g., acute lymphoblastic
leukemia, acute myeloid leukemia, chronic lymphocytic leukemia,
chronic myelogenous leukemia), liver cancer, lung cancer, lymphoma
(e.g., AIDS-related lymphoma, Burkitt lymphoma, cutaneous T cell
lymphoma, Hogkin lymphoma, Non-Hogkin lymphoma, primary central
nervous system lymphoma), malignant mesothelioma, multiple myeloma,
myelodysplastic syndrome, nasal cavity cancer, paranasal sinus
cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer,
oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic
cancer, penile cancer, pituitary tumors, prostate cancer,
retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland
cancer, sarcoma, skin cancer (e.g., basal cell carcinoma,
melanoma), small intestine cancer, stomach cancer, teratoid tumor,
testicular cancer, throat cancer, thymus cancer, thyroid cancer,
unusual childhood cancers, urethral cancer, uterine cancer, uterine
sarcoma, vaginal cancer, vulvar cancer, Waldenstrom
macrogloblulinemia, and Wilms tumor. In some embodiments, the
symptom(s) associated thereof include, but are not limited to,
anemia, loss of appetite, irritation of bladder lining, bleeding
and bruising (thrombocytopenia), changes in taste or smell,
constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair
loss (alopecia), infection, infertility, lymphedema, mouth sores,
nausea, pain, peripheral neuropathy, tooth decay, urinary tract
infections, and/or problems with memory and concentration.
[0773] The method may comprise preparing a pharmaceutical
composition with at least one genetically engineered species,
strain, or subtype of bacteria described herein, and administering
the pharmaceutical composition to a subject in a therapeutically
effective amount. The genetically engineered microorganisms may be
administered locally, e.g., intratumorally or peritumorally into a
tissue or supplying vessel, or systemically, e.g., intravenously by
infusion or injection. In some embodiments, the genetically
engineered bacteria are administered intravenously, intratumorally,
intra-arterially, intramuscularly, intraperitoneally, orally, or
topically. In some embodiments, the genetically engineered
microorganisms are administered intravenously, i.e.,
systemically.
[0774] In certain embodiments, administering the pharmaceutical
composition to the subject reduces cell proliferation, tumor
growth, and/or tumor volume in a subject. In some embodiments, the
methods of the present disclosure may reduce cell proliferation,
tumor growth, and/or tumor volume by at least about 10%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as
compared to levels in an untreated or control subject. In some
embodiments, reduction is measured by comparing cell proliferation,
tumor growth, and/or tumor volume in a subject before and after
administration of the pharmaceutical composition. In some
embodiments, the method of treating or ameliorating a cancer in a
subject allows one or more symptoms of the cancer to improve by at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
more.
[0775] Before, during, and after the administration of the
pharmaceutical composition, cancerous cells and/or biomarkers in a
subject may be measured in a biological sample, such as blood,
serum, plasma, urine, peritoneal fluid, and/or a biopsy from a
tissue or organ. In some embodiments, the methods may include
administration of the compositions of the invention to reduce tumor
volume in a subject to an undetectable size, or to less than about
1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or
90% of the subject's tumor volume prior to treatment. In other
embodiments, the methods may include administration of the
compositions of the invention to reduce the cell proliferation rate
or tumor growth rate in a subject to an undetectable rate, or to
less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,
75%, 80%, or 90% of the rate prior to treatment.
[0776] The genetically engineered bacteria may be destroyed, e.g.,
by defense factors in tissues or blood serum (Sonnenborn et al.
(2009) Microbial Ecology in Health and Disease 21: 122-58), or by
activation of a kill switch, several hours or days after
administration. Thus, the pharmaceutical composition comprising the
genetically engineered bacteria comprising a heterologous gene
encoding a substrate transporter may be re-administered at a
therapeutically effective dose and frequency. In alternate
embodiments, the genetically engineered bacteria are not destroyed
within hours or days after administration and may propagate and
colonize the tumor.
[0777] The pharmaceutical composition may be administered alone or
in combination with one or more additional therapeutic agents,
e.g., a chemotherapeutic drug such a methotrexate. An important
consideration in selecting the one or more additional therapeutic
agents is that the agent(s) should be compatible with the
genetically engineered bacteria of the invention, e.g., the
agent(s) must not kill the bacteria. In some studies, the efficacy
of anticancer immunotherapy, e.g., CTLA-4 or PD-1 inhibitors,
requires the presence of particular bacterial strains in the
microbiome (Ilda et al., 2013; Vetizou et al., 2015; Sivan et al.,
2015). In some embodiments, the pharmaceutical composition is
administered with one or more commensal or probiotic bacteria,
e.g., Bifidobacterium or Bacteroides.
[0778] In some embodiments, the genetically engineered
microorganisms may be administered as part of a regimen, which
includes other treatment modalities or combinations of other
modalities. Non-limiting examples of these modalities or agents are
conventional therapies (e.g., radiotherapy, chemotherapy), other
immunotherapies, stem cell therapies, and targeted therapies,
(e.g., BRAF or vascular endothelial growth factor inhibitors;
antibodies or compounds), bacteria described herein, and oncolytic
viruses. Therapies also include related to antibody-immune
engagement, including Fc-mediated ADCC therapies, therapies using
bispecific soluble scFvs linking cytotoxic T cells to tumor cells
(e.g., BiTE), and soluble TCRs with effector functions.
Immunotherapies include vaccines (e.g., viral antigen, tumor
associated antigen, neoantigen, or combinations thereof),
checkpoint inhibitors, cytokine therapies, adoptive cellular
therapy (ACT). ACT includes but is not limited to, tumor
infiltrating lymphocyte (TIL) therapies, native or engineered TCR
or CAR-T therapies, natural killer cell therapies, and dendritic
cell vaccines or other vaccines of other antigen presenting cells.
Targeted therapies include antibodies and chemical compounds, and
include for example antiangiogenic strategies and BRAF
inhibition.
[0779] The immunostimulatory activity of bacterial DNA is mimicked
by synthetic oligodeoxynucleotides (ODNs) expressing unmethylated
CpG motifs (see, e.g., Bode et al. (2011) Expert Rev Vaccines
10(4): 499-511). CpG DNA as a vaccine adjuvant. When used as
vaccine adjuvants, CpG ODNs improve the function of professional
antigen-presenting cells and boost the generation of humoral and
cellular vaccine-specific immune responses. In some embodiments,
CpG can be administered in combination with the genetically
engineered baceteria of the invention.
[0780] In one embodiment, the genetically engineered micororganisms
are administered in combination with tumor cell lysates.
[0781] The dosage of the pharmaceutical composition and the
frequency of administration may be selected based on the severity
of the symptoms and the progression of the cancer. The appropriate
therapeutically effective dose and/or frequency of administration
can be selected by a treating clinician.
[0782] Treatment In Vivo
[0783] The genetically engineered bacteria may be evaluated in
vivo, e.g., in an animal model. Any suitable animal model of a
disease or condition associated with cancer may be used, e.g., a
tumor syngeneic or xenograft mouse models (see, e.g., Yu et al.,
2015). The genetically engineered bacteria may be administered to
the animal systemically or locally, e.g., via oral administration
(gavage), intravenous, or subcutaneous injection or via
intratumoral injection, and treatment efficacy determined, e.g., by
measuring tumor volume.
[0784] Non-limiting examples of animal models include mouse models,
as described in Dang et al., 2001, Heap et al., 2014 and Danino et
al., 2015).
[0785] Pre-clinical mouse models determine which immunotherapies
and combination immunotherapies will generate the optimal
therapeutic index (maximal anti-tumor efficacy and minimal immune
related adverse events (irAEs)) in different cancers.
[0786] Implantation of cultured cells derived from various human
cancer cell types or a patient's tumor mass into mouse tissue sites
has been widely used for generations of cancer mouse models
(xenograft modeling). In xenograft modeling, human tumors or cell
lines are implanted either subcutaneously or orthotopically into
immune-compromised host animals (e.g., nude or SCID mice) to avoid
graft rejection. Because the original human tumor microenvironment
is not recapitulated in such models, the activity of anti-cancer
agents that target immune modulators may not be accurately measured
in these models, making mouse models with an intact immune system
more desirable.
[0787] Accordingly, implantation of murine cancer cells in a
syngeneic immunocompetent host (allograft) are used to generate
mouse models with tumor tissues derived from the same genetic
background as a given mouse strain. In syngeneic models, the host
immune system is normal, which may more closely represent the real
life situation of the tumor's micro-environment. The tumor cells or
cancer cell lines are implanted either subcutaneously or
orthotopically into the syngeneic immunocompetent host animal
(e.g., mouse). Representative murine tumor cell lines, which can be
used in syngeneic mouse models for immune checkpoint benchmarking
include, but are not limited to the cell lines listed in Table
32.
TABLE-US-00009 TABLE 32 Selected cell lines for use in syngeneic
mouse models Cancer Types Cell LInes Bladder MBT-2 Breast 4T1,
EMT6, JC Colon CT-26, Colon26, MC38 Kidney Renca Leukemia L1210,
C1498 Mastocytoma P815 P815 Neuroblastoma Neuro-2-A Neuro-2a
Myeloma MPC-11 Liver H22 Lung LL/2, KLN205 Lymphoma A20, EL4,
P388D1, L15178-R, E.G7- OVA Melanoma B16-BL6, B16-F10, S91
Pancreatic Pan02 Prostate RM-1 Fibrosarcom WHI-164 Plasmacytoma
J558
[0788] Additional cell lines include, but are not limited to those
in Table 33, which are described with respect to CTLA-4
benchmarking in Joseph F. Grosso and Maria N. Jure-Kunkel et al.,
2013, the contents of which is herein incorporated by reference in
its entirety.
TABLE-US-00010 TABLE 33 Murine cell lines and CTLA-4 antibodies for
syngenic mouse models Murine Tumor type/Mouse Anti-CTLA-4 Ab/Tx
Tumor strain regimen Brain SMA-560 9H10; d7* (100 .mu.g), d10
Glioma/Vm/Dk) (50 .mu.g), d13 (50 .mu.g) post- implant GL-261 9H10;
d0 (100 .mu.g), d3 (50 .mu.g), Glioma/C57BL/6) d6 (50 .mu.g),
Ovarian OV-HM/C57BL/6 .times. UC10-4F10-11; 1 mg/mouse C3H/He)
Bladder MB49/C57BL/6 9D9; d7, d10, d13 (200 .mu.g each) Sarcoma
Meth-A/BALB/c 9H10; d6 (100 .mu.g), d9 (50 .mu.g), d12 (50 .mu.g)
MC38, 11A1 9H10; d14 (100 .mu.g), d17 BALB/c, C57BL/6 (50 .mu.g),
d20 (50 .mu.g) Breast TSA/BALB/c (62 9H10; d12, d14, d16 (200 .mu.g
each) 4T1 BALB/c 9H10; d14, d18, d21 (200 .mu.g each) 4T1 BALB/c
9H10; d14, d18, d21 (200 .mu.g each) 4T1 BALB/c UC10-4F10-11; d7,
d11, d15, d19 (100 .mu.g each) SM1/BALB/c 9H10; d4, d7, d10 (100
.mu.g each) EMT6/BALB/c UC10-4F10-11; d4, d8, d12 (400 .mu.g each)
Ixa: d3, d7, d11 Colon MC38/C57BL/6 UC10-4F10-11; d7, d11, d16 (100
.mu.g each) MC38 K4G4, L1B11, L3D10 CT26 BALB/c 9H10; d10 (100
.mu.g), d13 (50 .mu.g), d15 (50 .mu.g) CT26 BALB/c UC10-4F10-11;
d5, d9, d13 (400 .mu.g each) Ixa: d4, d8, d12 MC38/C57BL/6
UC10-4F10-11; d14, d21, d28 (800 .mu.g each) Lymphoma BW5147.3/AKR
UC10-4F10-11; d-1 (250 .mu.g), d0 (250 .mu.g), d4 [100 .mu.g), d8
(100 .mu.g), dl2 (100 .mu.g) EL4/C57BL/6 9H10; d3, d5 (100 .mu.g
each) Fibrosarcoma SA1N/A/J 9H10; every 4 days (200 .mu.g each)
SA1N UC10-4F10-11; d12, d16, d20 (400 .mu.g each) Ixa: d11, d15,
d15 Prostata TRAMP 9H10; d7, d10, d13 (100 .mu.g C1[pTC1]/C57BU6
each) TRAMP 9H10; d4, d7, d10 (100 .mu.g C2/C57BL/6 each)
TRAMP/C57BL 9H10; 14-16 week old mice d7, d10, d16 post-tR tx (100
.mu.g each) TRAMP 9H10; d29, d33, d40, d50 C2/C57BL/6 (100 .mu.g
each) d29 = 1d post- cryoablation Melanoma B16/C57BL/6 9H10; d0,
d3, d6 (200 .mu.g each) B16/C57BL/6 9H10; d6 (100 .mu.g), d8 [50
.mu.g), d10 (50 .mu.g) B16/C57BL/6 9D9; d3, d6, d9 B16/C57BL/6
9H10; d3, d6, d9 (100 .mu.g each) B16.F10/C57BL/6 9H10; d5 (100
.mu.g), d7 (50 .mu.g), d9 (50 .mu.g) Lung M109/BALB/c UC10-4F10-11;
d4, d8, d12(400 .mu.g each) Ixa: d3, d7, d11 Plasmacytoma
MOPC-315/BALB/ UC10-4F10-11; 20 mm cANnCrlBr tumors tx daily for 10
days (100 .mu.g each)
[0789] For tumors derived from certain cell lines, ovalbumin can be
added to further stimulate the immune response, thereby increasing
the response baseline level.
[0790] Examples of mouse strains that can be used in syngeneic
mouse models, depending on the cell line include C57BL/6, FVB/N,
Balb/c, C3H, HeJ, C3H/HeJ, NOD/ShiLT, A/J, 129S1/SvlmJ, NOD.
Additionally, several further genetically engineered mouse strains
have been reported to mimic human tumorigenesis at both molecular
and histologic levels. These genetically engineered mouse models
also provide excellent tools to the field and additionally, the
cancer cell lines derived from the invasive tumors developed in
these models are also good resources for cell lines for syngeneic
tumor models Examples of genetically engineered strains are
provided in Table 34.
TABLE-US-00011 TABLE 34 Exemplary genetic engineered mouse strains
of interest Animal strain Strain background Predicted cancer type
C57BL/6- C57BL/6 Prostate cancer Tg(TRAMP)8247Ng/JNju
FVB/N-Tg.quadrature.MMTV- FVB/N Breast cancer PyVT)634Mul/Jnju
C57BL/6J-Apc.sup.Min/JNju C57BL/6 Colorectal cancer STOCK
Ptch1.sup.tm1Mps/JNju C57BL/6JNju Medulloblastoma
NOD-Prkdc.sup.em26Cd52Il2rg.sup.em26Cd22Nju NOD/ShiLt Not specific
C57BL/6J-Apc.sup.Min/JNju C57BL/6 Colorectal cancer BALB/cJNju
BALB/c Lung cancer C3H/HeJNju (Urethane C3H/HeJ Lung cancer induced
lung cancer model) A/JNju A/J Lung cancer A/Jnju (Urethane induced
A/J Lung cancer lung cancer model) C3H/HeJSlac C3H/HeJ Lung cancer
129S1/SvImJNju (Urethane 129S1/SvImJ Lung cancer induced lung
cancer model) Kras.sup.LSL-G12D/WT C57BL/6 Lung cancer
Kras.sup.LSL-G12D/WT;P53.sup.KO/KO C57BL/6 Lung cancer Pdx1-cre;
Kras.sup.LSL-G12D/WT;P53.sup.KO/KO C57BL/6 Pancreatic cancer
Kras.sup.LSL-G12D/WT;P16.sup.KO/KO C57BL/6; Pancreaticc cancer;
FVB/N Lung cancer Kras.sup.LSL-G12D/WT; PTEN.sup.CKO/CKO C57BL/6
Ovarian cancer; Prostate cancer; Brain cancer
Pbsn-cre;Kras.sup.LSL-G12D/WT;PTEN.sup.CKO/CKO C57BL/6 Prostate
cancer P53.sup.KO/KO;PTEN.sup.CKO/CKO C57BL/6 Prostate cancer
Pbsn-cre;PTEN.sup.CKO/CKO C57BL/6 Prostate cancer NOD NOD Leukemia
B6.Cg- C57BL/6 B cell Lymphoma Tg(IghMyc)22Bri/JNju
PTEN.sup.CKO/CKO C57BL/6 Ovarian cancer (Female); Prostate cancer
(Male); Tes/s cancer (Male) NASH-HCC (Streptozotocin C57BL/6
Hepatocellular and high-fat diet induced liver Carcinoma cancer
model) BALB/c nude BALB/c Not specific C3H/He C3H/He Hepatocellular
Carcinoma B6N C57BL/6 Not specific B6/N-Akr1c12.sup.tm1aNju C57BL/6
Not specific P53 null from VitalStar C57BL/6 Not specific P53 null
from VitalStar C57BL/6 Not specific P53 null from VitalStar C57BL/6
Not specific Pdx1-cre;Kras.sup.LSL-G12D/WT;p53.sup.KO/KO C57BL/6
Pancrea/c cancer Kras.sup.LSL-G12D/WT;P16.sup.KO/KO C57BL/6;
Pancrea/c cancer; FVB/N Lung cancer
Kras.sup.LSL-G12D/WT;PTEN.sup.CKO/CKO C57BL/6 Ovarian cancer;
Kras.sup.LSL-G12D/WT;PTEN.sup.CKO/CKO C57BL/6 Prostate cancer;
Kras.sup.LSL-G12D/WT;PTEN.sup.CKO/CKO C57BL/6 Brain cancer
Pbsn-cre; Kras.sup.LSL-G12D/WT;PTEN.sup.CKO/CKO C57BL/6 Prostate
cancer P53.sup.KO/KO;PTEN.sup.CKO/CKO C57BL/6 Prostate cancer
Pbsn-cre;PTEN.sup.CKO/CKO C57BL/6 Prostate cancer
Kras.sup.LSL-G12D/WT C57BL/6 Lung cancer NOD NOD Leukemia B6.Cg-
C57BL/6 B cell Lymphoma Tg(IghMyc)22Bri/JNju PTENCKO/CKO C57BL/6
Ovarian cancer (Female); Prostate cancer (Male); Tes/s cancer
(Male) NASH-HCC (Streptozotocin C57BL/6 Hepatocellular and high-fat
diet induced Carcinoma liver cancer model) BALB/c nude BALB/c Not
specific C3H/He C3H/He Hepatocellular Carcinoma B6N C57BL/6 Not
specific B6/N-Akr1c12.sup.tm1aNju C57BL/6 Not specific P53 null
from VitalStar C57BL/6 Not specific P53 null from VitalStar C57BL/6
Not specific P53 null from VitalStar C57BL/6 Not specific
Kras.sup.LSL-G12D/WT;P53.sup.KO/KO C57BL/6 Not specific
[0791] Often antibodies directed against human proteins do not
detect their murine counterparts. In studying antibodies, including
those directed against human immune checkpoint molecules, it is
necessary to take this in consideration. For example, Ipilimumab
did not show cross-reactivity with or binding to CTLA-4 from rats,
mice or rabbits.
[0792] In some cases, mice transgenic for the gene of interest can
used to overcome this issue, as was done for ipilimumab. However,
in syngeneic mouse models without a human transgene, mouse protein
reactive antibodies must be used to test therapeutic antibody
strategies. For example, suitable CTLA-4 antibodies for expression
by the genetically engineered bacteria of interest include, but are
not limited to, 9H10, UC10-4F10-11, 9D9, and K4G4 (Table 33).
[0793] More recently, "humanized" mouse models have been developed,
in which immunodeficient mice are reconstituted with a human immune
system, and which have helped overcome issues relating to the
differences between the mouse and human immune systems, allowing
the in vivo study of human immunity. Severely immunodeficient mice
which combine the IL2receptor null and the severe combined immune
deficiency mutation (scid) (NOD-scid IL2Rgnull mice) lack mature T
cells, B cells, or functional NK cells, and are deficient in
cytokine signaling. These mice can be engrafted with human
hematopoietic stem cells and peripheral-blood mononuclear cells.
CD34+ hematopoietic stem cells (hu-CD34) are injected into the
immune deficient mice, resulting in multi-lineage engraftment of
human immune cell populations including very good T cell maturation
and function for long-term studies. This model has a research span
of 12 months with a functional human immune system displaying
T-cell dependent inflammatory responses with no donor cell immune
reactivity towards the host. Patient derived xenografts can readily
be implanted in these models and the effects of immune modulatory
agents studied in an in vivo setting more reflective of the human
tumor microenvironment (both immune and non-immune cell-based)
(Baia et al., 2015).
[0794] Human cell lines of interest for use in the humanized mouse
models include but are not limited to HCT-116 and HT-29 colon
cancer cell lines.
[0795] A rat F98 glioma model and the utility of spontaneous canine
tumors, as described in Roberts et al 2014, the contents of each of
which are herein incorporated by reference in their entireties.
Locally invasive tumors generated by implantation of F98 rat glioma
cells engineered to express luciferase were intratumorally injected
with C. novyi-NT spores, resulting in germination and a rapid fall
in luciferase activity. C. novyi-NT germination was demonstrated by
the appearance of vegetative forms of the bacterium. In these
studies, C. novyi-NT precisely honed to the tumor sparing
neighboring cells.
[0796] Canine soft tissue sarcomas for example are common in many
breeds and have clinical, histopathological, and genetically
features similar to those in humans (Roberts et al, 2014; Staedtke
et al., 2015), in particular, in terms of genetic alterations and
spectrum of mutations. Roberts et al. conducted a study in dogs, in
which C. novyi-NT spores were intrtatumorally injected
(1.times.10.sup.8 C. novyi-NT spores) into spontaneously occurring
solid tumors in one to 4 treatment cycles and followed for 90 days.
A potent inflammatory response was observed, indicating that the
intrattumoral injections mounted an innate immune response.
[0797] In some embodiments, the genetically engineered
microorganisms of the invention are administered systemically,
e.g., orally, subcutaneously, intraveneously or intratumorally into
any of the models described herein to assess anti-tumor efficacy
and any treatment related adverse side effects.
EXAMPLES:
Example 1
Phenylalanine Transporter--Integration of PheP into the Bacterial
Chromosome
[0798] In some embodiments, it may be advantageous to increase
phenylalanine transport into the cell, thereby enhancing
phenylalanine metabolism. Therefore, a second copy of the native
high affinity phenylalanine transporter, PheP, driven by an
inducible promoter, was inserted into the Nissle genome through
homologous recombination. Organization of the construct is shown in
FIG. 4. The pheP gene was placed downstream of the P.sub.tet
promoter, and the tetracycline repressor, TetR, was divergently
transcribed (see, e.g., FIG. 4). This sequence was synthesized by
Genewiz (Cambridge, Mass.). To create a vector capable of
integrating the synthesized TetR-PheP construct into the
chromosome, Gibson assembly was first used to add 1000 bp sequences
of DNA homologous to the Nissle lacZ locus into the R6K origin
plasmid pKD3. This targets DNA cloned between these homology arms
to be integrated into the lacZ locus in the Nissle genome. Gibson
assembly was used to clone the TetR-PheP fragment between these
arms. PCR was used to amplify the region from this plasmid
containing the entire sequence of the homology arms, as well as the
pheP sequence between them. This PCR fragment was used to transform
electrocompetent Nissle-pKD46, a strain that contains a
temperature-sensitive plasmid encoding the lambda red recombinase
genes. After transformation, cells were grown for 2 hrs before
plating on chloramphenicol at 20 .mu.g/mL at 37.degree. C. Growth
at 37.degree. C. cures the pKD46 plasmid. Transformants containing
anhydrous tetracycline (ATC)-inducible pheP were lac-minus (lac-)
and chloramphenicol resistant.
Example 2
Effect of the Phenylalanine Transporter on Phenylalanine
Degradation
[0799] To determine the effect of the phenylalanine transporter on
phenylalanine degradation, phenylalanine degradation and
trans-cinnamate accumulation achieved by genetically engineered
bacteria expressing PAL1 or PAL3 on low-copy (LC) or high-copy (HC)
plasmids in the presence or absence of a copy of pheP driven by the
Tet promoter integrated into the chromosome was assessed.
[0800] For in vitro studies, all incubations were performed at
37.degree. C. Cultures of E. coli Nissle transformed with a plasmid
comprising the PAL gene driven by the Tet promoter were grown
overnight and then diluted 1:100 in LB. The cells were grown with
shaking (200 rpm) to early log phase. Anhydrous tetracycline (ATC)
was added to cultures at a concentration of 100 ng/mL to induce
expression of PAL, and bacteria were grown for another 2 hrs.
Bacteria were then pelleted, washed, and resuspended in minimal
media, and supplemented with 4 mM phenylalanine. Aliquots were
removed at 0 hrs, 2 hrs, and 4 hrs for phenylalanine quantification
(FIG. 8A), and at 2 hrs and 4 hrs for cinnamate quantification
(FIG. 8B), by mass spectrometry, as described in Examples 24-26. As
shown in FIG. 8, expression of pheP in conjunction with PAL
significantly enhances the degradation of phenylalanine as compared
to PAL alone or pheP alone. Notably, the additional copy of pheP
permitted the complete degradation of phenylalanine (4 mM) in 4 hrs
(FIG. 8A). FIG. 8B depicts cinnamate levels in samples at 2 hrs and
4 hrs post-induction. Since cinnamate production is directly
correlated with phenylalanine degradation, these data suggest that
phenylalanine disappearance is due to phenylalanine catabolism, and
that cinnamate may be used as an alternative biomarker for strain
activity. PheP overexpression improves phenylalanine metabolism in
engineered bacteria.
[0801] In conclusion, in conjunction with pheP, even low-copy
PAL-expressing plasmids are capable of almost completely
eliminating phenylalanine from a test sample (FIGS. 8A and 8B).
Furthermore, without wishing to be bound by theory, in some
embodiments, that incorporate pheP, there may be additional
advantages to using a low-copy PAL-expressing plasmid in
conjunction in order to enhance the stability of PAL expression
while maintaining high phenylalanine metabolism, and to reduce
negative selection pressure on the transformed bacterium. In
alternate embodiments, the phenylalanine transporter is used in
conjunction with a high-copy PAL-expressing plasmid.
Example 3
Phenylalanine Degradation in Recombinant E. Coli with and without
pheP Overexpression
[0802] The SYN-PKU304 and SYN-PKU305 strains contain low-copy
plasmids harboring the PAL3 gene, and a copy of pheP integrated at
the lacZ locus. The SYN-PKU308 and SYN-PKU307 strains also contain
low-copy plasmids harboring the PAL3 gene, but lack a copy of pheP
integrated at the lacZ locus. In all four strains, expression of
PAL3 and pheP (when applicable) is controlled by an oxygen
level-dependent promoter.
[0803] To determine rates of phenylalanine degradation in
engineered E. coli Nissle with and without pheP on the chromosome,
overnight cultures of SYN-PKU304 and SYN-PKU307 were diluted 1:100
in LB containing ampicillin, and overnight cultures of SYN-PKU308
and SYN-PKU305 were diluted 1:100 in LB containing kanamycin. All
strains were grown for 1.5 hrs before cultures were placed in a Coy
anaerobic chamber supplying 90% N.sub.2, 5% CO.sub.2, and 5%
H.sub.2. After 4 hrs of induction, bacteria were pelleted, washed
in PBS, and resuspended in 1 mL of assay buffer. Assay buffer
contained M9 minimal media supplemented with 0.5% glucose, 8.4%
sodium bicarbonate, and 4 mM of phenylalanine.
[0804] For the activity assay, starting counts of colony-forming
units (cfu) were quantified using serial dilution and plating.
Aliquots were removed from each cell assay every 30 min for 3 hrs
for phenylalanine quantification by mass spectrometry.
Specifically, 150 .mu.L of bacterial cells were pelleted and the
supernatant was harvested for LC-MS analysis, with assay media
without cells used as the zero-time point. FIG. 10 shows the
observed phenylalanine degradation for strains with pheP on the
chromosome (SYN-PKU304 and SYN-PKU305; left), as well as strains
lacking pheP on the chromosome (SYN-PKU308 and SYN-PKU307; right).
These data show that pheP overexpression is important in order to
increase rates of phenylalanine degradation in synthetic
probiotics.
[0805] Strains Used in the Experiments
TABLE-US-00012 PAL Activity (umol/hr/10{circumflex over ( )}9
Strain Name Strain Name Genotype cells) SYN025 SYN-PKU101 Low copy
pSC101- ND Ptet::PAL1, ampicillin resistant SYN026 SYN-PKU102 High
copy pColE1- ND Ptet::PAL1, ampicillin resistant, SYN065 SYN-PKU201
Low copy pSC101- ND Ptet::PAL3, ampicillin resistant SYN063
SYN-PKU202 High copy pColE1- ND Ptet::PAL3, ampicillin resistant,
SYN107 SYN-PKU203 lacZ::Ptet-pheP::cam 0 SYN108 SYN-PKU401 Low copy
pSC101- 1.1 Ptet::PAL1, ampicillin resistant, chromosomal
lacZ::Ptet-pheP::cam SYN109 SYN-PKU402 High copy pColE1- 0.8
Ptet::PAL1, ampicillin resistant, chromosomal lacZ::Ptet-pheP::cam
SYN110 SYN-PKU302 Low Copy pSC101- 2.2 Ptet::PAL3, ampicillin
resistant; chromosomal lacZ::Ptet-pheP::cam SYN111 SYN-PKU303 High
copy pColE1- 7.1 Ptet::PAL3, ampicillin resistant, chromosomal
lacZ::Ptet-pheP::cam SYN340 SYN-PKU304 Low Copy pSC101- 3
PfnrS::PAL3, ampicillin resistant; chromosomal
lacZ::PfnrS-pheP::cam SYN958 SYN-PKU305 Low Copy pSC101- 3
PfnrS::PAL3, kanamycin resistant; chromosomal lacZ::PfnrS-pheP::cam
SYN959 SYN-PKU307 Low Copy pSC101- 0.3 PfnrS::PAL3, ampicillin
resistant; SYN837 SYN-PKU308 Low Copy pSC101- 0.3 PfnrS::PAL3,
kanamycin resistant;
Example 4
Construction of Plasmids Encoding Branched Chain Amino Acid
Importers and Branched Chain Amino Acid Catabolism Enzyme
[0806] The kivD gene of lactococcus lactis IFPL730 was synthesized
(Genewiz), fused to the Tet promoter, cloned into the high-copy
plasmid pUC57-Kan by Gibson assembly and transformed into E. coli
DH5.alpha. to generate the plasmid pTet-kivD. The bkd operon of
Pseudomonas aeruginosa PAO1 fused to the Tet promoter was
synthesized (Genewiz) and cloned into the high-copy plasmid
pUC57-Kan to generate the plasmid pTet-bkd. The bkd operon of
Pseudomonas aeruginosa PAO1 fused to the ldh gene from PAO1 and the
Tet promoter was synthesized (Genewiz) and cloned into the
high-copy plasmid pUC57-Kan to generate the plasmid pTet-ldh-bkd.
The livKHMGF operon from E. coli Nissle fused to the Tet promoter
was synthesized (Genewiz), cloned into the pKIKO-lacZ plasmid by
Gibson assembly and transformed into E. coli PIR1 as described in
Example 3 to generate the pTet-livKHMGF.
Example 5
Generation of Recombinant Bacterial Cell Comprising a Genetic
Modification that Reduces Export of a Branched Chain Amino Acid
[0807] E. coli Nissle was transformed with the pKD46 plasmid
encoding the lambda red proteins under the control of an
arabinose-inducible promoter as follows. An overnight culture of E.
coli Nissle grown at 37.degree. C. was diluted 1:100 in 4 mL of
lysogeny broth (LB) and grown at 37.degree. C. until it reached an
OD.sub.600 of 0.4-0.6. 1 mL of the culture was then centrifuged at
13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the
supernatant was removed. The cells were then washed three times in
pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10%
glycerol. The electroporator was set to 1.8 kV. 1 uL of a pKD46
miniprep was added to the cells, mixed by pipetting, and pipetted
into a sterile, chilled 1 mm cuvette. The cuvette was placed into
the sample chamber, and the electric pulse was applied. 500 uL of
room-temperature SOC media was immediately added, and the mixture
was transferred to a culture tube and incubated at 30.degree. C.
for 1 hr. The cells were spread out on an LB plate containing 100
ug/mL carbenicillin and incubated at 30.degree. C.
[0808] .DELTA.leuE deletion construct with 77 bp and a 100 bp
flanking leuE homology regions and a kanamycin resistant cassette
flanked by FRT recombination site was generated by PCR,
column-purified and transformed into E. coli Nissle pKD46 as
follows. An overnight culture of E. coli Nissle pKD46 grown in 100
ug/mL carbenicillin at 30.degree. C. was diluted 1:100 in 5 mL of
LB supplemented with 100 ug/mL carbenicillin, 0.15% arabinose and
grown until it reaches an OD.sub.600 of 0.4-0.6. The bacteria were
aliquoted equally in five 1.5 mL microcentrifuge tubes, centrifuged
at 13,000 rpm for 1 min and the supernatant was removed. The cells
were then washed three times in pre-chilled 10% glycerol and
combined in 50 uL pre-chilled 10% glycerol. The electroporator was
set to 1.8 kV. 2 uL of a the purified .DELTA.leuE deletion PCR
fragment are then added to the cells, mixed by pipetting, and
pipetted into a sterile, chilled 1 mm cuvette. The cuvette was
placed into the sample chamber, and the electric pulse was applied.
500 uL of room-temperature SOC media was immediately added, and the
mixture was transferred to a culture tube and incubated at
37.degree. C. for 1 hr. The cells were spread out on an LB plate
containing 50 ug/mL kanamycin. Five kanamycin-resistant
transformants were then checked by colony PCR for the deletion of
the leuE locus.
[0809] The kanamycin cassette was then excised from the .DELTA.leuE
deletion strain as follows. .DELTA.leuE was transformed with the
pCP20 plasmid encoding the Flp recombinase gene. An overnight
culture of .DELTA.leuE grown at 37.degree. C. in LB with 50 ug/mL
kanamycin was diluted 1:100 in 4 mL of LB and grown at 37.degree.
C. until it reaches an OD.sub.600 of 0.4-0.6. 1 mL of the culture
was then centrifuged at 13,000 rpm for 1 min in a 1.5 mL
microcentrifuge tube and the supernatant was removed. The cells
were then washed three times in pre-chilled 10% glycerol and
resuspended in 40 uL pre-chilled 10% glycerol. The electroporator
was set to 1.8 kV. 1 uL of a pCP20 miniprep was added to the cells,
mixed by pipetting, and pipetted into a sterile, chilled 1 mm
cuvette. The dry cuvette was placed into the sample chamber, and
the electric pulse was applied. 500 uL of room-temperature SOC
media was immediately added, and the mixture was transferred to a
culture tube and incubated at 30.degree. C. for 1 hr. The cells
were spread out on an LB plate containing 100 ug/mL carbenicillin
and incubated at 30.degree. C. Eight transformants were then
streaked on an LB plate and were incubated overnight at 43.degree.
C. One colony per transformant was picked and resuspended in 10 uL
LB and 3 uL of the suspension were pipetted on LB, LB with 50 ug/mL
Kanamycin or LB with 100 ug/mL carbenicillin The LB and LB
Kanamycin plates were incubated at 37.degree. C. and the LB
Carbenicillin plate was incubated at 30.degree. C. Colonies showing
growth on LB alone were selected and checked by PCR for the
excision of the Kanamycin cassette.
Example 6
Generation of Recombinant Bacteria Comprising an Importer of a
Branched Chain Amino Acid and/or a Branched Chain Amino Acid
Catabolism Enzyme and Lacking an Exporter of a Branched Chain Amino
Acid
[0810] pTet-kivD, pTet-bkd, pTet-ldh-bkd and pTet-livKHFGF plasmids
described above were transformed into E. coli Nissle (pTet-kivD),
Nissle (pTet-kivD, pTet-bkd, pTet-ldh-bkd), DH5.alpha. (pTet-kivD,
pTet-bkd, pTet-ldh-bkd) or PIR1 (pTet-livKHMGF). All tubes,
solutions, and cuvettes were pre-chilled to 4.degree. C. An
overnight culture of E. coli (Nissle, .DELTA.leuE, DH5.alpha. or
PIR1) was diluted 1:100 in 4 mL of LB and grown until it reached an
OD.sub.600 of 0.4-0.6. 1 mL of the culture was then centrifuged at
13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the
supernatant was removed. The cells were then washed three times in
pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10%
glycerol. The electroporator was set to 1.8 kV. 1 uL of a
pTet-kivD, pTet-bkd, pTet-ldh-bkd or pTet-livKHMGF miniprep was
added to the cells, mixed by pipetting, and pipetted into a
sterile, chilled 1mm cuvette. The dry cuvette was placed into the
sample chamber, and the electric pulse was applied. 500 uL of
room-temperature SOC media was immediately added, and the mixture
was transferred to a culture tube and incubated at 37.degree. C.
for 1 hr. The cells were spread out on an LB plate containing 50
ug/mL Kanamycin for pTet-kivD, pTet-bkd and pTet-ldh-bkd or 100
ug/mL carbenicillin for pTet-livKHMGF.
Example 7
Generation of Recombinant Bacteria Comprising an Importer of a
Branched Chain Amino Acid and a Genetic Modification that Reduces
Export of a Branched Chain Amino Acid
[0811] E. coli Nissle .DELTA.leuE was transformed with the pKD46
plasmid encoding the lambda red proteins under the control of an
arabinose-inducible promoter as follows. An overnight culture of E.
coli Nissle .DELTA.leuE grown at 37.degree. C. was diluted 1:100 in
4 mL of LB and grown at 37.degree. C. until it reached an
OD.sub.600 of 0.4-0.6. 1 mL of the culture was then centrifuged at
13,000 rpm for 1 min in a 1.5 mL microcentrifuge tube and the
supernatant was removed. The cells were then washed three times in
pre-chilled 10% glycerol and resuspended in 40 uL pre-chilled 10%
glycerol. The electroporator was set to 1.8 kV. 1 uL of a pKD46
miniprep was added to the cells, mixed by pipetting, and pipetted
into a sterile, chilled 1 mm cuvette. The dry cuvette was placed
into the sample chamber, and the electric pulse was applied. 500 uL
of room-temperature SOC media was immediately added, and the
mixture was transferred to a culture tube and incubated at
30.degree. C. for 1 hr. The cells were spread out on an LB plate
containing 100 ug/mL carbenicillin and incubated at 30.degree.
C.
[0812] The DNA fragment used to integrate Tet-livKHMGF into E. coli
Nissle lacZ was amplified by PCR from the pTet-livKHMGF plasmid,
column-purified and transformed into .DELTA.leuE pKD46 as follows.
An overnight culture of the E. coli Nissle .DELTA.leuE pKD46 strain
grown in LB at 30.degree. C. with 100 ug/mL carbenicillin was
diluted 1:100 in 5 mL of lysogeny broth (LB) supplemented with 100
ug/mL carbenicillin, 0.15% arabinose and grown at 30.degree. C.
until it reached an OD.sub.600 of 0.4-0.6. The bacteria were
aliquoted equally in five 1.5 mL microcentrifuge tubes, centrifuged
at 13,000 rpm for 1 min and the supernatant was removed. The cells
were then washed three times in pre-chilled 10% glycerol and
combined in 50 uL pre-chilled 10% glycerol. The electroporator was
set to 1.8 kV. 2 uL of a the purified Tet-livKHMGF PCR fragment
were then added to the cells, mixed by pipetting, and pipetted into
a sterile, chilled 1 mm cuvette. The dry cuvette was placed into
the sample chamber, and the electric pulse was applied. 500 uL of
room-temperature SOC media was immediately added, and the mixture
was transferred to a culture tube and incubated at 37.degree. C.
for 1 hr. The cells were spread out on an LB plate containing 20
ug/mL chloramphenicol, 40 ug/mL X-Gal and incubated overnight at
37.degree. C. White chloramphenicol resistant transformants were
then checked by colony PCR for integration of Tet-livKHMGF into the
lacZ locus.
Example 8
Functional Assay Demonstrating that the Recombinant Bacterial Cells
Decrease Branched Chain Amino Acid Concentration
[0813] For in vitro studies, all incubations were performed at
37.degree. C. Cultures of E. coli Nissle .DELTA.leuE,
.DELTA.leuE+pTet-kivD, .DELTA.leuE+pTet-bkd,
.DELTA.leuE+pTet-ldh-bkd, .DELTA.leuE lacZ:Tet-livKHMGF,
.DELTA.leuE lacZ:Tet-livKHMGF+pTet-kivD, .DELTA.leuE
lacZ:Tet-livKHMGF+pTet-bkd, .DELTA.leuE
lacZ:Tet-livKHMGF+pTet-ldh-bkd were grown overnight in LB, LB 50
ug/mL Kanamycin or LB 50 ug/mL Kanamycin 20 ug/mL chloramphenicol
and then diluted 1:100 in LB. The cells were grown with shaking
(250 rpm) to early log phase with the appropriate antibiotics.
Anhydrous tetracycline (ATC) was added to cultures at a
concentration of 100 ng/mL to induce expression of KivD, Bkd, Ldh
and LivKHFMG, and bacteria were grown for another 3 hours. Bacteria
were then pelleted, washed, and resuspended in minimal media, and
supplemented with 0.5% glucose and 2 mM leucine. Aliquots were
removed at 0 h, 1.5 h, 6 h and 18 h for leucine quantification by
liquid chromatography-mass spectrometry (LCMS) using a Thermo TSQ
Quantum Max triple quadrupole instrument. Briefly, 100 uL aliquots
were centrifuged at 4,500 rpm for 10 min 10 uL of the supernatant
was resuspended in 90 uL water with 1 ug/mL L-leucine-5,5,5-d.sub.3
(isotope used as internal standard). 10 uL of the samples was then
resuspended in 90 uL 10% acetonitrile, 0.1% formic acid and placed
in the LCMS autosampler. A C18 column 100.times.2mm, 3 um particles
was used (Luna, Phenomenex). The mobile phases used were water 0.1%
formic acid (solvent A) and acetonitrile 0.1% (solvent B). The
gradient used was:
[0814] 0 min: 95%A, 5%B
[0815] 0.5 min: 95%A, 5%B
[0816] 1 min: 10%A, 90%B
[0817] 2.5 min: 10%A, 90%B
[0818] 2.51 min: 95%A, 5%B
[0819] 3.5 min: 95%A, 5%B
[0820] The Q1/Q3 transitions used for leucine and
L-leucine-5,5,5-d.sub.3 were 132.1/86.2 and 135.1/89.3
respectively.
[0821] Leucine was rapidly graded by the expression of kivD in the
Nissle .DELTA.leuE strain. After 6 h of incubation, leucine
concentration droped by over 99% in the presence of ATC. This
effect was even more pronounced in the case of .DELTA.leuE
expressing both kivD and the leucine transporter livKHMGF where
leucine is undetectable after 6 h of incubation. The expression of
the bkd complex also leads rapidly to the degradation of leucine.
After 6 h of incubation, 99% of leucine was degraded. The
expression of the leucine transporter livKHMGF, in parallel with
the expression of ldh and bkd leads to the complete degradation of
leucine after 18 h.
Example 9
Simultaneous Degradation of Branched Chain Amino Acids by
Recombinant Bacteria Expressing a Branched Chain Amino Acid
Catabolism Enzyme and an Importer of a Branched Chain Amino
Acid
[0822] In these studies, all incubations were performed at
37.degree. C. Cultures of E. coli Nissle, Nissle+pTet-kivD,
.DELTA.leuE+pTet-kivD, .DELTA.leuE lacZ:Tet-livKHMGF+pTet-kivD were
grown overnight in LB, LB 50 ug/mL Kanamycin or LB 50 ug/mL
Kanamycin 20 ug/mL chloramphenicol and then diluted 1:100 in LB.
The cells were grown with shaking (250 rpm) to early log phase with
the appropriate antibiotics. Anhydrous tetracycline (ATC) was added
to cultures at a concentration of 100 ng/mL to induce expression of
KivD and LivKHFMG, and bacteria were grown for another 3 hours.
Bacteria were then pelleted, washed, and resuspended in minimal
media, and supplemented with 0.5% glucose and the three branched
chain amino acids (leucine, isoleucine and valine, 2 mM each).
Aliquots were removed at 0 h, 1.5 h, 6 h and 18 h for leucine,
isoleucine and valine quantification by liquid chromatography-mass
spectrometry (LCMS) using a Thermo TSQ Quantum Max triple
quadrupole instrument. Briefly, 100 uL aliquots were centrifuged at
4,500 rpm for 10 min 10 uL of the supernatant was resuspended in 90
uL water with lug/mL L-leucine-5,5,5-d.sub.3 (isotope used as
internal standard). 10 uL of the samples was then resuspended in
water, 0.1% formic acid and placed in the LCMS autosampler. A C18
column 100.times.2mm, 3 um particles was used (Luna, Phenomenex).
The mobile phases used were water 0.1% formic acid (solvent A) and
acetonitrile 0.1% (solvent B). The gradient used was:
[0823] 0 min: 100%A, 0%B
[0824] 0.5 min: 100%A, 0%B
[0825] 1.5 min: 10%A, 90%B
[0826] 3.5 min: 10%A, 90%B
[0827] 3.51 min: 100%A, 0%B
[0828] 4.5 min: 100%A, 0%B
[0829] The Q1/Q3 transitions used are:
[0830] Leucine: 132.1/86.2
[0831] L-leucine-5,5,5-d.sub.3: 135.1/89.3
[0832] Isoleucine: 132.1/86.2
[0833] Valine: 118.1/72
[0834] As shown in FIGS. 32A-32C, leucine, isoleucine and valine
were all degraded by the expression of kivD in E. coli Nissle. At
18 h, 96.8%, 67.2% and 52.1% of leucine, isoleucine and valine
respectively were degraded in Nissle expressing kivD in the
presence of ATC. The efficiency of leucine and isoleucine
degradation was further improved by expressing kivD in the
.DELTA.leuE background strain with a 99.8% leucine and 80.6%
isoleucine degradation at 18 h Finally, an additional increase in
leucine and isoleucine degradation was achieved by expressing the
leucine transporter livKHMGF in the Nissle .DELTA.leuE pTet-kivD
strain with a 99.98% leucine and 95.5% isoleucine degradation at 18
h. No significant improvement in valine degradation was observed in
the .DELTA.leuE deletion strain expressing livKHMGF.
Example 10
Increase of BCAA Import by Overexpressing the High Affinity BCAA
Transporters livKHMGF and livJHMGF In Vitro
Study Objective
[0835] BCAA accumulate to toxic levels in MSUT) patients [0836]
Different synthetic probiotic E. coli Nissle strains were
engineered to efficiently import BCAA into the bacterial cell to be
degraded [0837] The objective of this study was to determine if
expressing the two BCAA transporters livKHMGF and livJHMGF increase
the import of valine, a BCAA naturally secreted to high levels by
E. coli Nissle.
Description of the Different Probiotic Strains
[0837] [0838] All strains are derived from the human probiotic:
strain E. coli Nissle 1917. A .DELTA.leuE deletion strain (deleted
for the leucine exporter leuE) was generated by lambda
red-recombination [0839] A copy of the high-affinity leucine ABC
transporter livKHMGF under the control of a tetracycline-inducible
promoter (Ptet) was inserted into the lacZ locus of the .DELTA.leuE
deletion strain by lambda-red recombination to generate the
.DELTA.leuE, lacZ:Ptet-livKHMGF strain. In this strain, the BCAA
transporter livKHMGF can get induced in the presence of
anhydrotetracycline (ATC) [0840] Finally, the endogenous promoter
of livJ was swapped with the constitutive promoter Ptac by
lambda-red recombination to generate the .DELTA.leuE,
lacZ:Ptet-livKHMGF, Ptac-livJ strain. In this strain, livJ is
constitutively induced. In the presence of ATC, both BCAA
transporters livKHMGF and livJHMGF are expressed
Experimental Procedure
[0840] [0841] The three strains tested (.DELTA.leuE; .DELTA.leuE,
lacZ:Ptet-livKHMGF; .DELTA.leuE, lacZ:Ptet-livKHMGF, Ptac-livJ)
were grown overnight at 37.degree. C. and 250 rpm in 4 mL of LB
[0842] Cells were diluted 100 fold in 4 mL LB and grown for 2 h at
37.degree. C. and 250 rpm [0843] Cells were split in two 2 mL
culture tubes [0844] One 2 mL culture tube was induced with
100ng/mL anhydrotetracycline (ATC) to activate the Ptet promoter
[0845] After 1 h induction, 1 mL of cells was spun down at maximum
speed for 30 seconds in a microcentrifuge [0846] The supernatant
was removed and the pellet re-suspended in 1 mL M9 medium 0.5%
glucose [0847] The cells were spun down again at maximum speed for
30 seconds and resuspended in 1 mL M9 medium 0.5% glucose [0848]
The cells were transferred to a culture tube and incubated at at
37.degree. C. and 250 rpm for 5.5 h [0849] 150 .mu.L of cells were
collected at 0 h, 2 h and 5.5 h [0850] The concentration of valine
in the cell supernatant at the different time points was determined
by LC-MS/MS.
Results
[0851] The natural secretion of valine by E. coli Nissle is
observed for the .DELTA.leuE strain. The secretion of valine is
strongly reduced for .DELTA.leuE, lacZ:Ptet-livKHMGF in the
presence of ATC. This strongly suggests that the secreted valine is
efficiently imported back into the cell by livKHMGF. The secretion
of valine is abolished in the .DELTA.leuE, lacZ:Ptet-livKHMGF,
Ptac-livJ strain, with or without ATC. This strongly suggests that
the constitutive expression of livJ is sufficient to import back
the entire amount of valine secreted by the cell via the livJHMGF
transporter. E. coli Nissle was engineered to efficiently import
BCAA, in this case valine, using both an inducible promoter (Ptet),
and a constitutive promoter (Ptac), controlling the expression of
livKHMGF and livJ respectively.
Example 11
Generation of Bacterial Strains with Enhance Ability to Transport
Biomolecules
[0852] Due to their ease of culture, short generation times, very
high population densities and small genomes, microbes can be
evolved to unique phenotypes in abbreviated timescales. Adaptive
laboratory evolution (ALE) is the process of passaging microbes
under selective pressure to evolve a strain with a preferred
phenotype. Most commonly, this is applied to increase utilization
of carbon/energy sources or adapting a strain to environmental
stresses (e.g., temperature, pH), whereby mutant strains more
capable of growth on the carbon substrate or under stress will
outcompete the less adapted strains in the population and will
eventually come to dominate the population.
[0853] This same process can be extended to any essential
metabolite by creating an auxotroph. An auxotroph is a strain
incapable of synthesizing an essential metabolite and must
therefore have the metabolite provided in the media to grow. In
this scenario, by making an auxotroph and passaging it on
decreasing amounts of the metabolite, the resulting dominant
strains should be more capable of obtaining and incorporating this
essential metabolite.
[0854] For example, if the biosynthetic pathway for producing an
amino acid is disrupted a strain capable of high-affinity capture
of said amino acid can be evolved via ALE. First, the strain is
grown in varying concentrations of the auxotrophic amino acid,
until a minimum concentration to support growth is established. The
strain is then passaged at that concentration, and diluted into
lowering concentrations of the amino acid at regular intervals.
Over time, cells that are most competitive for the amino acid--at
growth-limiting concentrations--will come to dominate the
population. These strains will likely have mutations in their amino
acid-transporters resulting in increased ability to import the
essential and limiting amino acid.
[0855] Similarly, by using an auxotroph that cannot use an upstream
metabolite to form an amino acid, a strain can be evolved that not
only can more efficiently import the upstream metabolite, but also
convert the metabolite into the essential downstream metabolite.
These strains will also evolve mutations to increase import of the
upstream metabolite, but may also contain mutations which increase
expression or reaction kinetics of downstream enzymes, or that
reduce competitive substrate utilization pathways.
[0856] In the previous examples, a metabolite innate to the microbe
was made essential via mutational auxotrophy and selection was
applied with growth-limiting supplementation of the endogenous
metabolite. However, phenotypes capable of consuming non-native
compounds can be evolved by tying their consumption to the
production of an essential compound. For example, if a gene from a
different organism is isolated which can produce an essential
compound or a precursor to an essential compound this gene can be
recombinantly introduced and expressed in the heterologous host.
This new host strain will now have the ability to synthesize an
essential nutrient from a previously non-metabolizable substrate.
Hereby, a similar ALE process can be applied by creating an
auxotroph incapable of converting an immediately downstream
metabolite and selecting in growth-limiting amounts of the
non-native compound with concurrent expression of the recombinant
enzyme. This will result in mutations in the transport of the
non-native substrate, expression and activity of the heterologous
enzyme and expression and activity of downstream native enzymes. It
should be emphasized that the key requirement in this process is
the ability to tether the consumption of the non-native metabolite
to the production of a metabolite essential to growth.
[0857] Once the basis of the selection mechanism is established and
minimum levels of supplementation have been established, the actual
ALE experimentation can proceed. Throughout this process several
parameters must be vigilantly monitored. It is important that the
cultures are maintained in an exponential growth phase and not
allowed to reach saturation/stationary phase. This means that
growth rates must be check during each passaging and subsequent
dilutions adjusted accordingly. If growth rate improves to such a
degree that dilutions become large, then the concentration of
auxotrophic supplementation should be decreased such that growth
rate is slowed, selection pressure is increased and dilutions are
not so severe as to heavily bias subpopulations during passaging.
In addition, at regular intervals cells should be diluted, grown on
solid media and individual clones tested to confirm growth rate
phenotypes observed in the ALE cultures.
[0858] Predicting when to halt the stop the ALE experiment also
requires vigilance. As the success of directing evolution is tied
directly to the number of mutations "screened" throughout the
experiment and mutations are generally a function of errors during
DNA replication, the cumulative cell divisions (CCD) acts as a
proxy for total mutants which have been screened. Previous studies
have shown that beneficial phenotypes for growth on different
carbon sources can be isolated in about 10.sup.11.2 CCD.sup.1. This
rate can be accelerated by the addition of chemical mutagens to the
cultures--such as N-methyl-N-nitro-N-nitrosoguanidine (NTG)--which
causes increased DNA replication errors. However, when continued
passaging leads to marginal or no improvement in growth rate the
population has converged to some fitness maximum and the ALE
experiment can be halted.
[0859] At the conclusion of the ALE experiment, the cells should be
diluted, isolated on solid media and assayed for growth phenotypes
matching that of the culture flask. Best performers from those
selected are then prepped for genomic DNA and sent for whole genome
sequencing. Sequencing with reveal mutations occurring around the
genome capable of providing improved phenotypes, but will also
contain silent mutations (those which provide no benefit but do not
detract from desired phenotype). In cultures evolved in the
presence of NTG or other chemical mutagen, there will be
significantly more silent, background mutations. If satisfied with
the best performing strain in its current state, the user can
proceed to application with that strain. Otherwise the contributing
mutations can be deconvoluted from the evolved strain by
reintroducing the mutations to the parent strain by genome
engineering techniques. See Lee, D.-H., Feist, A. M., Barrett, C.
L. & Palsson, B. O. Cumulative Number of Cell Divisions as a
Meaningful Timescale for Adaptive Laboratory Evolution of
Escherichia coli. PLoS ONE 6, e26172 (2011).
[0860] These methods were used to generate E. Coli Nissle mutants
that consume kynurenine and over-produce tryptophan as described
elsewhere herein.
Example 12
Engineered Bacteria Engineered to Efficiently Import KYN
[0861] In the tumor microenvironment the amino acid tryptophan
(TRP) and its degradation product kynurenine (KYN) play pivotal
roles as immunomodulatory signals. Tumors often degrade TRP (which
has proinflammatory properties) into KYN, which possesses
anti-inflammatory characteristics, thereby promoting evasion from
immune surveillance.
[0862] E. coli Nissle can be engineered to efficiently import KYN
and convert it to TRP. While Nissle does not typically utilize KYN,
by introducing the Kynureninase (KYNase) from Pseudomonas
fluorescens (kynU) on a medium-copy plasmid under the control of
the tetracycline promoter (Ptet) a new strain with this plasmid
(Ptet-KYNase) is able to convert L-kynurenine into
anthranilate.
[0863] E. coli naturally utilizes anthranilate in its TRP
biosynthetic pathway. Briefly, the TrpE (in complex with TrpD)
enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and
TrpB then catalyze a five-step reaction ending with the
condensation of an indole with serine to form tryptophan. By
replacing the TrpE enzyme via lambda-RED recombineering, the
subsequent strain of Nissle (.DELTA.trpE::Cm) is an auxotroph
unable to grow in minimal media without supplementation of TRP or
anthranilate. By expressing kynureninase in .DELTA.trpE::Cm
(KYNase-trpE), this auxotrophy can be alternatively rescued by
providing KYN.
[0864] Leveraging the growth-limiting nature of KYN in KYNase-trpE,
adaptive laboratory evolution was employed to evolve a strain
capable of increasingly efficient utilization of KYN. First a lower
limit of KYN concentration was established and mutants were evolved
by passaging in lowering concentrations of KYN. While this can
select for mutants capable of increasing KYN import, the bacterial
cells still prefer to utilize free, exogenous TRP. In the tumor
environment, dual-therapeutic functions can be provided by
depletion of KYN and increasing local concentrations of TRP.
Therefore, to evolve a strain which prefers KYN over TRP, a toxic
analogue of TRP--5-fluoro-L-tryptophan (ToxTRP)--can be
incorporated into the ALE experiment. The resulting best performing
strain is then whole genome sequenced in order to deconvolute the
contributing mutations. Lambda-RED can be performed in order to
reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR,
transcriptional attenuators) to up-regulate TrpABCDE expression and
increase chorismate production. The resulting strain is now
insensitive to external TRP, efficiently converts KYN into TRP, and
also now overproduces TRP.
[0865] Kynureninase Protein Sequences
TABLE-US-00013 Description ID Sequence Pseudomonas P83788
MTTRNDCLALDAQDSLAPLRQQFALPEGVIYLDGNS kynureninase
LGARPVAALARAQAVIAEEWGNGLIRSWNSAGWRD
LSERLGNRLATLIGARDGEVVVTDTTSINLFKVLSAA
LRVQATRSPERRVIVTETSNFPTDLYIAEGLADMLQQ
GYTLRLVDSPEELPQAIDQDTAVVMLTHVNYKTGYM
HDMQALTALSHECGALAIWDLAHSAGAVPVDLHQA
GADYAIGCTYKYLNGGPGSQAFVWVSPQLCDLVPQP
LSGWFGHSRQFAMEPRYEPSNGIARYLCGTQPITSLA
MVECGLDVFAQTDMASLRRKSLALTDLFIELVEQRC
AAHELTLVTPREHAKRGSHVSFEHPEGYAVIQALIDR
GVIGDYREPRIMRFGFTPLYTTFTEVWDAVQILGEILD RKTWAQAQFQVRHSVT* Human
Q16719 MEPSSLELPADTVQRIAAELKCHPTDERVALHLDEED
KLRHFRECFYIPKIQDLPPVDLSLVNKDENAIYFLGNS
LGLQPKMVKTYLEEELDKWAKIAAYGHEVGKRPWI
TGDESIVGLMKDIVGANEKEIALMNALTVNLHLLML
SFFKPTPKRYKILLEAKAFPSDHYAIESQLQLHGLNIE
ESMRMIKPREGEETLRIEDILEVIEKEGDSIAVILFSGV
HFYTGQHFNIPAITKAGQAKGCYVGFDLAHAVGNVE
LYLHDWGVDFACWCSYKYLNAGAGGIAGAFIHEKH
AHTIKPALVGWFGHELSTRFKMDNKLQLIPGVCGFRI
SNPPILLVCSLHASLEIFKQATMKALRKKSVLLTGYLE
YLIKHNYGKDKAATKKPVVNIITPSHVEERGCQLTITF
SVPNKDVFQELEKRGVVCDKRNPNGIRVAPVPLYNS FHDVYKFTNLLTSILDSAETKN*
Shewanella Q8E973 MLLNVKQDFCLAGPGYLLNHSVGRPLKSTEQALKQA
FFAPWQESGREPWGQWLGVIDNFTAALASLFNGQPQ
DFCPQVNLSSALTKIVMSLDRLTRDLTRNGGAVVLM
SEIDFPSMGFALKKALPASCELRFIPKSLDVTDPNVW
DAHICDDVDLVFVSHAYSNTGQQAPLAQIISLARERG
CLSLVDVAQSAGILPLDLAKLQPDFMIGSSVKWLCSG
PGAAYLWVNPAILPECQPQDVGWFSHENPFEFDIHDF
RYHPTALRFWGGTPSIAPYAIAAHSIEYFANIGSQVM
REHNLQLMEPVVQALDNELVSPQEVDKRSGTIILQFG
ERQPQILAALAAANISVDTRSLGIRVSPHIYNDEADIA RLLGVIKANR* *designates the
position of the stop codon
[0866] Selected Codon-Optimized Sequences for Kynureninase
TABLE-US-00014 Kynureninase protein sequences Kynureninase protein
sequences Ptet-
atctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagttat
kynU(Pseudomonas)
tttaccactccctatcagtgatagagaaaagtgaattatataaaagtgggaggtgccc
gaatgacgacccgaaatgattgcctagcgttggatgcacaggacagtctggctccgctg
cgccaacaatttgcgctgccggagggtgtgatatacctggatggcaattcgctgggcgca
cgtccggtagctgcgctggctcgcgcgcaggctgtgatcgcagaagaatggggcaacg
ggttgatccgttcatggaactctgcgggctggcgtgatctgtctgaacgcctgggtaatcg
cctggctaccctgattggtgcgcgcgatggggaagtagttgttactgataccacctcgatt
aatctgtttaaagtgctgtcagcggcgctgcgcgtgcaagctacccgtagcccggagcg
ccgtgttatcgtgactgagacctcgaatttcccgaccgacctgtatattgcggaagggttg
gcggatatgctgcaacaaggttacactctgcgtttggtggattcaccggaagagctgcca
caggctatagatcaggacaccgcggtggtgatgctgacgcacgtaaattataaaaccggt
tatatgcacgacatgcaggctctgaccgcgttgagccacgagtgtggggctctggcgatt
tgggatctggcgcactctgctggcgctgtgccggtggacctgcaccaagcgggcgcgg
actatgcgattggctgcacgtacaaatacctgaatggcggcccgggttcgcaagcgtttgt
ttgggtttcgccgcaactgtgcgacctggtaccgcagccgctgtctggttggttcggccat
agtcgccaattcgcgatggagccgcgctacgaaccttctaacggcattgctcgctatctgt
gcggcactcagcctattactagcttggctatggtggagtgcggcctggatgtgtttgcgca
gacggatatggcttcgctgcgccgtaaaagtctggcgctgactgatctgttcatcgagctg
gttgaacaacgctgcgctgcacacgaactgaccctggttactccacgtgaacacgcgaa
acgcggctctcacgtgtcttttgaacaccccgagggttacgctgttattcaagctctgattg
atcgtggcgtgatcggcgattaccgtgagccacgtattatgcgtttcggtttcactcctctgt
atactacttttacggaagtttgggatgcagtacaaatcctgggcgaaatcctggatcgtaag
acttgggcgcaggctcagtttcaggtgcgccactctgttacttaaaaataaaacgaaag
gctcagtcgaaagactgggcctttcgttttatctgttg Ptet-kynU(Human)
atctaatctagacatcattaattcctaatttttgttgacactctatcattgatagagttat
tttaccactccctatcagtgatagagaaaagtgaatatcaagacacgaggaggtaag
attatggagccttcatctttagaactgccagcggacacggtgcagcgcatcgcggcgga
actgaagtgccatccgactgatgagcgtgtggcgctgcatctggacgaagaagataaac
tgcgccactttcgtgaatgatttatattcctaaaattcaagacttgccgccggtagatttgagt
ctcgttaacaaagatgaaaacgcgatctactttctgggcaactctctgggtctgcaaccaa
aaatggttaaaacgtacctggaggaagaactggataaatgggcaaaaatcgcggcttatg
gtcacgaagtgggcaagcgtccttggattactggcgacgagtctattgtgggtttgatgaa
agatattgtgggcgcgaatgaaaaggaaattgcactgatgaatgctctgaccgttaatctg
cacctgctgatgctgtctttttttaaaccgaccccgaaacgctacaaaatactgctggaagc
gaaagcgtttccgtcggatcactatgctatagaaagtcaactgcagttgcatggtctgaata
tcgaggaatctatgcgcatgattaaaccgcgtgagggtgaagaaacgctgcgtattgaag
acattctggaagttattgaaaaagaaggtgattctatcgcagttatactgttttctggcgtgca
cttttatacaggtcagcacttcaatatcccggcaatcactaaagcggggcaggcaaaagg
ctgctatgttggttttgacctggcgcatgcagtggggaatgttgaactgtatctgcacgattg
gggcgttgatttcgcgtgttggtgtagctacaaatatctgaacgctggcgcgggtggcatt
gctggcgcttttattcacgaaaaacacgcgcacaccattaaaccggctctggttggctggt
tcggtcatgagctgagtactcgctttaaaatggataacaaactgcaattgattccgggtgttt
gcggcttccgtatcagcaatccgccgattctgctggtttgcagcctgcacgctagtctgga
aatctttaagcaggcgactatgaaagcgctgcgcaaaaaatctgtgctgctgaccggctat
ctggagtatctgatcaaacacaattatggcaaagataaagctgcaactaaaaaaccggta
gtgaacattatcaccccctcacacgtggaggagcgcggttgtcagctgactattactttca
gtgtacctaataaagatgtgttccaggaactggaaaaacgcggcgttgtttgtgataaacg
taacccgaatggtattcgcgtggctcctgtgccgctgtacaattcattccacgatgtttataa
attcaccaacctgctgacttctattctcgacagtgctgagactaaaaattaaaaataaaac
gaaaggctcagtcgaaagactgggcctttcgttttatctgttg ptet-
atctaatctagacatcattaattectaatttttgttgacactctatcattgatagagttat
kynU(Shewanella)
tttaccactccctatcagtgatagagaaaagtgaatggttcaccaccacaaggaggg
attatgctgctgaatgtaaaacaggacttttgcctggcaggcccgggctacctgctgaatc
actcggttggccgtccgctgaaatcaactgagcaagcgctgaaacaagcattttttgctcc
gtggcaagagagcggtcgtgaaccgtggggccagtggctgggtgttattgataatttcac
tgctgcgctggcatctctgtttaatggtcaaccgcaggatttttgtccgcaggttaacctgag
cagcgcgctgactaaaattgtgatgtcactggatcgtctgactcgcgatctgacccgcaat
ggcggtgctgttgtgctgatgtctgaaatcgatttcccatctatgggcttcgcgttgaaaaa
agcgctgccagcgagctgcgaactgcgttttatcccgaaaagtctggacgtgactgatcc
gaacgtatgggatgcacacatctgtgatgatgtagacctggtttttgtgtctcacgcctatag
taatacgggccaacaggctccgctggcgcaaatcatctctctggcgcgtgaacgtggct
gcctgtcactggtggatgtagcgcaatcagcggggattttgccgctggatctggcgaaac
tgcaaccggacttcatgatcggcagttcggttaaatggctgtgctcgggccctggtgcgg
catatctgtgggttaatccggcgattctgccggaatgtcagccgcaggatgtgggctggtt
ttcacatgagaatccctttgaattcgacatccacgatttccgctaccacccgactgcactgc
gcttttggggtggtacgccgtcgatcgcgccttatgcgatcgcggcgcactcgatcgaat
attttgccaatatcggctcgcaagtgatgcgtgaacacaacctgcaactgatggaaccggt
ggttcaggcgctggacaatgaactggtgagcccgcaggaagtggataaacgctcaggc
actattattctgcaattcggtgaacgtcaaccgcaaattctggcggctctggctgcggcga
acatttcggtggacactcgttctttggggattcgtgttagtccgcacatttataatgatgagg
cggacattgcgcgcctgctgggtgtgatcaaagcaaatcgctaaaaataaaacgaaagg
ctcagtcgaaagactgggcctttcgttttatctgttg
[0867] The ptet-promoter is in bold, designed Ribosome binding site
is underlined, codon-optimized protein coding sequence is in plain
text, and the terminator is in italics.
[0868] Generation of E.Coli Mutants with increased ability to
consume L-Kynurenine
Example 13
Results
[0869] Adaptive Laboratory Evolution was used to produce mutant
bacterial strains that consume Kynurenine and produce tryptophan.
First, a AtrpE strain was constructed that expresses kynureninase
and is capable of converting L-kynurenine to anthranilate to rescue
the auxotrophic tryptophan background (KYNase). E. coli Nissle can
be engineered to efficiently import KYN and convert it to TRP.
While Nissle does not typically utilize KYN, by introducing the
Kynureninase (KYNase) from Pseudomonas fluorescens (kynU) on a
medium-copy plasmid under the control of the tetracycline promoter
(Ptet) a new strain with this plasmid (Ptet-KYNase) is able to
convert L-kynurenine into anthranilate.
[0870] E. coli naturally utilizes anthranilate in its TRP
biosynthetic pathway. Briefly, the TrpE (in complex with TrpD)
enzyme converts chorismate into anthranilate. TrpD, TrpC, TrpA and
TrpB then catalyze a five-step reaction ending with the
condensation of an indole with serine to form tryptophan. By
replacing the TrpE enzyme via lambda-RED recombineering, the
subsequent strain of Nissle (AtrpE::Cm) is an auxotroph unable to
grow in minimal media without supplementation of TRP or
anthranilate. By expressing kynureninase in .DELTA.trpE::Cm
(KYNase-trpE), this auxotrophy can be alternatively rescued by
providing KYN.
[0871] First a lower limit of KYN concentration was established and
mutants were evolved by passaging in lowering concentrations of
KYN. While this can select for mutants capable of increasing KYN
import, the bacterial cells still prefer to utilize free, exogenous
TRP. In the tumor environment, dual-therapeutic functions can be
provided by depletion of KYN and increasing local concentrations of
TRP. Therefore, to evolve a strain which prefers KYN over TRP, a
toxic analogue of TRP--5-fluoro-L-tryptophan (ToxTRP)--can be
incorporated into the ALE experiment. The resulting best performing
strain is then whole genome sequenced in order to deconvolute the
contributing mutations. Lambda-RED can be performed in order to
reintroduce TrpE, to inactivate Trp regulation (trpR, tyrR,
transcriptional attenuators) to up-regulate TrpABCDE expression and
increase chorismate production. The resulting strain is now
insensitive to external TRP, efficiently converts KYN into TRP, and
also now overproduces TRP.
[0872] To establish the minimum concentration of L-kynurenine and
maximum concentration of 5-fluoro-L-tryptophan (ToxTrp) capable of
sustaining growth of the KYNase strain, using a checkerboard assay,
the following protocol was used. Using a 96-well plate with M9
minimal media with glucose, KYNU is supplemented decreasing across
columns in 2-fold dilutions from 2000 ug/mL down to .about.1 ug/mL.
In the rows, ToxTrp concentration decreases by 2-fold from 200
ug/mL down to .about.1.5 ug/mL. In one plate, Anhydrous
Tetracycline (aTc) was added to a final concentration of 100 ng/uL
to induce production of the KYNase. From an overnight culture cells
were diluted to an OD600=0.5 in 12 mL of TB (plus appropriate
antibiotics and inducers, where applicable) and grown for 4 hours.
100 uL of cells were spun down and resuspended to an OD600=1.0.
These were diluted 2000-fold and 25 uL was added to each well to
bring the final volumes in each well to 100 uL. Cells were grown
for roughly 20 hours with static incubation at 37C then growth was
assessed by OD600, making sure readings fell within linear range
(0.05-1.0).
[0873] Once identified, the highest concentrations of ToxTrp and
lowest concentration of kynurenine capable of supporting growth
becomes the starting point for ALE. The ALE parental strain was
chosen by culturing the KYNase strain on M9 minimal media
supplemented with glucose and L-kynurenine (referred to as M9+KYNU
from here on). A single colony was selected, resuspended in 20 uL
of sterile phosphate-buffered saline solution. This colony was then
used to inoculate three cultures of M9+KYNU, grown into
late-logarithmic phase and optical density determined at 600 nm.
These cultures were then diluted to 10.sup.3 in 4 rows of a 96-well
deep-well plate with 1 mL of M9+KYNU. Each one of the four rows has
a different ToxTrp (increasing 2-fold), while each column has
decreasing concentrations of KYNU (by 2-fold). Each morning and
evening this plate is diluted back to 10.sup.3 using the well in
which the culture has grown to just below saturation so that the
culture is always in logarithmic growth. This process is repeated
until a change in growth rate is no longer detected. Once no growth
rate increases are detected (usually around 10.sup.11 Cumulative
Cell Divisions) the culture is plated onto M9+KYNU. Phillips, R. S.
Structure and mechanism of kynureninase. Archives of Biochemistry
and Biophysics 544, 69-74 (2014). Individual colonies are selected
and screened in M9+KYNU+ToxTrp media to confirm increased growth
rate phenotype. Once mutants with significantly increased growth
rate on M9+KYNU are isolated, genomic DNA can be isolated and sent
for whole genome sequencing to reveal the mutations responsible for
phenotype.
[0874] All culturing is done shaking at 350 RPM at 37.degree.
C.
TABLE-US-00015 Rich Min Min + STRAIN Media Media Anthranilate Min +
KYNU + aTc SYN094 + + + + trpE + - + - trpE + - + + pseudoKYNase
trpE hKYNase + - + -
[0875] In a preliminary assay, wildtype Nissle (SYN094), Nissle
with a deletion of trpE, and trpE mutants expressing either the
human kynureninase (hKYNase) or the Pseudomonas fluorescens
kynureninase (pseudoKYNase) from a Ptet promoter on a medium-copy
plasmid were grown in either rich media, minimal media (min media),
minimal media with 5 mM anthranilate (Min+anthranilate) or minimal
media with 10 mM kynurenine and 100 ng/uL aTc (Min+KYNU+aTc). These
were grown in 1 mL of media in a deep well plate with shaking at
37.degree. C. A positive for growth (+) in the above table
indicates a change in optical density of >5-fold from
inoculation.
[0876] The results show that in a mutant trpE (which is typically
used in the tryptophan biosynthetic pathway to convert chorismate
into anthranilate) background, Nissle is unable to grow in minimal
media without supplementation with anthranilate (or tryptophan).
When minimal media was supplemented with KYNU, the trpE mutant was
also unable to grow. However, when the pseudoKYNase was expressed
in the trpE tryptophan-auxotroph the cells were able to grow in
Min+KYNU. This indicates that Nissle is able to import L-kynurenine
from the media and convert it into anthranilate using the
pseudoKYNase. The hKYNase homolog was unable to support growth on
M9+KYNU, most likely due to differences in substrate specificity as
it has been documented that the human kynureninase prefers
3-hydroxykynurenine as a substrate. Lee, D.-H., Feist, A. M.,
Barrett, C. L. & Palsson, B. O. Cumulative Number of Cell
Divisions as a Meaningful Timescale for Adaptive Laboratory
Evolution of Escherichia coli. PLoS ONE 6, e26172 (2011).
[0877] Moving forward with the knowledge that Nissle is able to
grow on KYNU supplemented minimal media in a trpE auxotroph by
importing and converting kynurenine, the next step was to establish
the minimal concentrations of kynurenine capable of supporting
growth. Additionally, in our selection experiment if
5-fluoro-L-tryptophan (ToxTrp) was employed the concentrations of
both KYNU and ToxTrp capable of still sustaining growth. A growth
assay was performed in 96-well plates using SYN094, trpE and trpE
pseudoKYNase with and without induction of pseudoKYNase expression
using 100 ng/uL aTc. These strains were inoculated at very dilute
concentrations into M9 minimal media with varying concentrations of
KYNU across columns (2-fold dilutions starting at 2000 ug/mL) and
varying concentrations of ToxTrp across rows (2-fold dilutions
starting at 200 ug/mL). On a separate plate, the strains were grown
in M9+KYNU (at the same concentrations) in the absence of
ToxTrp.
[0878] The results of the initial checkerboard assay are displayed
in FIGS. 36-38 as a function of optical density at 600 nm
(normalized to a media blank). In FIGS. 36 and 37, the X-axis shows
decreasing KYNU concentration from left-to-right, while the Z-axis
shows decreasing ToxTrp concentration from front-to-back with the
very back row representing media with no ToxTrp. In FIG. 38. the
control strains SYN094 and trpE are shown in M9+KYNU without any
ToxTrp, as there was no growth detected from either strain at any
concentration of ToxTrp. The results of the assay show that
expression of the pseudoKYNase provides protection against toxicity
of ToxTrp. More importantly, growth is permitted between 250-62.5
ug/mL of KYNU and 6.3-1.55 ug/mL of ToxTrp.
[0879] Together these experiments establish that expression of the
Pseudomonas fluorescens kynureninase is sufficient to rescue a trpE
auxotrophy in the presence of kynurenine. In addition, the
pseudoKYNase is also capable of providing increased resistance to
the toxic tryptophan, 5-fluoro-L-tryptophan Using the information
attained here it is possible to proceed to an adapative laboratory
evolution experiment to select for mutants with highly efficient
and selective conversion of kynurenine to tryptophan.
TABLE-US-00016 Sequence Listing SEQ ID Gene or NO: Operon Sequence
6 kivD
ATGTATACAGTAGGAGATTACCTATTAGACCGATTACACGAGTTAGGAATTGAAGAAATTTTT
(Lactococcus
GGAGTCCCTGGAGACTATAACTTACAATTTTTAGATCAAATTATTTCCCACAAGGATATGAAA
lactis
TGGGTCGGAAATGCTAATGAATTAAATGCTTCATATATGGCTGATGGCTATGCTCGTACTAAA
IFPL730)
AAAGCTGCCGCATTTCTTACAACCTTTGGAGTAGGTGAATTGAGTGCAGTTAATGGATTAGCA
GGAAGTTACGCCGAAAATTTACCAGTAGTAGAAATAGTGGGATCACCTACATCAAAAGTTCAA
AATGAAGGAAAATTTGTTCATCATACGCTGGCTGACGGTGATTTTAAACACTTTATGAAAATG
CACGAACCTGTTACAGCAGCTCGAACTTTACTGACAGCAGAAAATGCAACCGTTGAAATTGAC
CGAGTACTTTCTGCACTATTAAAAGAAAGAAAACCTGTCTATATCAACTTACCAGTTGATGTT
GCTGCTGCAAAAGCAGAGAAACCCTCACTCCCTTTGAAAAAGGAAAACTCAACTTCAAATACA
AGTGACCAAGAAATTTTGAACAAAATTCAAGAAAGCTTGAAAAATGCCAAAAAACCAATCGTG
ATTACAGGACATGAAATAATTAGTTTTGGCTTAGAAAAAACAGTCACTCAATTTATTTCAAAG
ACAAAACTACCTATTACGACATTAAACTTTGGTAAAAGTTCAGTTGATGAAGCCCTCCCTTCA
TTTTTAGGAATCTATAATGGTACACTCTCAGAGCCTAATCTTAAAGAATTCGTGGAATCAGCC
GACTTCATCTTGATGCTTGGAGTTAAACTCACAGACTCTTCAACAGGAGCCTTCACTCATCAT
TTAAATGAAAATAAAATGATTTCACTGAATATAGATGAAGGAAAAATATTTAACGAAAGAATC
CAAAATTTTGATTTTGAATCCCTCATCTCCTCTCTCTTAGACCTAAGCGAAATAGAATACAAA
GGAAAATATATCGATAAAAAGCAAGAAGACTTTGTTCCATCAAATGCGCTTTTATCACAAGAC
CGCCTATGGCAAGCAGTTGAAAACCTAACTCAAAGCAATGAAACAATCGTTGCTGAACAAGGG
ACATCATTCTTTGGCGCTTCATCAATTTTCTTAAAATCAAAGAGTCATTTTATTGGTCAACCC
TTATGGGGATCAATTGGATATACATTCCCAGCAGCATTAGGAAGCCAAATTGCAGATAAAGAA
AGCAGACACCTTTTATTTATTGGTGATGGTTCACTTCAACTTACAGTGCAAGAATTAGGATTA
GCAATCAGAGAAAAAATTAATCCAATTTGCTTTATTATCAATAATGATGGTTATACAGTCGAA
AGAGAAATTCATGGACCAAATCAAAGCTACAATGATATTCCAATGTGGAATTACTCAAAATTA
CCAGAATCGTTTGGAGCAACAGAAGATCGAGTAGTCTCAAAAATCGTTAGAACTGAAAATGAA
TTTGTGTCTGTCATGAAAGAAGCTCAAGCAGATCCAAATAGAATGTACTGGATTGAGTTAATT
TTGGCAAAAGAAGGTGCACCAAAAGTACTGAAAAAAATGGGCAAACTATTTGCTGAACAAAAT
AAATCATAA 7 Tet-bkd
gtaaaacgacggccagtgaattcgTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCC
construct
GCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTC
sequence
GATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGC
(Gene coding
GACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGC
regions are
ATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGT
shown in
TTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAG
uppercase)
TAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAA
AGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCG
CTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACG
GGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACT
TTTATCTAATCTAGACATcattaattcctaatttttgttgacactctatcattgatagagtta
ttttaccactccctatcagtgatagagaaaagtgaactctagaaataattttgtttaacttta
agaaggagatatacatATGAGTGATTACGAGCCGTTGCGTCTGCATGTCCCGGAGCCCACCGG
GCGTCCTGGCTGCAAGACCGACTTTTCCTATCTGCACCTGTCCCCCGCCGGCGAGGTACGCAA
GCCGCCGGTGGATGTCGAGCCCGCCGAAACCAGCGACCTGGCCTACAGCCTGGTACGTGTGCT
CGACGACGACGGCCACGCCGTCGGTCCCTGGAATCCGCAGCTCAGCAACGAACAACTGCTGCG
CGGCATGCGGGCGATGCTCAAGACCCGCCTGTTCGACGCGCGCATGCTCACCGCGCAACGGCA
GAAAAAGCTTTCCTTCTATATGCAATGCCTCGGCGAGGAAGCCATCGCCACCGCCCACACCCT
GGCCCTGCGCGACGGCGACATGTGCTTTCCGACCTATCGCCAGCAAGGCATCCTGATCACCCG
CGAATACCCGCTGGTGGACATGATCTGCCAGCTTCTCTCCAACGAGGCCGACCCGCTCAAGGG
CCGCCAGCTGCCGATCATGTACTCGAGCAAGGAGGCAGGTTTCTTCTCCATCTCCGGCAACCT
CGCCACCCAGTTCATCCAGGCGGTCGGCTGGGGCATGGCCTCGGCGATCAAGGGCGACACGCG
CATCGCCTCGGCCTGGATCGGCGACGGCGCCACCGCCGAGTCGGACTTCCACACCGCCCTCAC
CTTCGCCCATGTCTACCGCGCGCCGGTAATCCTCAACGTGGTCAACAACCAGTGGGCGATCTC
CACCTTCCAGGCCATCGCCGGCGGCGAAGGCACCACCTTCGCCAACCGTGGCGTGGGCTGCGG
GATCGCCTCGCTGCGGGTCGACGGCAATGACTTCCTGGCGGTCTACGCCGCCTCCGAGTGGGC
CGCCGAGCGCGCCCGGCGCAACCTCGGGCCGAGCCTGATCGAATGGGTCACCTACCGCGCCGG
CCCGCACTCGACTTCGGACGACCCGTCCAAGTACCGCCCCGCCGACGACTGGACCAACTTCCC
GCTGGGCGACCCGATCGCCCGCCTGAAGCGGCACATGATCGGCCTCGGCATCTGGTCGGAGGA
ACAGCACGAAGCCACCCACAAGGCCCTCGAAGCCGAAGTACTGGCCGCGCAGAAACAGGCGGA
GAGCCATGGCACCCTGATCGACGGCCGGGTGCCGAGCGCCGCCAGCATGTTCGAGGACGTCTA
TGCAGAACTGCCGGAGCATCTGCGCCGGCAACGCCAGGAGCTCGGGGTATGAATGCCATGAAC
CCGCAACACGAGAACGCCCAGACGGTCACCAGCATGACCATGATCCAGGCGCTGCGCTCGGCG
ATGGACATCATGCTCGAGCGCGACGACGACGTGGTGGTATTCGGCCAGGACGTCGGCTACTTC
GGCGGCGTGTTCCGCTGCACCGAAGGCCTGCAGAAGAAATACGGCACCTCGCGGGTGTTCGAT
GCGCCGATCTCCGAGAGCGGCATCATCGGCGCCGCGGTCGGCATGGGTGCCTACGGCCTGCGC
CCGGTGGTGGAGATCCAGTTCGCCGACTACGTCTACCCGGCCTCCGACCAGTTGATCTCCGAG
GCGGCGCGCCTGCGCTATCGCTCGGCCGGCGACTTCATCGTGCCGATGACCGTACGCATGCCC
TGTGGCGGCGGCATCTACGGCGGGCAAACGCACAGCCAGAGCCCGGAGGCGATGTTCACCCAG
GTCTGCGGCCTGCGCACGGTGATGCCGTCCAACCCCTACGACGCCAAGGGCCTGCTGATCGCC
TGCATCGAGAACGACGACCCGGTGATCTTCCTCGAGCCCAAGCGCCTCTACAACGGCCCGTTC
GATGGCCACCACGACCGCCCGGTGACGCCCTGGTCCAAGCATCCGGCCAGCCAGGTGCCGGAC
GGCTACTACAAGGTGCCGCTGGACAAGGCGGCGATCGTCCGCCCCGGCGCGGCGCTGACCGTG
CTGACCTACGGCACCATGGTCTACGTGGCCCAGGCCGCGGCCGACGAAACCGGCCTGGACGCC
GAGATCATCGACCTGCGCAGCCTCTGGCCGCTGGACCTGGAAACCATCGTCGCCTCGGTGAAG
AAGACCGGCCGCTGCGTCATCGCCCACGAGGCGACCCGCACCTGTGGGTTCGGCGCCGAGCTG
ATGTCGCTGGTGCAGGAGCACTGCTTCCACCACCTGGAGGCGCCGATCGAGCGCGTCACCGGT
TGGGACACCCCCTACCCGCATGCCCAGGAGTGGGCGTATTTCCCCGGCCCCGCGCGCGTCGGC
GCGGCATTCAAGCGTGTGATGGAGGTCTGAATGGGTACCCATGTGATCAAGATGCCGGACATC
GGGGAAGGCATCGCCGAGGTCGAACTGGTGGAGTGGCATGTCCAGGTCGGCGACTCGGTCAAT
GAAGACCAGGTCCTCGCCGAGGTGATGACCGACAAGGCCACGGTGGAGATTCCCTCGCCGGTG
GCCGGACGCATCCTCGCCCTCGGCGGCCAGCCGGGCCAGGTGATGGCGGTGGGCGGCGAACTG
ATCCGCCTGGAGGTGGAAGGCGCCGGCAACCTCGCCGAGAGTCCGGCCGCGGCGACGCCGGCC
GCGCCCGTCGCCGCCACCCCGGAGAAACCGAAGGAAGCCCCGGTCGCGGCGCCGAAAGCCGCC
GCCGAAGCGCCGCGCGCCTTGCGCGACAGCGAGGCGCCACGGCAGCGGCGCCAGCCCGGCGAA
CGCCCGCTGGCCTCCCCCGCGGTGCGCCAGCGCGCCCGCGACCTGGGCATCGAGTTGCAGTTC
GTGCAGGGCAGCGGTCCCGCCGGACGCGTCCTCCACGAGGACCTCGATGCCTACCTGACCCAG
GATGGCAGCGTCGCGCGCAGCGGCGGCGCCGCGCAGGGGTATGCCGAGCGACACGACGAACAG
GCGGTGCCGGTGATCGGCCTGCGTCGCAAGATCGCCCAGAAGATGCAGGACGCCAAGCGACGC
ATCCCGCATTTCAGCTATGTCGAGGAAATCGACGTCACCGATCTGGAAGCCCTGCGCGCCCAT
CTCAACCAGAAATGGGGTGGCCAGCGCGGCAAGCTGACCCTGCTGCCGTTCCTGGTCCGCGCC
ATGGTCGTGGCGCTGCGCGACTTCCCGCAGTTGAACGCGCGCTACGACGACGAGGCCGAGGTG
GTCACCCGCTACGGCGCGGTGCACGTCGGCATCGCCACCCAGAGCGACAACGGCCTGATGGTG
CCGGTGCTGCGCCACGCCGAATCGCGCGACCTCTGGGGCAACGCCAGCGAAGTGGCGCGCCTG
GCCGAAGCCGCACGCAGCGGCAAGGCGCAACGCCAGGAGCTGTCCGGCTCGACCATCACCCTG
AGCAGCCTCGGCGTGCTCGGCGGGATCGTCAGCACACCGGTGATCAACCATCCGGAGGTGGCC
ATCGTCGGCGTCAACCGCATCGTCGAGCGACCGATGGTGGTCGGCGGCAACATCGTCGTGCGC
AAGATGATGAACCTCTCCTCCTCCTTCGACCACCGGGTGGTCGACGGGATGGACGCGGCGGCC
TTCATCCAGGCCGTGCGCGGCCTGCTCGAACATCCCGCCACCCTGTTCCTGGAGTAAgcgATG
AGCCAGATCCTGAAGACTTCCCTGCTGATCGTCGGCGGCGGTCCCGGCGGCTACGTCGCGGCG
ATCCGTGCCGGGCAACTGGGCATTCCCACCGTACTGGTGGAGGGCGCCGCCCTCGGCGGCACC
TGTCTGAACGTCGGCTGCATCCCGTCGAAGGCGCTGATCCACGCCGCCGAGGAATACCTCAAG
GCCCGCCACTATGCCAGCCGGTCGGCGCTGGGCATCCAGGTACAGGCGCCGAGCATCGACATC
GCCCGCACCGTGGAATGGAAGGACGCCATCGTCGACCGCCTCACCAGCGGCGTCGCCGCGCTG
CTGAAGAAACACGGGGTCGATGTCGTCCAGGGCTGGGCGAGGATCCTCGACGGCAAAAGCGTG
GCGGTCGAACTCGCCGGCGGCGGCAGCCAGCGCATCGAGTGCGAGCATCTGCTGCTGGCCGCC
GGCTCGCAGAGCGTCGAGCTACCGATCCTGCCGCTGGGCGGCAAGGTGATCTCCTCCACCGAG
GCGCTGGCGCCCGGCAGCCTGCCCAAGCGCCTGGTGGTGGTCGGCGGCGGCTACATCGGCCTG
GAGCTGGGTACCGCCTACCGCAAGCTCGGCGTCGAGGTGGCGGTGGTGGAAGCGCAACCACGC
ATCCTGCCGGGCTACGACGAAGAACTGACCAAGCCGGTGGCCCAGGCCTTGCGCAGGCTGGGC
GTCGAGCTGTACCTCGGGCACAGCCTGCTGGGCCCGAGCGAGAACGGCGTGCGGGTCCGCGAC
GGCGCCGGCGAGGAGCGCGAGATCGCCGCCGACCAGGTACTGGTGGCGGTCGGCCGCAAGCCG
CGCAGCGAAGGCTGGAACCTGGAAAGCCTGGGCCTGGACATGAACGGCCGGGCGGTGAAGGTC
GACGACCAGTGCCGCACCTCGATGCGCAATGTCTGGGCCATAGGCGATCTCGCCGGCGAGCCG
ATGCTCGCGCACCGGGCCATGGCCCAGGGCGAGATGGTCGCCGAGCTGATCGCCGGCAAGCGT
CGCCAGTTCGCCCCGGTGGCGATCCCCGCGGTGTGCTTCACCGATCCGGAAGTGGTGGTCGCC
GGGTTGTCCCCGGAGCAGGCGAAGGATGCCGGCCTGGACTGCCTGGTGGCGAGCTTCCCGTTC
GCCGCCAACGGTCGCGCCATGACCCTGGAGGCCAACGAAGGCTTCGTCCGCGTGGTGGCGCGT
CGCGACAACCACCTGGTCGTCGGCTGGCAGGCGGTGGGCAAGGCGGTTTCGGAACTGTCCACG
GCCTTCGCCCAGTCGCTGGAGATGGGCGCCCGCCTGGAAGACATCGCCGGCACCATCCACGCC
CATCCGACCCTCGGCGAAGCGGTCCAGGAAGCCGCCCTGCGCGCGCTGGGACACGCCCTGCAC
ATCTGA 8 Tet-ldh-bkd
gtaaaacgacggccagtgaattcgTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCC
construct
GCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTC
(gene coding
GATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGC
regions are
GACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGC
shown in
ATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGT
uppercase)
TTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAG
TAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAA
AGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCG
CTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACG
GGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACT
TTTATCTAATCTAGACATcattaattcctaatttttgttgacactctatcattgatagagtta
ttttaccactccctatcagtgatagagaaaagtgaactctagaaataattttgtttaacttta
agaaggagatatacatATGTTCGACATGATGGACGCGGCCCGGCTCGAGGGTCTgCACCTCGC
CCAAGACCCGGCCACGGGACTCAAGGCCATTATCGCCATCCACAGCACGCGACTCGGCCCGGC
GCTGGGTGGTTGTCGCTACCTGCCTTACCCCAACGACGAAGCCGCCATCGGCGACGCCATCCG
CCTGGCCCAGGGCATGAGCTACAAGGCGGCCCTGGCCGGGCTGGAGCAGGGCGGCGGCAAGGC
GGTGATCATCCGCCCGCCGCACCTGGACAATCGCGGCGCGCTGTTCGAGGCCTTCGGGCGCTT
CATCGAAAGCCTCGGCGGACGCTACATCACTGCGGTGGACAGCGGTACCTCCAGCGCCGACAT
GGACTGCATCGCCCAGCAGACCCGCCACGTCACCAGCACCACCCAGGCCGGCGACCCCTCGCC
GCATACCGCCCTCGGCGTGTTCGCCGGGATTCGCGCCAGCGCCCAGGCGCGCCTCGGCAGCGA
CGACCTGGAAGGCCTGCGGGTCGCGGTGCAGGGGCTCGGCCACGTCGGCTACGCATTGGCCGA
GCAACTGGCGGCGGTCGGCGCCGAGCTGCTGGTCTGCGACCTCGATCCCGGCCGGGTGCAACT
GGCCGTCGAGCAGCTCGGTGCCCATCCGCTGGCGCCGGAGGCATTGCTCTCCACCCCTTGCGA
CATCCTCGCGCCCTGCGGCCTGGGCGGCGTGCTCACCAGCCAGAGCGTCAGCCAGTTGCGCTG
CGCGGCGGTGGCCGGGGCGGCGAACAACCAGTTGGAGCGGCCGGAGGTCGCCGACGAGCTGGA
GGCGCGCGGCATCCTCTATGCGCCGGACTACGTGATCAACTCCGGCGGCCTGATCTACGTCGC
CCTCAAGCACCGCGGCGCCGATCCGCACAGCATCACCGCGCACCTGGCGCGGATTCCCGCGCG
GCTCACCGAGATCTATGCCCATGCCCAGGCCGACCACCAGTCGCCGGCGCGGATCGCCGACCG
TCTGGCGGAACGGATTCTCTACGGCCCGCAGTGAgaaggagatatacatATGAGTGATTACGA
GCCGTTGCGTCTGCATGTCCCGGAGCCCACCGGGCGTCCTGGCTGCAAGACCGACTTTTCCTA
TCTGCACCTGTCCCCCGCCGGCGAGGTACGCAAGCCGCCGGTGGATGTCGAGCCCGCCGAaAC
CAGCGACCTGGCCTACAGCCTGGTACGTGTGCTCGACGACGACGGCCACGCCGTCGGTCCCTG
GAATCCGCAGCTCAGCAACGAACAACTGCTGCGCGGCATGCGGGCGATGCTCAAGACCCGCCT
GTTCGACGCGCGCATGCTCACCGCGCAACGGCAGAAAAAGCTTTCCTTCTATATGCAATGCCT
CGGCGAGGAAGCCATCGCCACCGCCCACACCCTGGCCCTGCGCGACGGCGACATGTGCTTTCC
GACCTATCGCCAGCAAGGCATCCTGATCACCCGCGAATACCCGCTGGTGGACATGATCTGCCA
GCTTCTCTCCAACGAGGCCGACCCGCTCAAGGGCCGCCAGCTGCCGATCATGTACTCGAGCAA
GGAGGCAGGTTTCTTCTCCATCTCCGGCAACCTCGCCACCCAGTTCATCCAGGCGGTCGGCTG
GGGCATGGCCTCGGCGATCAAGGGCGACACGCGCATCGCCTCGGCCTGGATCGGCGACGGCGC
CACCGCCGAGTCGGACTTCCACACCGCCCTCACCTTCGCCCATGTCTACCGCGCGCCGGTAAT
CCTCAACGTGGTCAACAACCAGTGGGCGATCTCCACCTTCCAGGCCATCGCCGGCGGCGAAGG
CACCACCTTCGCCAACCGTGGCGTGGGCTGCGGGATCGCCTCGCTGCGGGTCGACGGCAATGA
CTTCCTGGCGGTCTACGCCGCCTCCGAGTGGGCCGCCGAGCGCGCCCGGCGCAACCTCGGGCC
GAGCCTGATCGAATGGGTCACCTACCGCGCCGGCCCGCACTCGACTTCGGACGACCCGTCCAA
GTACCGCCCCGCCGACGACTGGACCAACTTCCCGCTGGGCGACCCGATCGCCCGCCTGAAGCG
GCACATGATCGGCCTCGGCATCTGGTCGGAGGAACAGCACGAAGCCACCCACAAGGCCCTCGA
AGCCGAAGTACTGGCCGCGCAGAAACAGGCGGAGAGCCATGGCACCCTGATCGACGGCCGGGT
GCCGAGCGCCGCCAGCATGTTCGAGGACGTCTATGCAGAACTGCCGGAGCAtCTGCGCCGGCA
ACGCCAGGAGCTCGGGGTATGAATGCCATGAACCCGCAACACGAGAACGCCCAGACGGTCACC
AGCATGACCATGATCCAGGCGCTGCGCTCGGCGATGGACATCATGCTCGAGCGCGACGACGAC
GTGGTGGTATTCGGCCAGGACGTCGGCTACTTCGGCGGCGTGTTCCGCTGCACCGAAGGCCTG
CAGAAGAAATACGGCACCTCGCGGGTGTTCGATGCGCCGATCTCCGAGAGCGGCATCATCGGC
GCCGCGGTCGGCATGGGTGCCTACGGCCTGCGCCCGGTGGTGGAGATCCAGTTCGCCGACTAC
GTCTACCCGGCCTCCGACCAGTTGATCTCCGAGGCGGCGCGCCTGCGCTATCGCTCGGCCGGC
GACTTCATCGTGCCGATGACCGTACGCATGCCCTGTGGCGGCGGCATCTACGGCGGGCAAACG
CACAGCCAGAGCCCGGAGGCGATGTTCACCCAGGTCTGCGGCCTGCGCACGGTGATGCCGTCC
AACCCCTACGACGCCAAGGGCCTGCTGATCGCCTGCATCGAGAACGACGACCCGGTGATCTTC
CTCGAGCCCAAGCGCCTCTACAACGGCCCGTTCGATGGCCACCACGACCGCCCGGTGACGCCC
TGGTCCAAGCATCCGGCCAGCCAGGTGCCGGACGGCTACTACAAGGTGCCGCTGGACAAGGCG
GCGATCGTCCGCCCCGGCGCGGCGCTGACCGTGCTGACCTACGGCACCATGGTCTACGTGGCC
CAGGCCGCGGCCGACGAaACCGGCCTGGACGCCGAGATCATCGACCTGCGCAGCCTCTGGCCG
CTGGACCTGGAAACCATCGTCGCCTCGGTGAAGAAGACCGGCCGCTGCGTCATCGCCCACGAG
GCGACCCGCACCTGtGGGTTCGGCGCCGAGCTGATGTCGCTGGTGCAGGAGCACTGCTTCCAC
CACCTGGAGGCGCCGATCGAGCGCGTCACCGGTTGGGACACCCCCTACCCGCATGCCCAGGAG
TGGGCGTATTTCCCCGGCCCCGCGCGCGTCGGCGCGGCATTCAAGCGTGTGATGGAGGTCTGA
ATGGGTACCCATGTGATCAAGATGCCGGACATCGGGGAAGGCATCGCCGAGGTCGAACTGGTG
GAGTGGCATGTCCAGGTCGGCGACTCGGTCAATGAAGACCAGGTCCTCGCCGAGGTGATGACC
GACAAGGCCACGGTGGAGATTCCCTCGCCGGTGGCCGGACGCATCCTCGCCCTCGGCGGCCAG
CCGGGCCAGGTGATGGCGGTGGGCGGCGAACTGATCCGCCTGGAGGTGGAAGGCGCCGGCAAC
CTCGCCGAGAGTCCGGCCGCGGCGACGCCGGCCGCGCCCGTCGCCGCCACCCCGGAGAAACCG
AAGGAAGCCCCGGTCGCGGCGCCGAAAGCCGCCGCCGAAGCGCCGCGCGCCTTGCGCGACAGC
GAGGCGCCACGGCAGCGGCGCCAGCCCGGCGAACGCCCGCTGGCCTCCCCCGCGGTGCGCCAG
CGCGCCCGCGACCTGGGCATCGAGTTGCAGTTCGTGCAGGGCAGCGGTCCCGCCGGACGCGTC
CTCCACGAGGACCTCGATGCCTACCTGACCCAGGATGGCAGCGTCGCGCGCAGCGGCGGCGCC
GCGCAGGGGTATGCCGAGCGACACGACGAACAGGCGGTGCCGGTGATCGGCCTGCGTCGCAAG
ATCGCCCAGAAGATGCAGGACGCCAAGCGACGCATCCCGCATTTCAGCTATGTCGAGGAAATC
GACGTCACCGATCTGGAAGCCCTGCGCGCCCATCTCAACCAGAAATGGGGTGGCCAGCGCGGC
AAGCTGACCCTGCTGCCGTTCCTGGTCCGCGCCATGGTCGTGGCGCTGCGCGACTTCCCGCAG
TTGAACGCGCGCTACGACGACGAGGCCGAGGTGGTCACCCGCTACGGCGCGGTGCACGTCGGC
ATCGCCACCCAGAGCGACAACGGCCTGATGGTGCCGGTGCTGCGCCACGCCGAATCGCGCGAC
CTCTGGGGCAACGCCAGCGAAGTGGCGCGCCTGGCCGAAGCCGCACGCAGCGGCAAGGCGCAA
CGCCAGGAGCTGTCCGGCTCGACCATCACCCTGAGCAGCCTCGGCGTGCTCGGCGGGATCGTC
AGCACACCGGTGATCAACCATCCGGAGGTGGCCATCGTCGGCGTCAACCGCATCGTCGAGCGA
CCGATGGTGGTCGGCGGCAACATCGTCGTGCGCAAGATGATGAACCTCTCCTCCTCCTTCGAC
CACCGGGTGGTCGACGGGATGGACGCGGCGGCCTTCATCCAGGCCGTGCGCGGCCTGCTCGAA
CATCCCGCCACCCTGTTCCTGGAGTAAgcgATGAGCCAGATCCTGAAGACTTCCCTGCTGATC
GTCGGCGGCGGTCCCGGCGGCTACGTCGCGGCGATCCGTGCCGGGCAACTGGGCATTCCCACC
GTACTGGTGGAGGGCGCCGCCCTCGGCGGCACCTGtCTGAACGTCGGCTGCATCCCGTCGAAG
GCGCTGATCCACGCCGCCGAGGAATACCTCAAGGCCCGCCACTATGCCAGCCGGTCGGCGCTG
GGCATCCAGGTACAGGCGCCGAGCATCGACATCGCCCGCACCGTGGAATGGAAGGACGCCATC
GTCGACCGCCTCACCAGCGGCGTCGCCGCGCTGCTGAAGAAACACGGGGTCGATGTCGTCCAG
GGCTGGGCGAGGATCCTCGACGGCAAAAGCGTGGCGGTCGAACTCGCCGGCGGCGGCAGCCAG
CGCATCGAGTGCGAGCAtCTGCTGCTGGCCGCCGGCTCGCAGAGCGTCGAGCTACCGATCCTG
CCGCTGGGCGGCAAGGTGATCTCCTCCACCGAGGCGCTGGCGCCCGGCAGCCTGCCCAAGCGC
CTGGTGGTGGTCGGCGGCGGCTACATCGGCCTGGAGCTGGGTACCGCCTACCGCAAGCTCGGC
GTCGAGGTGGCGGTGGTGGAAGCGCAACCACGCATCCTGCCGGGCTACGACGAAGAACTGACC
AAGCCGGTGGCCCAGGCCTTGCGCAGGCTGGGCGTCGAGCTGTACCTCGGGCACAGCCTGCTG
GGCCCGAGCGAGAACGGCGTGCGGGTCCGCGACGGCGCCGGCGAGGAGCGCGAGATCGCCGCC
GACCAGGTACTGGTGGCGGTCGGCCGCAAGCCGCGCAGCGAAGGCTGGAACCTGGAAAGCCTG
GGCCTGGACATGAACGGCCGGGCGGTGAAGGTCGACGACCAGTGCCGCACCTCGATGCGCAAT
GTCTGGGCCATAGGCGATCTCGCCGGCGAGCCGATGCTCGCGCACCGGGCCATGGCCCAGGGC
GAGATGGTCGCCGAGCTGATCGCCGGCAAGCGTCGCCAGTTCGCCCCGGTGGCGATCCCCGCG
GTGTGCTTCACCGATCCGGAAGTGGTGGTCGCCGGGTTGTCCCCGGAGCAGGCGAAGGATGCC
GGCCTGGACTGCCTGGTGGCGAGCTTCCCGTTCGCCGCCAACGGTCGCGCCATGACCCTGGAG
GCCAACGAAGGCTTCGTCCGCGTGGTGGCGCGTCGCGACAACCACCTGGTCGTCGGCTGGCAG
GCGGTGGGCAAGGCGGTtTCGGAACTGTCCACGGCCTTCGCCCAGTCGCTGGAGATGGGCGCC
CGCCTGGAAGACATCGCCGGCACCATCCACGCCCATCCGACCCTCGGCGAAGCGGTCCAGGAA
GCCGCCCTGCGCGCGCTGGGACACGCCCTGCACATCTGA 9 Tet-livKHMGF
ccagtgaattcgTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAA
construct
TTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCG
(gene coding
TAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTC
regions are
TTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATT
shown in
CTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTT
uppercase)
TTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCT
AAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTG
AGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTAC
ATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACC
TTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCT
AGACATcattaattcctaatttttgttgacactctatcattgatagagttattttaccactcc
ctatcagtgatagagaaaagtgaactctagaaataattttgtttaactttaagaaggagatat
acatATGAAACGGAATGCGAAAACTATCATCGCAGGGATGATTGCACTGGCAATTTCACACAC
CGCTATGGCTGACGATATTAAAGTCGCCGTTGTCGGCGCGATGTCCGGCCCGATTGCCCAGTG
GGGCGATATGGAATTTAACGGCGCGCGTCAGGCAATTAAAGACATTAATGCCAAAGGGGGAAT
TAAGGGCGATAAACTGGTTGGCGTGGAATATGACGACGCATGCGACCCGAAACAAGCCGTTGC
GGTCGCCAACAAAATCGTTAATGACGGCATTAAATACGTTATTGGTCATCTGTGTTCTTCTTC
TACCCAGCCTGCGTCAGATATCTATGAAGACGAAGGTATTCTGATGATCTCGCCGGGAGCGAC
CAACCCGGAGCTGACCCAACGCGGTTATCAACACATTATGCGTACTGCCGGGCTGGACTCTTC
CCAGGGGCCAACGGCGGCAAAATACATTCTTGAGACGGTGAAGCCCCAGCGCATCGCCATCAT
TCACGACAAACAACAGTATGGCGAAGGGCTGGCGCGTTCGGTGCAGGACGGGCTGAAAGCGGC
TAACGCCAACGTCGTCTTCTTCGACGGTATTACCGCCGGGGAGAAAGATTTCTCCGCGCTGAT
CGCCCGCCTGAAAAAAGAAAACATCGACTTCGTTTACTACGGCGGTTACTACCCGGAAATGGG
GCAGATGCTGCGCCAGGCCCGTTCCGTTGGCCTGAAAACCCAGTTTATGGGGCCGGAAGGTGT
GGGTAATGCGTCGTTGTCGAACATTGCCGGTGATGCCGCCGAAGGCATGTTGGTCACTATGCC
AAAACGCTATGACCAGGATCCGGCAAACCAGGGCATCGTTGATGCGCTGAAAGCAGACAAGAA
AGATCCGTCCGGGCCTTATGTCTGGATCACCTACGCGGCGGTGCAATCTCTGGCGACTGCCCT
TGAGCGTACCGGCAGCGATGAGCCGCTGGCGCTGGTGAAAGATTTAAAAGCTAACGGTGCAAA
CACCGTGATTGGGCCGCTGAACTGGGATGAAAAAGGCGATCTTAAGGGATTTGATTTTGGTGT
CTTCCAGTGGCACGCCGACGGTTCATCCACGGCAGCCAAGTGAtcatcccaccgcccgtaaaa
tgcgggcgggtttagaaaggttaccttATGTCTGAGCAGTTTTTGTATTTCTTGCAGCAGATG
TTTAACGGCGTCACGCTGGGCAGTACCTACGCGCTGATAGCCATCGGCTACACCATGGTTTAC
GGCATTATCGGCATGATCAACTTCGCCCACGGCGAGGTTTATATGATTGGCAGCTACGTCTCA
TTTATGATCATCGCCGCGCTGATGATGATGGGCATTGATACCGGCTGGCTGCTGGTAGCTGCG
GGATTCGTCGGCGCAATCGTCATTGCCAGCGCCTACGGCTGGAGTATCGAACGGGTGGCTTAC
CGCCCGGTGCGTAACTCTAAGCGCCTGATTGCACTCATCTCTGCAATCGGTATGTCCATCTTC
CTGCAAAACTACGTCAGCCTGACCGAAGGTTCGCGCGACGTGGCGCTGCCGAGCCTGTTTAAC
GGTCAGTGGGTGGTGGGGCATAGCGAAAACTTCTCTGCCTCTATTACCACCATGCAGGCGGTG
ATCTGGATTGTTACCTTCCTCGCCATGCTGGCGCTGACGATTTTCATTCGCTATTCCCGCATG
GGTCGCGCGTGTCGTGCCTGCGCGGAAGATCTGAAAATGGCGAGTCTGCTTGGCATTAACACC
GACCGGGTGATTGCGCTGACCTTTGTGATTGGCGCGGCGATGGCGGCGGTGGCGGGTGTGCTG
CTCGGTCAGTTCTACGGCGTCATTAACCCCTACATCGGCTTTATGGCCGGGATGAAAGCCTTT
ACCGCGGCGGTGCTCGGTGGGATTGGCAGCATTCCGGGAGCGATGATTGGCGGCCTGATTCTG
GGGATTGCGGAGGCGCTCTCTTCTGCCTATCTGAGTACGGAATATAAAGATGTGGTgTCATTC
GCCCTGCTGATTCTGGTGCTGCTGGTGATGCCGACCGGTATTCTGGGTCGCCCGGAGGTAGAG
AAAGTATGAAACCGATGCATATTGCAATGGCGCTGCTCTCTGCCGCGATGTTCTTTGTGCTGG
CGGGCGTCTTTATGGGCGTGCAACTGGAGCTGGATGGCACCAAACTGGTGGTCGACACGGCTT
CGGATGTCCGTTGGCAGTGGGTGTTTATCGGCACGGCGGTGGTCTTTTTCTTCCAGCTTTTGC
GACCGGCTTTCCAGAAAGGGTTGAAAAGCGTTTCCGGACCGAAGTTTATTCTGCCCGCCATTG
ATGGCTCCACGGTGAAGCAGAAACTGTTCCTCGTGGCGCTGTTGGTGCTTGCGGTGGCGTGGC
CGTTTATGGTTTCACGCGGGACGGTGGATATTGCCACCCTGACCATGATCTACATTATCCTCG
GTCTgGGGCTGAACGTGGTTGTTGGTCTTTCTGGTCTGCTGGTGCTGGGGTACGGCGGTTTTT
ACGCCATCGGCGCTTACACTTTTGCGCTGCTCAATCACTATTACGGCTTGGGCTTCTGGACCT
GCCTGCCGATTGCTGGATTAATGGCAGCGGCGGCGGGCTTCCTGCTCGGTTTTCCGGTGCTGC
GTTTGCGCGGTGACTATCTGGCGATCGTTACCCTCGGTTTCGGCGAAATTGTGCGCATATTGC
TGCTCAATAACACCGAAATTACCGGCGGCCCGAACGGAATCAGTCAGATCCCGAAACCGACAC
TCTTCGGACTCGAGTTCAGCCGTACCGCTCGTGAAGGCGGCTGGGACACGTTCAGTAATTTCT
TTGGCCTGAAATACGATCCCTCCGATCGTGTCATCTTCCTCTACCTGGTGGCGTTGCTGCTGG
TGGTGCTAAGCCTGTTTGTCATTAACCGCCTGCTGCGGATGCCGCTGGGGCGTGCGTGGGAAG
CGTTGCGTGAAGATGAAATCGCCTGCCGTTCGCTGGGCTTAAGCCCGCGTCGTATCAAGCTGA
CTGCCTTTACCATAAGTGCCGCGTTTGCCGGTTTTGCCGGAACGCTGTTTGCGGCGCGTCAGG
GCTTTGTCAGCCCGGAATCCTTCACCTTTGCCGAATCGGCGTTTGTGCTGGCGATAGTGGTGC
TCGGCGGTATGGGCTCGCAATTTGCGGTGATTCTGGCGGCAATTTTGCTGGTGGTGTCGCGCG
AGTTGATGCGTGATTTCAACGAATACAGCATGTTAATGCTCGGTGGTTTGATGGTGCTGATGA
TGATCTGGCGTCCGCAGGGCTTGCTGCCCATGACGCGCCCGCAACTGAAGCTGAAAAACGGCG
CAGCGAAAGGAGAGCAGGCATGAGTCAGCCATTATTATCTGTTAACGGCCTGATGATGCGCTT
CGGCGGCCTGCTGGCGGTGAACAACGTCAATCTTGAACTGTACCCGCAGGAGATCGTCTCGTT
AATCGGCCCTAACGGTGCCGGAAAAACCACGGTTTTTAACTGTCTGACCGGATTCTACAAACC
CACCGGCGGCACCATTTTACTGCGCGATCAGCACCTGGAAGGTTTACCGGGGCAGCAAATTGC
CCGCATGGGCGTGGTGCGCACCTTCCAGCATGTGCGTCTGTTCCGTGAAATGACGGTAATTGA
AAACCTGCTGGTGGCGCAGCATCAGCAACTGAAAACCGGGCTGTTCTCTGGCCTGTTGAAAAC
GCCATCCTTCCGTCGCGCCCAGAGCGAAGCGCTCGACCGCGCCGCGACCTGGCTTGAGCGCAT
TGGTTTGCTGGAACACGCCAACCGTCAGGCGAGTAACCTGGCCTATGGTGACCAGCGCCGTCT
TGAGATTGCCCGCTGCATGGTGACGCAGCCGGAGATTTTAATGCTCGACGAACCTGCGGCAGG
TCTTAACCCGAAAGAGACGAAAGAGCTGGATGAGCTGATTGCCGAACTGCGCAATCATCACAA
CACCACTATCTTGTTGATTGAACACGATATGAAGCTGGTGATGGGAATTTCGGACCGAATTTA
CGTGGTCAATCAGGGGACGCCGCTGGCAAACGGTACGCCGGAGCAGATCCGTAATAACCCGGA
CGTGATCCGTGCCTATTTAGGTGAGGCATAAGATGGAAAAAGTCATGTTGTCCTTTGACAAAG
TCAGCGCCCACTACGGCAAAATCCAGGCGCTGCATGAGGTGAGCCTGCATATCAATCAGGGCG
AGATTGTCACGCTGATTGGCGCGAACGGGGCGGGGAAAACCACCTTGCTCGGCACGTTATGCG
GCGATCCGCGTGCCACCAGCGGGCGAATTGTGTTTGATGATAAAGACATTACCGACTGGCAGA
CAGCGAAAATCATGCGCGAAGCGGTGGCGATTGTCCCGGAAGGGCGTCGCGTCTTCTCGCGGA
TGACGGTGGAAGAGAACCTGGCGATGGGCGGTTTTTTTGCTGAACGCGACCAGTTCCAGGAGC
GCATAAAGTGGGTGTATGAGCTGTTTCCACGTCTGCATGAGCGCCGTATTCAGCGGGCGGGCA
CCATGTCCGGCGGTGAACAGCAGATGCTGGCGATTGGTCGTGCGCTGATGAGCAACCCGCGTT
TGCTACTGCTTGATGAGCCATCGCTCGGTCTTGCGCCGATTATCATCCAGCAAATTTTCGACA
CCATCGAGCAGCTGCGCGAGCAGGGGATGACTATCTTTCTCGTCGAGCAGAACGCCAACCAGG
CGCTAAAGCTGGCGGATCGCGGCTACGTGCTGGAAAACGGCCATGTAGTGCTTTCCGATACTG
GTGATGCGCTGCTGGCGAATGAAGCGGTGAGAAGTGCGTATTTAGGCGGGTAA 10 livJ
ATGAACATAAAGGGTAAAGCGTTACTGGCAGGATGTATCGCGCTGGCATTCAGCAATATGGCT
(Escherichia
CTGGCAGAAGATATTAAAGTCGCGGTCGTGGGCGCAATGTCCGGTCCGGTTGCGCAGTACGGT
coli)
GACCAGGAGTTTACCGGCGCAGAGCAGGCGGTTGCGGATATCAACGCTAAAGGCGGCATTAAA
GGCAACAAACTGCAAATCGTAAAATATGACGATGCCTGTGACCCGAAACAGGCGGTTGCGGTG
GCGAACAAAGTCGTTAACGACGGCATTAAATATGTGATTGGTCACCTCTGTTCTTCATCAACG
CAGCCTGCGTCTGACATCTACGAAGACGAAGGCATTTTAATGATCACCCCAGCGGCAACCGCG
CCGGAGCTGACCGCCCGTGGCTATCAGCTGATCCTGCGCACCACCGGCCTGGACTCCGACCAG
GGGCCGACGGCGGCGAAATATATTCTTGAGAAAGTGAAACCGCAGCGTATTGCTATCGTTCAC
GACAAACAGCAATACGGCGAAGGTCTGGCGCGAGCGGTGCAGGACGGCCTGAAGAAAGGCAAT
GCAAACGTGGTGTTCTTTGATGGCATCACCGCCGGGGAAAAAGATTTCTCAACGCTGGTGGCG
CGTCTGAAAAAAGAGAATATCGACTTCGTTTACTACGGCGGTTATCACCCGGAAATGGGGCAA
ATCCTGCGTCAGGCACGCGCGGCAGGGCTGAAAACTCAGTTTATGGGGCCGGAAGGTGTGGCT
AACGTTTCGCTGTCTAACATTGCGGGCGAATCAGCGGAAGGGCTGCTGGTGACCAAGCCGAAG
AACTACGATCAGGTTCCGGCGAACAAACCCATTGTTGACGCGATCAAAGCGAAAAAACAGGAC
CCAAGTGGCGCATTCGTTTGGACCACCTACGCCGCGCTGCAATCTTTGCAGGCGGGCCTGAAT
CAGTCTGACGATCCGGCTGAAATCGCCAAATACCTGAAAGCGAACTCCGTGGATACCGTAATG
GGACCGCTGACCTGGGATGAGAAAGGCGATCTGAAAGGCTTTGAGTTCGGCGTATTTGACTGG
CACGCCAACGGCACGGCGACCGATGCGAAGTAA 11 leucine
GTGTTCGCTGAATACGGGGTTCTGAATTACTGGACCTATCTGGTTGGGGCCATTTTTATTGTG
exporter
TTGGTGCCAGGGCCAAATACCCTGTTTGTACTCAAAAATAGCGTCAGTAGCGGTATGAAAGGC
gene leuE
GGTTATCTTGCGGCCTGTGGTGTATTTATTGGCGATGCGGTATTGATGTTTCTGGCATGGGCT
(Escherichia
GGAGTGGCGACATTAATTAAGACCACCCCGATATTATTCAACATCGTACGTTATCTTGGTGCG
coli Nissle
TTTTATTTGCTCTATCTGGGGAGTAAAATTCTCTACGCGACCCTGAAAGGTAAAAATAGCGAG
1917)
ACCAAATCCGATGAGCCCCAATACGGTGCCATTTTTAAACGCGCGTTAATTTTGAGCCTGACT
AATCCGAAAGCCATTTTGTTCTATGTGTCGTTTTTCGTACAGTTTATCGATGTTAATGCCCCA
CATACGGGAATTTCATTCTTTATTCTGGCGACGACGCTGGAACTGGTGAGTTTCTGCTATTTG
AGCTTCCTGATTATTTCTGGGGCTTTTGTCACGCAGTACATACGTACCAAAAAGAAACTGGCT
AAAGTGGGCAACTCACTGATTGGTTTGATGTTCGTGGGTTTCGCCGCCCGACTGGCGACGCTG
CAATCCTGA 12 Arginine ATGATGAAAG TGTTAATCGT GGAATCTGAA TTTCTGCACC
AGGATACGTG decarboxylase GGTCGGTAAC GCTGTTGAAC GTCTGGCCGA
TGCTTTAAGC CAGCAAAATG (Escherichia TGACAGTTAT CAAATCCACC TCTTTTGACG
ATGGCTTTGC CATTCTGTCA coli) AGCAATGAAG CCATCGATTG TCTGATGTTC
TCGTACCAGA TGGAACACCC CGATGAGCAC CAAAATGTTC GTCAGCTGAT CGGCAAACTT
CACGAACGTC AACAGAACGT ACCGGTCTTT CTGTTAGGCG ACCGCGAAAA GGCCTTGGCG
GCTATGGATC GCGATCTGCT GGAGTTGGTC GACGAGTTTG CCTGGATTCT CGAGGATACG
GCGGATTTTA TTGCCGGTCG CGCAGTCGCC GCCATGACGC GCTACCGCCA ACAGCTGCTC
CCGCCGCTGT TTTCTGCCCT GATGAAATAC TCGGACATTC ACGAATACAG CTGGGCAGCT
CCCGGGCACC AGGGCGGCGT TGGCTTCACG AAAACCCCAG CTGGTCGCTT TTATCATGAC
TACTACGGCG AGAATTTATT TCGTACCGAC ATGGGCATTG AACGTACCAG CCTGGGCTCG
CTGCTGGACC ACACGGGCGC TTTTGGGGAA TCAGAGAAAT ATGCAGCACG CGTGTTCGGT
GCGGACCGCA GTTGGTCCGT CGTGGTGGGC ACCAGTGGTA GCAACCGCAC CATTATGCAG
GCGTGCATGA CCGATAATGA TGTGGTAGTG GTGGATCGCA ATTGTCATAA GAGCATCGAA
CAAGGCTTGA TGCTGACTGG CGCTAAACCA GTCTATATGG TGCCGTCCCG TAATCGCTAT
GGTATTATCG GCCCGATTTA TCCTCAGGAG ATGCAGCCGG AAACCCTCCA GAAGAAAATC
TCAGAGTCCC CGTTAACTAA AGATAAAGCT GGGCAAAAAC CGAGTTATTG TGTAGTAACT
AATTGTACGT ATGATGGTGT TTGCTATAAC GCTAAGGAGG CCCAAGATCT TCTGGAAAAA
ACAAGTGATC GTCTTCATTT TGATGAAGCT TGGTACGGTT ATGCGCGTTT CAACCCTATT
TACGCCGACC ACTATGCGAT GCGTGGTGAA CCTGGGGATC ATAATGGCCC TACTGTGTTT
GCCACCCATT CTACGCATAA ACTCCTGAAT GCGTTGTCAC AGGCGAGTTA CATCCACGTA
CGCGAAGGCC GTGGCGCTAT TAATTTTAGC CGCTTTAACC AGGCCTATAT GATGCACGCG
ACGACAAGTC CGCTGTATGC GATTTGCGCG TCCAACGATG TTGCGGTCAG CATGATGGAC
GGCAACAGCG GTCTGTCGTT AACCCAGGAA GTGATTGATG AAGCGGTCGA CTTTCGCCAG
GCGATGGCCC GTCTGTACAA AGAATTCACC GCCGATGGCT CGTGGTTCTT CAAACCCTGG
AATAAAGAAG TCGTGACTGA CCCGCAGACG GGCAAAACTT ATGATTTTGC AGATGCCCCG
ACGAAGCTTC TTACTACGGT CCAGGATTGC TGGGTGATGC ACCCGGGGGA GTCTTGGCAT
GGCTTCAAAG ATATCCCTGA TAACTGGTCT ATGCTCGACC CAATCAAAGT TTCAATTTTA
GCTCCAGGCA TGGGCGAAGA TGGCGAACTG GAAGAGACGG GGGTACCAGC TGCGTTGGTT
ACCGCCTGGT TAGGCCGCCA TGGTATTGTT CCAACACGTA CCACTGATTT TCAGATTATG
TTTCTGTTCA GTATGGGTGT GACGCGCGGT AAATGGGGGA CGCTGGTCAA CACTCTCTGC
TCCTTTAAAC GCCATTATGA TGCGAACACG CCCCTGGCGC AAGTCATGCC AGAGCTGGTG
GAACAATACC CTGATACTTA TGCGAACATG GGTATCCACG ATCTGGGAGA TACTATGTTC
GCCTGGCTTA AAGAAAATAA CCCGGGGGCC CGCCTGAACG AAGCATATAG TGGCCTGCCC
ATGGCGGAAA TTACTCCGCG TGAAGCCTAT AATGCCATCG TTGATAATAA CGTCGAATTA
GTATCCATCG AGAACCTCCC CGGTCGTATT GCGGCAAATA GCGTAATCCC GTACCCGCCG
GGTATTCCCA TGCTGCTCAG CGGCGAAAAC TTCGGTGATA AAAATTCCCC GCAAGTTTCT
TATCTGCGCA GCCTGCAATC GTGGGACCAT CACTTTCCCG GGTTTGAGCA TGAAACTGAA
GGGACAGAGA TCATCGATGG CATTTATCAT GTGATGTGCG TCAAGGCG 13 ArgT
ATGAAAAAAA GCATCCTCGC GCTGTCACTG TTAGTGGGTC TCAGCGCCGC (Escherichia
GGCCAGCAGC TATGCTGCTC TTCCTGAAAC GGTGCGCATC GGGACGGATA coli)
CCACTTATGC ACCGTTTAGC AGCAAAGATG CTAAAGGAGA CTTCGTAGGG TTTGATATCG
ATTTAGGCAA CGAGATGTGC AAACGTATGC AAGTGAAATG TACCTGGGTG GCTTCAGACT
TTGATGCATT AATCCCGAGT TTGAAAGCAA AAAAAATTGA CGCAATTATT TCGAGCCTGA
GCATTACAGA TAAGCGCCAA CAAGAAATTG CCTTCTCAGA TAAATTATAT GCCGCTGATT
CGCGTCTTAT CGCGGCTAAA GGCTCCCCTA TCCAACCAAC GTTGGACAGC CTGAAGGGGA
AACATGTAGG GGTTCTGCAA GGGTCCACGC AGGAAGCTTA CGCCAATGAA ACCTGGCGTT
CGAAAGGGGT CGATGTGGTG GCGTACGCCA ATCAGGACTT GGTGTATTCC GATCTGGCCG
CAGGTCGTCT GGACGCAGCT CTGCAGGACG AAGTGGCGGC GAGTGAGGGT TTCCTGAAAC
AGCCAGCAGG CAAAGATTTT GCGTTCGCCG GCTCGAGTGT AAAGGATAAA AAATATTTCG
GGGATGGCAC GGGTGTCGGT TTACGCAAAG ATGATGCAGA ACTGACCGCG GCGTTTAATA
AAGCCCTTGG CGAACTGCGC CAAGACGGCA CATATGATAA AATGGCGAAA AAGTACTTTG
ACTTCAATGT TTATGGTGAT 14 artP ATGTCTATTC AATTAAATGG CATCAACTGT
TTCTACGGTG CACATCAAGC (Escherichia CTTATTTGAC ATCACGCTTG ATTGCCCGCA
AGGGGAGACA CTGGTGCTGC coli) TGGGCCCGAG TGGAGCCGGC AAATCGTCGT
TGCTGCGGGT GTTGAACCTG TTGGAGATGC CGCGCTCAGG CACCCTGAAT ATCGCGGGCA
ACCATTTCGA TTTTACGAAA ACACCGTCCG ATAAAGCTAT TCGTGATCTT CGTCGCAACG
TCGGCATGGT GTTTCAGCAG TATAATTTAT GTGCTCATCT GACGGTTCAG CAAAATCTGA
TCGAAGCACC GTGTCGTGTG TTGGGCCTGA GCAAAGACCA AGCCCTGGCC AGCGCAGAAA
AATTATTAGA GCGCCTGCGC TTGAAACCAT ATTCGGATCG GTACCCACTT CACTTAAGCG
GGGGCCAGCA ACAGCGCGTT GCCATCGCTC GTGCGCTGAT GATGGAGCCG CAAGTTCTCC
TTTTTGATGA ACCTACCGCA GCGCTTGATC CGGAGATCAC GGCGCAGATC GTCAGCATCA
TTCGTGAACT CGCTGAGACG AATATTACAC AAGTTATTGT GACACATGAG GTAGAAGTGG
CTCGCAAGAC CGCGTCTCGC GTAGTGTATA TGGAAAACGG TCATATCGTG GAGCAAGGGG
ACGCCTCATG TTTTACAGAG CCGCAGACAG AGGCATTCAA AAATTATCTG AGCCAC 15
artI ATGAAAAAAG TGCTTATTGC CGCCCTGATT GCGGGCTTCT CTCTGTCTGC
(Escherichia CACCGCGGCC GAAACCATCC GTTTTGCCAC TGAAGCGTCA TATCCCCCTT
coli) TCGAAAGCAT TGACGCCAAC AACCAAATTG TCGGTTTCGA CGTTGACCTC
GCGCAGGCCC TGTGCAAAGA AATTGATGCC ACCTGCACCT TCTCTAACCA AGCGTTTGAC
TCATTGATTC CTTCGCTGAA ATTTCGTCGC GTGGAAGCCG TCATGGGCGG CATGGATATC
ACCCCCGAGC GCGAAAAACA GGTCTTGTTT ACTACACCGT ACTACGACAA CTCGGCTTTG
TTTGTCGGCC AGCAAGGCAA GTATACTTCT GTCGACCAGC TGAAAGGTAA AAAAGTCCGT
TCAGTCCAGA ACGGCACCAC TCACCAGAAA TTCATCATGG ACAAACATCC TGAGATCACT
ACCGTGCCGT ATGATTCTTA CCAGAACGCG AAGTTAGATC TGGAAAATGG TCGGATTGAT
GGCGTCTTTG GCGACACCGC TGTGGTACAT GAATGGCTGA AAGACAATCC TAAATTAGTG
GTTGTGGGAG ATAAGGTTAC GGATAAGGAT TATTTTGGCA CCGGTCTCGG CATTGCAGTC
CGCCAAGGTA ATACCGAATT GCAACAGAAA TTGAATACCG CGCTGGAAAA AGTGAAAAAA
GACGGTACAT ACGAAACCAT TTACAACAAA TGGTTTCAAA AA 16 artQ ATGAACGAAT
TCTTCCCTCT CGCGTCTGCG GCAGGTATGA CCGTGGGTTT (Escherichia GGCGGTTTGT
GCGCTGATTG TCGGTCTCGC TCTGGCAATG TTCTTTGCCG coli) TATGGGAGTC
AGCGAAATGG CGTCCGGTCG CCTGGGCAGG TTCCGCCCTG GTAACCATTC TGCGTGGTCT
GCCAGAGATC CTGGTAGTTC TGTTTATCTA CTTTGGCTCT TCTCAGTTAC TGTTAACACT
GTCTGACGGG TTTACGATTA ACCTGGGTTT TGTCCAGATT CCGGTCCAGA TGGATATTGA
AAATTTCGAC GTCTCCCCTT TTCTCTGTGG CGTCATCGCG CTGAGCTTGC TCTACGCTGC
ATATGCATCA CAGACCCTTC GTGGTGCATT AAAAGCGGTG CCAGTAGGAC AGTGGGAAAG
CGGCCAGGCC CTTGGCCTGA GCAAGAGCGC AATTTTTTTC CGCCTTGTTA TGCCGGCCGA
TGTCCGCCAT GCGTTACCAG GTCTGGGTAA TCAATGGCTG GTGTTGTTGA AAGACACCGC
CCTTGTCTCG CTGATTAGCG TGAACGATTT AATGCTGCAA ACCAAATCGA TTGCAACCCG
CACTCAGGAA CCGTTTACCT GGTACATCGT GGCGGCAGCA ATCTATCTGG TGATCACACT
TCTGAGCCAG TATATTTTAA AACGTATTGA CCTGCGTGCC ACCCGCTTTG AGCGCCGCCC
TAGC 17 artM ATGTTTGAAT ATCTGCCGGA ACTGATGAAA GGTTTGCATA CTAGTCTGAC
(Escherichia GCTGACCGTC GCGAGTCTGA TCGTTGCGCT TATCCTGGCA CTGATCTTCA
coli) CCATTATTCT GACTCTCAAG ACCCCGGTCC TGGTGTGGCT GGTCCGCGGT
TACATTACCT TATTCACCGG GACCCCGCTC TTGGTTCGCA TTTTTCTTAT TTACTATGGT
CCGGGTCAGT TTCCGACCTT GCAAGAATAT CCTGCGTTAT GGCACCTGCT GTCTGAACCG
TGGCTGTGCG CTCTGATTGC TCTGAGTGTT AACTCGGCGG CCTATACGAC ACAGCTGTTC
TACGGTGCTA TTCGTGCGAT CCCAGAAGGT CAATGGCAGT CTTGTAGCGC ACTGGGCATG
TCAAAGAAAG ATACTCTTGC TATTCTGCTG CCGTACGCTT TTAAACGCTC TCTGAGCTCG
TACAGCAATG AAGTTGTCCT GGTTTTCAAA AGCACTAGCT TAGCGTATAC GATCACGCTG
ATGGAAGTCA TGGGTTATAG CCAGTTATTA TATGGTCGCA CGTACGACGT CATGGTGTTT
GGTGCAGCGG GCATTATCTA TCTTGTAGTT
AATGGATTAC TGACGTTAAT GATGCGCTTG ATCGAACGCA AAGCCGTGGC ATTCGAGCGG
CGTAAT 18 artJ ATGAAAAAAT TGGTGCTTGC AGCACTGCTG GCCAGTTTCA
CTTTCGGCGC (Escherichia TTCGGCGGCC GAAAAGATTA ATTTCGGTGT CAGCGCAACT
TACCCACCGT coli) TCGAAAGCAT CGGTGCGAAC AATGAGATTG TAGGATTTGA
TATCGATCTG GCCAAAGCGT TATGCAAACA AATGCAAGCG GAGTGCACTT TTACCAATCA
TGCGTTTGAT AGCCTGATCC CGTCGCTGAA GTTCCGTAAA TACGACGCCG TGATTTCGGG
GATGGACATC ACCCCTGAGC GCTCGAAACA GGTGAGCTTC ACCACTCCAT ATTATGAAAA
CTCAGCGGTG GTGATTGCGA AAAAAGACAC CTATAAAACA TTTGCCGACC TGAAAGGGAA
ATGTATTGGT ATGGAGAACG GCACCACCCA TCAGAAGTAT ATTCAAGACC AGCACCCGGA
GGTTAAGACC GTAAGCTACG ACTCCTACCA GAATGCTTTC ATTGATTTAA AAAATGGTCG
TATTGATGGT GTATTCGGAG ATACAGCCGT GGTGAATGAG TGGCTGAAAA CCAATCCGCA
GTTGGGTGTT GCGACCGAAA AAGTGACAGA TCCACAATAC TTTGGGACTG GCCTGGGCAT
CGCGGTGCGC CCGGATAACA AAGCCCTGTT GGAGAAACTG AACAACGCGT TAGCTGCGAT
TAAAGCGGAT GGGACCTATC AGAAGATTTC AGACCAATGG TTCCCGCAA 19 ArgO
ATGTTCTCGT ACTATTTCCA AGGCTTAGCA CTGGGTGCGG CCATGATCTT (Escherichia
ACCGCTGGGC CCACAAAACG CTTTTGTTAT GAACCAGGGA ATCCGCCGGC coli)
AGTACCATAT CATGATTGCG CTGCTGTGTG CCATCTCGGA TCTGGTCCTG ATTTGCGCCG
GTATTTTTGG CGGGTCGGCG TTACTTATGC AAAGCCCTTG GCTGCTGGCG CTGGTAACGT
GGGGCGGCGT AGCATTTCTG CTTTGGTATG GATTCGGCGC CTTCAAAACT GCGATGAGTT
CGAATATCGA GCTTGCGAGT GCTGAGGTAA TGAAACAGGG CCGTTGGAAA ATTATTGCGA
CCATGTTAGC CGTGACTTGG TTGAACCCGC ACGTGTACCT GGATACTTTT GTGGTGTTGG
GTTCACTCGG TGGGCAATTA GATGTGGAAC CGAAACGCTG GTTTGCCTTG GGCACAATCT
CGGCCAGTTT TTTGTGGTTC TTCGGGCTGG CGCTGCTGGC CGCGTGGCTG GCACCACGTT
TACGCACCGC CAAGGCCCAG CGCATCATCA ACTTAGTCGT GGGCTGTGTG ATGTGGTTCA
TTGCTCTGCA ACTGGCGCGC GATGGCATTG CGCACGCCCA GGCCCTGTTC TCA 20 [001]
mono- ATGATGCGCT TTGGCATCAT TAAAGAACGT AAGAACCCGC CAGATCGTCG
functional TGTAGTGTTT ACACCGTCCG AACTGATCAA ACTGAAAGAA CAGTTTCCGC
lysine- TGGCCGAAAT TAAGGTGGAA TCCTCAGATA TTCGCATTTT TTCTGATGAT
ketoglutarate GAGTATCGTA AACTTGGATT TGAAGTAACC GATGACCTGA
GTGATTGTGA reductase TGTCTTGATT GGCGTGAAAG AAGTACCGAT CGATGCCCTG
CTGCCCGGGA (Flavobacterium AAAAGTATTT TTTTTTCTCT CACACAATTA
AAAAACAGCC TTACAATAAA limnosedimin AAACTGCTGA TCGCCTGCTT GGAAAAAAAC
ATCCGTCTGA TTGATCATGA is JC2902) GACGATCGTG AATGAAGATA ATCATCGTTT
GATTGGGTTC GGCCGTTACG CAGGTATCGT GGGGGCCTAT AACGGTTTCC GTGCTTTTGG
TATTAAGTAC GAGCTCTTTA ACCTGCCCAA AGCGGAAACC TTAGCGGACA AAACGGCACT
TGTGGAACGC CTGCGTCGGC CGATGCTGCC GCCAATCAAA ATTGTGTTGA CCGGTCACGG
CAAAGTAGGT ATGGGTGCAA AAGAGATTCT GGATGCCATG AAAATCAAAC AAGTTTCCGT
GGAGGACTAC TTAACAAAAA CCTATGACAA GCCGGTGTAT ACGCAGATCG ACGTTCTGGA
CTATAACAAG CGGAAAGATG GCAAACCGGC GGAACGTGAA CACTTTTATG CCAATCCGCA
GGAGTATGTC TCGGACTTCG AACGCTTTAC CAAGGTGTCG GATCTGTTCA TCGCAGGCCA
TTTCTATGGC AACGGTGCAC CGGTAATTCT GACTCGCACC ATGCTTAACG CTTCTGATAA
TAAAATTAAA GTAGTTGCGG ATATTAGCTG TGATGTCGGT GGCCCTATCG AATGTACGCT
GCGCAGCAGC ACCATCGCAG AGCCGTTTTA TGGTTATTAT CCTTCCGAAG GTAAAGAAGT
CGACGTCAAC CATCCGGGCG CGGTGGTTGT GATGGCGGTG GACAATCTGC CCTGCGAGCT
GCCTAAAGAT GCCAGCGAGG GTTTCGGAGA AATGTTTCTC AAACATGTGA TTCCAGCCTT
CTACAACAAC GATAAGGACG GCATTCTTGA GCGGGCCAAA ATCACCGAAA ACGGCAAATT
AACAAAACGC TTCTCCTACT TACAGGACTA TGTCGATGGT GAA 21 saccharopine
ATGCGTAATA TTTTGATTAT CGGCGCCGGT CGGTCCGCTT CCTCGCTGAT
dehydrogenase TCAGTACTTA TTGAATAAGT CCCAAGAAGA ACAGCTGCAT
TTAACCATTG (Flavobacterium CCGATTTATC ACTCGAACTG GCTCAGAAGA
AAACCAATAA CCATCCGAAC sp. GCTACCGCGC TGGCGCTGGA TATTTATAAT
AAGGATGAAC GTCGTGCGGC EM1321) CATCGAGAAA GCGGCCATTG TGATCAGCAT
GTTGCCAGCG CATCTGCATA [002] TCGAAATCGC CCGGGATTGC CTGTATTTTA
AAAAGAACCT TGTTACGGCG AGCTATATTA GTGACGCGAT GCAGGAGCTT GATGCGGAAG
TTAAAGAGAA CAAACTGATC TTTATGAATG AGGTCGGTTT AGACCCGGGT ATTGATCATA
TGAGCGCCAT GAAAGTCATC GATGAAATTC GGGAACAAGG CGGCAAAATG CTTCTCTTCG
AAAGTTTTTG CGGCGGCCTG GTGGCACCAG AATCAGATAA CAATTTATGG AACTATAAAT
TTACCTGGGC CCCACGTAAC GTAGTTCTGG CTGGCCAGGG TGGTGTGGCA AAATTCATTC
AAGAAGGCAC CTATAAATAT ATCCCGTATG ACAGCTTATT TCGCCGGACC GAGTTTCTGG
AAGTAGAAGG ATACGGGCGT TTCGAAGCTT ATTCGAATCG CGATTCTCTC AAATATCGGA
GTATTTATGG GCTCGATGAC GTTCTCACCC TGTTTCGTGG TACAATCCGT CGCGTTGGCT
TCTCCAAAGC TTGGAACATG TTTGTGCAAC TGGGCATGAC GGACGACAGC TATGTTATGG
AAGATTCTGA GAATATGTCC TATCGTCAAT TTATTAACTC ATTCCTGCCT TATCACCCAA
CCGATAGCGT TGAAATTAAG ACCCGTTTTT TGTTAAAAAT CGATCAGGAT GATATCATGT
GGGACAAACT GCTGGAACTG GATCTTTTCA ACGATAAAAA AATGGTTGGG TTGAAAAATG
CGACGCCGGC ACAGATCCTG GAGAAAATCC TGAACGATTC GTGGACCCTG CAACCGGAAG
ATAAAGATAT GATCGTGATG TATCATAAAT TTGGTTACCA GATCAACGGC GAAAAAGTGC
AGATGGATTC ACAGATGGTG TGTATCGGCC AGGACCAAAC GTATACCGCG ATGGCAAAAA
CCGTCGGCCT GCCTGTGGCA ATGGCAACTC TGCTGATTCT GAACGGTAAA ATCAAAACAA
CGGGAGTTCA GTTGCCAATC AATAAAGAAG TTTACCTGCC GGTCCTGGAG GAACTGGAGA
AATATGGCGT TGTGTTCAAA GAACAGATGC TCCCATATCT TGGATACAAA TATAGT 22
Lysine ATG AAG AAA AAT CAT TCC TTG CAG TCG CTT AAA AAC CAA GAT GAG
CGT aminotransferase TTC ATT TGG CAC TCG ATG AAG CCG TAT AAC CCC
GAC AAG ACG ATC GT (Bacillus T GTCACC AAG GCC GAA GGA TCA TGG ATT
ACA ACG AGT GAT GGA AAG AA methanolicus G TAT CTT GAC GCA ATG GCC
GGT CTT TGG TGC GTT AAC GTG GGG TAT G PB1) GA CGC AAAGAG CTT GCC
GAT GCC GCG TAC GAA CAG ATG ATG GAA ATG G [003] CA TAC TAT CCA CTG
ACT CAG TCA CAT GTA CCC GCC ATT CAG TTA GCG GAG AAG TTG AACGAT CTG
CTG GAA GAC GAA TAC GTA ATC TTT TTT AGC AAT TCG GGG AGT GAG GCG AAC
GAG GCT GCT TTT AAA ATT GCT CGT CAG TAT CAT CAA CAA AAAGGA GAC CAC
AAT CGC TAT AAG ATT GTT GCA CGC TAC CGT GCA TAT CAT GGG AAC TCA ATT
GGA GCC TTG GCA GCG ACA GG G CAG GCC CAG CGT AAA TATAAG TAT GAG CCT
CTG GCC TTT GGA TTC GT C CAT GTT GCC CCT CCT GAC TCC TAC CGT GAT
GAA ACT AAC GTA TCC G AT CCT TCG CAG TTG TCC GCA GTCAAA GAA ATT GAC
CGT GTA ATG ACG T GG GAG CTT TCG GAA ACT ATC GCC GCA ATG ATC ATG
GAA CCG ATT ATT ACT GGT GGA GGC ATC TTA GTG CCC CCAGAG GGG TAT ATG
AAA GCG GCT AAG GAG GTT TGT GAA AAG CAC GGG GCT CTT TTG ATT GTG GAC
GAG GTG ATT TGC GGG TTT GGT CGT ACG GGT AAG CCGTTC GGA TTC ATG AAC
TAT GGA GTC AAG CCG GAC ATT ATC ACC ATG GCT AAA GGC ATC ACC AGT GC
G TAT CTT CCG TTG TCA GCA ACT GCA GTC AAA AAGGAA ATC TAT GAT GC C
TTT AAA GGT GAG GAC GAA TAT GAG TTC TTC CGT CAT GTC AAC ACT T TC
GGA GGG TCA CCC GCC GCA TGT GCG CTG GCT ATC AAGAAC ATT CAG A TT TTG
GAG GAG GAA AAG CTG TTT GAC CGC TCG GGC GAC ATG GGC GAA AAA GTT TTA
ACA GAA CTT CAG AAC TTG TTA CGC GAT CAC CCCTAC GTT GGC GAC GTT CGT
GGA AAG GGT CTG TTA ATC GGA ATT GAA TTG GTT AAA GAC AAG CAG ACG AAA
GAG CCC TTA AAT ACA AGC AAA GTT GAC GAAGTA ATC GCT CTT TGT AAA CAG
GAA GGA CTT CTG ATT GGA AAA AAT GGC AT G ACC GTG GCA GGC TAT AAC
AAC GTC CTT ACA CTG TCC CCT CCG CTT A ATATC CCA GAG ACC GAC TTA GAC
TTT TTG ATC AAA GTA CTG ACG GCG T CC TTG GAG AAG ATT AAG 23 [004]
lysine ATG AAA AAT ATC GTA GTG ATC GGG GCA GGG AAT ATT GGC AGC GCC
ATT dehydrogenase GCG TGG ATG TTG GCA GCT AGC GGG GAT TAT CGC ATT
ACT GTA GCA GA (Agrobacterium C CGCAGC GCG GAT CAG TTA GCT AAT GTA
CCG GCT CAT GAA CGT GTC GA tumefaciens) C ATT GTT GAC ATT ACC GAC
CGC CCC GCG CTG GAA GCA CTG TTA AAA G GG AAA TTTGCG GTA CTT AGC GCC
GCT CCC ACC GAG TTT CAT TTG ACT G CC GGA ATT GCG GAA GCG GCC GTC
GCG GTA GGC ACG CAC TAC TTA GAC TTA ACA GAA GATGTG GAG TCT ACC CGC
AAG GTA AAA GCG CTG GCT GAG ACG GCC GAG ACA GCT TTA ATC CCC CAA TGT
GGG CTG GCA CCA GGT TTT ATT TCG ATT GTT GCTGCC GAT TTG GCT GTG AAG
TTT GAT AAA TTA GAT TCT GTT CGT ATG CGC GTC GGG GCG CTG CCT CAG TAT
CCC AGT AAC GC A TTG AAT TAC AAT TTG ACTTGG AGC ACA GAT GGC CTT ATC
AAC GAG TA C ATT GAG CCT TGC GAG GGG TTT GTA GAA GGT CGC TTG ACC
GCG GTC C CG GCT TTA GAG GAA CGC GAG GAATTT AGT CTT GAT GGG ATC ACC
TAC G AG GCA TTC AAC ACC TCG GGC GGA CTT GGG ACC TTG TGC GCC ACC
CTT GAG GGT AAG GTG CGC ACA ATG AAC TACCGT ACC ATC CGC TAT CCG GGT
CAT GTA GCA ATC ATG AAG GCA CTT CTT AAT GAC TTG AAC CTG CGT AAT CGC
CGT GAC GTT TTG AAA GAT CTT TTT GAAAAT GCA CTG CCT GGA ACG ATG CAA
GAT GTC GTA ATT GTT TTT GTA ACA GTG TGT GGC ACT CGC AA C GGA CGC
TTT CTG CAA GAG ACT TAT GCC AAT AAAGTG TAC GCG GGG CC T GTG TCA GGC
CGC ATG ATG TCC GCG ATC CAG ATC ACA ACA GCT GCT G GA ATT TGC ACA
GTC CTG GAT TTG TTG GCC GAA GGC GCGCTT CCG CAG A AG GGC TTC GTT CGT
CAA GAG GAG GTC GCA CTG CCT AAG TTT TTG GAA AAT CGT TTC GGA CGT TAT
TAT GGT TCT CAC GAA CCG CTT GCTCGT GTT GGT 24 lysine ATG GCA CAT
ACA GGC CGT ATG TTT AAG ATC GAA GCC GCG GAG ATC GTA racemase GTG
GCT CGC CTG CCG CTG AAA TTT CGT TTT GAG ACA TCT TTC GGT GT
(uncultured C CAGACA CAT AAA GTG GTG CCT TTA CTG ATC TTA CAT GGC
GAA GGT GT bacterium) T CAA GGG GTC GCG GAG GGG ACA ATG GAA GCT CGC
CCC ATG TAC CGC G [005] AA GAA ACGATT GCC GGA GCC CTT GAT TTG TTG
CGT GGA ACT TTT TTA C CT GCG ATT CTG GGC CAA ACC TTT GCC AAT CCA
GAA GCG GTA AGT GAT GCC CTG GGC TCTTAC CGC GGC AAT CGC ATG GCA CGC
GCT ATG GTG GAG ATG GCA GCT TGG GAC TTG TGG GCC CGC ACC CTT GGT GTG
CCT TTG GGC ACA CTG TTG GGT GGTCAC AAG GAA CAA GTC GAG GTG GGT GTA
TCG TTG GGA ATC CAG GCA GAT GAG CAA GCT ACA GTA GAC TTA GTG CGT CGT
CA T GTT GAA CAA GGA TAT CGTCGC ATT AAG TTG AAG ATT AAG CCT GGG TG
G GAC GTT CAA CCT GTA CGT GCG ACC CGT GAG GCA TTC ATG TTA AAC A CG
CTT AAT GTC GGC GCC TCT GGTTAC GCG GGC GCA GAA CTG GTT ACA T AC GTG
AAC CGC CAC CCC CAT ATG AAC ATT ACG GCG TTG ACC GTA TCA GCA CAG TCA
AAC GAT GCA GGG AAG TTAATC TCC GAT TTG CAT CCC CAA TTA AAG GGC ATC
GTT GAC TTA CCA TTG CAG CCG ATG TCC GAC ATC TCT GAA TTC AGC CCC GGG
GTA GAT GTA GTG TTCCTG GCT ACA GCT CAC GAA GTT TCA CAC GAC CTG GCC
CCG CAA TTT TTG GAG GCG GGT TGT GTG GT C TTT GAT CTG TCC GGC GCT
TTT CGC GTT AAC GATGCT ACA TTT TAC GA G AAG TAT TAC GGT TTC ACC CAC
CAA TAC CCA GAG CTG CTG GAA CAG G CG GCC TAC GGG CTT GCT GAG TGG
TGT GGC AAC AAA CTTAAG GAA GCT A AT CTT ATT GCA GTT CCT GGA TGT TAC
CCT ACC GCC GCA CAG CTG GCG CTG AAG CCG TTA ATT GAT GCT GAC CTG CTG
GAC CTG AAC CAATGG CCG GTG ATC AAT GCG ACC AGT GGC GTA TCT GGG GCG
GGT CGT AAA GCC GCA ATT TCA AAC TCC TTC TGC GAG GTT AGC TTA CAA CCG
25 Lysine ATG GAG CAG ACG AAG AAA TGG GGA TTT TGG TTA CTG ACG GCC
TTC GTC transporter GTG GGC AAC ATG GTG GGT AGT GGA ATC TTT TCT CTT
CCA TCC TCC CT yvsh G GCGAGC ATC GCG TCG CCT TTC GGA GCT ACG TCC
GCT TGG CTT CTG AC (Bacillus A GGT GCG GGG GTG TTA ATG ATC GCC TTA
GTA TTC GGA CAT TTG TCC A subtilis) TT CGT AAACCC GAA TTG ACT GCC
GGG CCT CAA TCA TAC GCC CGT GCA T [006] TG TTC AGC GAT CCA AAA AAG
GGG AAT GCG GCC GGG TTT ACT ATG GTT TGG GGT TAC TGGGTC GCG AGC TGG
ATC AGT AAC GTA GCA ATC ATT ACA TCT CTG GCG GGG TAT CTG ACC AGC TTC
TTC CCC ATC CTG GTA GAC AAA CGC GAA ATG TTT TCTATT GGG GGT CAA GAG
GTC ACC CTG GGG CAG CTG CTG ACT TTT GCC GTT TGC ACC ATT CTG TTG TGG
GGC ACC CAT GCG AT T TTG GTC GCA TCG ATC AATGGC GCA AGC AAG CTG AAT
TTT GTG ACC AC A TTA TCC AAG GTC TTG GGA TTC GTG TTT TTC ATT GTG
GCA GGG TTA T TC GTC TTC CAG ACG ACG CTT TTTGGT CAT TTC TAT TTC CCG
GTC CAA G GC GAG AAT GGA ACG AGC ATC GGT ATT GGG GGA CAG GTG CAT
AAC GCT GCG ATT TCT ACA CTT TGG GCT TTC GTCGGA ATC GAA AGC GCC GTT
ATC TTG TCT GGC CGC GCG CGC AGC CAG CGC GAT GTT AAA CGT GCT ACC ATT
ACC GGA CTT CTG ATT GCA CTG TCG ATC TATATT ATC GTC ACG TTA ATC ACG
ATG GGT GTT TTA CCC CAC GAC AAA TTA GTA GGA AGT GAA AAG CC A TTT
GTC GAT GTT TTA TAT GCA ATC GTC GGG AACGCT GGT TCA GTA AT C ATG GCA
CTG CTG GCC ATC TTG TGC CTT TTT GGA ACC ATG TTG GGG T GG ATT TTA
CTG GGC TCG GAG GTG CCC TAC CAA GCA GCCAAA GCT GGT G AT TTC CCC GCC
TTC TTT GCC AAA ACT AAT AAG AAA GGT TCT CCA GTG ATT GCG CTT ATC ATT
ACC AAT GTC ATG TCA CAG GTT TTC ATTTTT AGC GTG ATC AGT CGT ACA ATT
TCC GAT GCT TTT ACT TTT TTG ACT ACA GCG GCC ACG TTG GCC TAT CTG ATT
CCC TAC TTA GTT TCA GCG ATT TATAGT TTG AAA GTG GTT ATT AAA GGC GAA
ACC TAT GAC CAG TTG AAA GGC AG T CGT GTA CGT GAT GGT CTT ATC GCT
ATC TTG GCA TGT GCA TAC TCA G TCTTC GTA ATC GTG ACG GGT ACC GCC GAT
TTG ACG ACC TTT ATT TTA G GT ATT GGG CTT TTT TTT GTG GGC CTT ATC
GTG TAC CCA TTT GTC TCG AAG AAGTTT CAA AAG GAG AAG CAG GAA 26
Lysine ATG ACC ATG ATT GCT ATT GGC GGG TCG ATC GGC ACA GGG CTT TTC
GTT Transporter GCA TCC GGA GCA ACG ATT AGT CAA GCA GGT CCA GGC GGG
GCT CTG CT LysP G TCTTAT ATT CTT ATC GGC TTA ATG GTG TAT TTT CTG
ATG ACC TCT CT (Klebsiella) T GGA GAG CTG GCC GCT TTT ATG CCA GTC
TCC GGA TCG TTC GCT ACA T [007] AT GGG CAAAAC TAC GTA GAG GAG GGT
TTC GGG TTT GCG CTG GGT TGG A AT TAC TGG TAT AAT TGG GCT GTG ACG
ATC GCA GTT GAC TTG GTG GCT TCG CAG CTT GTGATG AGC TAT TGG TTC CCT
GAC ACT CCG GGC TGG ATT TGG TCT GCT TTG TTT TTG GGC ATC ATG TTC TTG
CTT AAC TGG ATC TCC GTT CGC GGG TTC GGTGAA GCT GAG TAC TGG TTC AGT
CTG ATT AAA GTT GCG ACC GTT ATT ATC TTC ATC ATC GTT GGC GTG ATG ATG
ATT GTC GG C ATT TTC AAA GGG GCG CAACCG GCT GGA TGG TCC AAC TGG GGT
ATC GC T GAC GCC CCA TTT GCG GGG GGC TTC TCG GCG ATG ATT GGC GTT
GCC A TG ATT GTC GGT TTT TCC TTT CAGGGT ACA GAG TTA ATT GGA ATT GCT
G CT GGT GAA TCC GAG AAT CCT GAG AAA AAT ATT CCA CGT GCG GTA CGT
CAG GTA TTC TGG CGC ATT TTA CTG TTTTAT GTT TTT GCA ATC TTG ATT ATC
TCG TTG ATC ATC CCT TAT ACT GAC CCA TCC TTA TTG CGT AAC GAT GTG AAG
GAT ATT TCC GTG TCT CCC TTC ACGTTG GTA TTT CAG TAT GCT GGG CTG CTT
AGT GCC GCT GCG ATC ATG AAC GCA GTC ATT CTT ACG GC T GTA CTG AGC
GCT GGA AAC TCG GGA ATG TAC GCTTCA ACA CGC ATG TT A TAT ACC TTG GCA
TGT GAC GGG AAA GCA CCG CGT ATC TTT AGC AAG C TT TCC CGT GGC GGT
GTG CCA CGC AAT GCT CTG TAT GCAACA ACT GTA A TT GCT GCC TTA TGC TTT
CTT ACC AGC ATG TTC GGC AAC CAA ACG GTT TAT CTG TGG TTG CTG AAC ACT
TCG GGA ATG ACA GGG TTC ATCGCC TGG CTG GGT ATT GCT ATT TCT CAC TAT
CGT TTC CGT CGC GGC TAC GTG CTG CAG GGG AAT GAT ATC AAT AAT CTT CCG
TAT CGT TCA GGA TTT TTTCCT CTT GGA CCC ATT TTT GCA TTT GTA TTG TGT
TTG ATT ATT ACT CTT GG C CAA AAT TAT GAG GCG TTC TTA AAA GAT ACT
ATC GAT TGG GGT GGG G TAGCC GCA ACC TAC ATC GGG ATT CCC TTG TTC CTT
GTT ATT TGG TTT G GA TAT AAG TTG GCT AAG GGT ACC CGC TTT GTC CGT
TAT TCC GAA ATG ACC TTCCCA GAT CGT TTT AAA CGC 27 Lysine ATG AGT
ATG GAA GTC TGG CTG GGG TTT TTT GCA GCG TGT TGG GTG ATT Exporter
AGT TTG TCA CCG GGA GCC GGA GCC ATC GCC TCT ATG TCA TCG GGT TT
(Pseudomonas) A CAATAT GGC TTC TGG CGT GGC TAC TGG AAT GCA CTT GGA
TTG CAG CT T GGT TTA ATT ATG CAA ATT GCA ATT ATC GCT GCG GGC GTC
GGA GCC G TC TTG GCGGCC TCG GCT ACG GCC TTC CAG GTA ATT AAA TGG TTC
GGA G TT GGG TAT CTT GTG TAT TTA GCA TAC AAA CAA TGG CGT GCA CTG
CCC ATG GAT ATG TCGGAT GAA AGC GGG GTG CGT CCA ATC GGC AAA CCA
TTA
TCG CTG GTA TTT CGT GGA TTT TTG GTG AAT ATC TCC AAC CCA AAA GCT TTA
GTA TTC ATG TTGGCC GTT TTA CCC CAG TTC CTG AAT CCC CAC GCC CCC TTG
TTA CCC CAA TAC GTG GCT ATC ACT GTG ACA ATG GTT ACA GT T GAC TTG
TTA GTG ATG GCCGGA TAC ACA GGT TTA GCA TCT CAT GTA TT A CGT ATG CTT
CGT ACC CCA AAA CAG CAA AAA CGC CTG AAC CGC ACC T TC GCC GGT TTA
TTC ATC GGA GCGGCC ACA TTC CTT GCC ACT TTG CGC C GC GCA CCA GTA 28
Asparaginase ATG CAG AAG AAA TCG ATC TAC GTC GCG TAC ACG GGC GGC
ACC ATT GGG (Escherichia ATG CAG CGT TCG GAG CAG GGT TAC ATC CCC
GTT TCC GGT CAC TTG CA coli) G CGCCAG CTG GCC TTG ATG CCC GAG TTC
CAT CGC CCC GAG ATG CCA GA T TTT ACC ATT CAT GAG TAC ACT CCA CTT
ATG GAT TCA TCG GAC ATG A CG CCG GAAGAC TGG CAA CAC ATT GCA GAA GAT
ATC AAG GCT CAC TAT G AT GAT TAT GAC GGC TTT GTT ATT TTA CAC GGT
ACT GAC ACA ATG GCA TAC ACA GCT TCTGCA CTT TCC TTT ATG CTT GAG AAC
CTT GGT AAG CCC GTG ATC GTG ACC GGG TCG CAG ATC CCC CTT GCC GAA TTG
CGC AGT GAC GGG CAG ATC AAT CTTCTT AAT GCG TTA TAT GTG GCC GCT AAC
TAT CCG ATC AAT GAA GTG ACT TTA TTC TTC AAT AAC CGC TTG TAC CGT GGA
AA C CGC ACT ACG AAA GCC CATGCT GAT GGC TTT GAC GCC TTT GCA TCC CC
A AAT CTG CCT CCC CTT TTG GAA GCC GGG ATT CAC ATC CGT CGT TTA A AT
ACA CCC CCC GCC CCA CAT GGAGAG GGG GAG CTT ATC GTA CAT CCA A TT ACC
CCT CAA CCT ATC GGA GTT GTA ACG ATT TAC CCT GGT ATT AGT GCC GAC GTA
GTC CGC AAT TTC CTT CGCCAG CCC GTG AAA GCA TTG ATC TTA CGT TCC TAC
GGT GTA GGG AAC GCG CCA CAG AAT AAG GCA TTT CTG CAA GAA TTA CAA GAG
GCA TCG GAT CGT GGTATC GTG GTA GTC AAC CTG ACA CAG TGC ATG TCA GGT
AAA GTT AAT ATG GGT GGA TAC GCA ACC GG G AAT GCA TTA GCT CAT GCA
GGG GTA ATT GGA GGCGCT GAT ATG ACG GT C GAA GCT ACC CTG ACG AAG CTT
CAT TAT CTG TTA TCC CAG GAG TTG G AC ACC GAG ACC ATT CGC AAA GCT
ATG TCT CAG AAC CTTCGC GGT GAG C TT ACT CCC GAT GAC 29 Asparagine
ATG CCG CCT CTG GAC ATC ACC GAC GAA CGC TTG ACT CGC GAA GAT ACA
transporter GGA TAT CAC AAA GGC CTT CAC TCC CGT CAG CTT CAG ATG ATC
GCT CT ansp2 T GGAGGT GCT ATT GGG ACC GGA CTT TTT CTG GGG GCA GGC
GGA CGT CT (Mycobacterium G GCT TCT GCC GGA CCG GGA TTA TTC TTG GTT
TAT GGT ATC TGT GGC A bovis) TT TTT GTCTTT CTT ATT CTT CGT GCC TTG
GGA GAA CTT GTG CTT CAC C GC CCT AGT TCA GGA TCA TTT GTA TCC TAC
GCG GGG GAA TTT TAT GGT GAA AAG GTC GCGTTC GTC GCG GGG TGG ATG TAT
TTT TTG AAT TGG GCA ATG ACT GGG ATT GTG GAC ACT ACA GCC ATC GCC CAC
TAT TGC CAC TAT TGG CGC GCT TTT CAACCA ATT CCA CAG TGG ACG TTG GCC
CTT ATT GCG TTG TTA GTT GTA TTA TCC ATG AAT CTG ATC TCC GTC CGC TTA
TTC GG G GAA CTT GAG TTT TGG GCCTCG CTT ATT AAA GTA ATT GCG CTT GTT
AC G TTC CTG ATT GTA GGG ACT GTA TTC CTG GCG GGG CGT TAC AAG ATT G
AC GGG CAA GAA ACT GGT GTA TCATTA TGG TCA TCT CAT GGC GGA ATC G TT
CCT ACG GGG TTA CTG CCC ATT GTC CTT GTG ACC TCT GGA GTT GTG TTC GCA
TAC GCA GCC ATC GAG CTG GTAGGA ATC GCA GCC GGG GAG ACG GCC GAA CCA
GCC AAA ATC ATG CCC CGC GCA ATC AAT TCG GTC GTC CTT CGT ATT GCG TGT
TTT TAT GTG GGA TCT ACGGTG CTT CTG GCG TTG CTT TTA CCA TAC ACG GCT
TAT AAG GAG CAC GTA AGT CCC TTC GTA ACA TT T TTC AGC AAA ATT GGA
ATT GAT GCC GCG GGG AGTGTA ATG AAC TTG GT A GTG CTT ACG GCA GCG TTA
TCT AGT TTG AAC GCT GGT TTG TAT TCC A CA GGA CGC ATC CTG CGC TCA
ATG GCG ATC AAC GGC AGCGGA CCA CGC T TT ACG GCA CCC ATG AGT AAA ACC
GGT GTT CCT TAT GGC GGT ATC TTG CTT ACA GCA GGT ATC GGT TTA TTG GGA
ATC ATT CTT AAT GCGATC AAA CCC TCG CAG GCG TTC GAA ATC GTT TTA CAC
ATC GCT GCT ACC GGC GTA ATC GCA GCC TGG GCT ACG ATC GTG GCT TGT CAG
TTG CGC TTA CATCGC ATG GCC AAC GCT GGC CAA CTT CAG CGC CCT AAG TTC
CGT ATG CCC TT G TCA CCT TTT AGC GGG TAC CTG ACT TTG GCG TTT CTG
GCG GGC GTG C TGATT CTG ATG TAT TTC GAT GAG CAG CAC GGG CCC TGG ATG
ATC GCC G CG ACA GTA ATT GGG GTT CCT GCC CTT ATT GGG GGT TGG TAC
TTG GTT CGT AACCGT GTG ACT GCC GTC GCT CAT CAC GCT ATT GAC CAC ACT
AAG AGT GTA GCT GTG GTT CAT TCG GCA GAT CCC ATT 30 Serine ATG TCC
ATT AAT CAG GAG GCG CTT CAC GTA CTG TTG AAG GAT CCT TTT ammonia ATT
CAT CGT CTT ATT GAT GCT GAG CCA GTG TTT TGG GCA AAT CCA GG lyase T
ATGAAG GAG GGG CTG CTT TTT CAC GCT GAC GAG TGG GAA AGT GAG AT
(Bacillus T GCC GAA GCA GAG AAA CGC TTG CGT CGT TTT GCG CCT TAT ATC
GCG G subtilis) AG GTT TTCCCA GAG ACC AAA GAT GCA AAG GGC ATG ATC
GAG TCT CCA C TG TTT GAG ATG CAA CAT ATG AAA AAG AAA CTG GAG GCA
GCA TAC CAA CAA CCT TTC CCCGGA CGT TGG CTG CTT AAG TGT GAC CAT GAA
CTT CCC ATT TCC GGG TCG ATT AAG GCC CGC GGA GGT ATC TAT GAA GTC TTG
AAA CAT GCC GAA AAG CTGGCT CTT CAG GAG GGG ATG CTT CAA GAG TCG GAC
GAT TAT CGT ATG TTG CAA GAA GAT CGT TTC GCG GCC TTC TTC AGC CG C
TAT TCT ATC GCA GTG GGCTCC ACG GGC AAT TTA GGT TTA AGT ATC GG G ATT
ATT GGC GCT GCT CTT GGT TTC CGC GTA ACA GTT CAC ATG AGT G CT GAC
GCT AAG CAA TGG AAG AAAGAT CTG TTA CGT CAG AAA GGG GTA A CC GTA ATG
GAA TAT GAG AGC GAT TAT TCA GAA GCA GTT AAA GAA GGT CGT CGC CAA GCA
GAA CAA GAC CCA TTCTGT TAC TTC ATT GAT GAT GAA CAT AGC CGT CAA TTG
TTC CTG GGC TAC GCA GTT GCC GCG TCC CGC CTT AAG ACA CAA CTG GAT TGC
ATG GAA ATC CAACCT GGT CCC GAA ACA CCC CTG TTC GTG TAT CTT CCC TGT
GGC GTA GGT GGG GGG CCA GGA GGT GT C GCT TTC GGG TTG AAA CTG CTG
TAT GGA GAT CACGTC CAT GTA TTT TT C GGA GAA CCG ACG CAA TCC CCG TGC
ATG CTT TTA GGC TTA TAT TCT G GC TTA CAC GAG CAG ATT TCA GTT CAA
GAC ATT GGA TTGGAC AAC CGT A CC GCG GCG GAT GGC TTG GCG GTA GGG CGT
CCC TCA GGA TTC GTA GGA AAA TTA ATC GAA CCA CTG CTG TCG GGC TGC TAT
ACT GTA GAAGAC GAT ACA CTG TAT GCT TTA CTG CAC ATG CTG GCA GCT TCG
GAA TCC AAG TAT CTT GAA CCT AGC GCC TTG GCG GGG ATG TTC GGC CCG ATC
CAG CTGTTC AGC ACA GAA GAA GGA CGT CGC TAT TCT CAG AAA CAT AAA ATG
GAG CA T GCG GTG CAC GTT ATC TGG GGG ACG GGG GGT AGC ATG GTG CCA
AAG G AGGAG ATG GCC GCA TAC AAC CGC ATC GGG GCG GAT CTG TTA AAA AAT
G AA ATG AAG AAG 31 SdaA ATG TCC CTT TCA GTG TTT GAT CTT TTT AAG
ATC GGA ATT GGT CCC TCG (Pseudomonas TCC TCT CAT ACC GTA GGA CCT
ATG CGT GCG GCC GCT CGT TTT GCC GA fluorescens A GGTCTG CGC CGC GAC
GAC CTG CTG AAC TGT ACT ACT AGC GTG AAA GT F113) C GAG CTG TAC GGA
TCT CTG GGC GCG ACT GGT AAA GGG CAC GGT TCG G AC AAA GCAGTG TTA CTG
GGA TTG GAG GGA GAA CAC CCT GAC ACT GTC G AC ACC GAG ACG GTT GAC
GCT CGT TTA CAG GCG ATC CGC AGT TCA GGC CGC CTG AAT TTATTG GGG GAG
CAT AGC ATT GAG TTT AAT GAA AAG CTG CAC TTG GCA ATG ATT CGC AAG CCG
TTA GCT TTC CAT CCG AAT GGC ATG ATT TTC CGT GCG TTTGAT GCT GCG GGC
TTA CAG GTA CGT TCC CGT GAG TAT TAC TCC GTC GGC GGA GGG TTC GTT GTA
GAC GAG GAC GCA GCG GG T GCC GAC CGT ATC GTC GAGGAT GCA ACA CCT TTG
ACA TTC CCC TTC AA G AGC GCG AAG GAT CTT TTA GGT CAT TGT TCT ACT
TAT GGT TTA AGC A TC AGC CAA GTC ATG CTT ACA AACGAG TCT GCG TGG CGT
CCG GAA GCG G AG ACC CGC GCA GGG CTT CTT AAA ATT TGG CAG GTG ATG
CAA GAC TGC GTT GCC GCG GGG TGT CGC AAT GAG GGCATC CTT CCA GGA GGT
CTT AAA GTA AAG CGC CGC GCG GCT GCG TTG CAT CGT CAA TTG TGT AAG AAC
CCC GAG GCT GCC CTG CGC GAT CCG TTA AGT GTATTA GAT TGG GTG AAT TTG
TAT GCG TTA GCG GTA AAT GAA GAG AAC GCC TAC GGT GGA CGC GTG GT C
ACG GCG CCC ACT AAT GGA GCC GCA GGA ATC ATTCCT GCC GTA TTG CA T TAC
TAC ATG CGC TTT ATT CCG GGG GCA TCT GAG GAC GGA GTA GTC C GC TTC
CTT CTT ACA GCG GCG GCA ATC GGG ATC TTG TATAAA GAG AAC G CC TCT ATT
AGT GGG GCT GAG GTT GGC TGT CAG GGC GAA GTA GGA GTG GCA TGC TCC ATG
GCA GCG GGG GCG TTG TGC GAA GTC TTG GGAGGC TCG GTC CAA CAA GTA GAA
AAC GCA GCA GAA ATC GGA ATG GAG CAT AAC CTT GGC TTG ACA TGT GAT CCT
ATC GGC GGG TTA GTA CAG GTC CCG TGTATC GAG CGT AAC GCA ATG GGA TCT
GTT AAA GCC ATT AAC GCA GTA CGC AT G GCT ATG CGC GGG GAC GGT CAC
CAT TTC GTC TCC CTT GAC AAA GTA A TTCGT ACC ATG CGT CAA ACT GGG GCC
GAC ATG AAA AGC AAG TAC AAG G AA ACC GCG CGT GGT GGA CTT GCT GTC
AAC ATC ATC GAG TGT 32 sdaB (Klebsiella ATG ATT AGT GTG TTT GAC ATC
TTT AAA ATC GGT ATC GGT CCG TCT TCT pneumoniae) TCC CAT ACG GTT GGT
CCC ATG AAA GCA GGG AAG CAG TTT ACC GAC GA C TTAATT GCT CGT GGA CTG
CTG GCA GAG GTC AGT AAG GTC GTG GTT GA T GTT TAT GGC TCC CTT TCA
TTG ACG GGC AAA GGT CAC CAT ACT GAC A TT GCT ATCATT ATG GGT CTG GCG
GGA AAC TTG CCA GAC ACC GTT GAC A TC GAC GCC ATC CCC GGC TTC ATC
CAA GAT GTT AAC ACT CAC GGA CGT CTG ATG TTA GCGAAT GGG CAG CAT GAA
GTT GAT TTC CCG GTA GAC CAG TGT ATG AAT TTT CAC GCT GAC AAC CTG TCC
TTG CAC GAG AAT GGA ATG CGT ATT ACG GCT CTTGCG GGA GAC AAA GTG TTG
TAC TCT CAG ACT TAC TAC TCA ATC GGC GGC GGA TTC ATT GTT GAT GAG GAA
CAT TTT GGC CA A ACA ACG GAG GCT CCT GTAGCC GTC CCA TAT CCA TAC AAA
AAC GCC GC T GAT TTG CAG CGT CAT TGC CGT GAA ACT GGT TTG AGT TTA
TCT GGA C TT ATG ATG CAA AAC GAA CTT GCATTG CAT AGC AAA GAA GCT CTG
GAA C AG CAC TTT GCT GCA GTT TGG GAG GTT ATG TCT GCC GGC ATT GAG
CGC GGC ATT ACA ACT GAA GGT GTG TTG CCTGGC AAA TTA CGT GTA CCC CGC
CGC GCC GCG GCA CTG CGT CGT ATG TTA GTC TCG CAA GAC ACG ACG AAC TCG
GAC CCT ATG GCT GTT GTA GAT TGG ATCAAT ATG TTC GCG TTG GCC GTC AAC
GAG GAG AAC GCG GCG GGC GGT CGC GTT GTT ACA GCC CCC AC A AAT GGC
GCG TGC GGA ATT GTT CCG GCC GTG CTGGCA TAT TAT GAC AA A TTT ATC CGC
AAA GTC AAC TCC AAC AGT CTG GCG CGT TAT ATG CTG G TG GCA AGT GCA
ATC GGC TCA CTT TAT AAG ATG AAT GCGAGC ATC TCC G GC GCA GAA GTT GGC
TGC CAA GGT GAA GTG GGG GTC GCC TGC TCT ATG GCA GCG GCT GGC TTG GCA
GAG CTG TTG GGC GGG TCG CCA GGGCAA GTG TGC ATT GCG GCT GAA ATT GCG
ATG GAG CAT AAC TTG GGC CTT ACG TGC GAT CCC GTA GCT GGC CAA GTG CAG
GTA CCG TGT ATC GAA CGC AATGCA ATT GCA GCC GTA AAA GCA GTA AAT GCG
GCT CGC ATG GCC TTA CGT CG T ACT TCC GAG CCC CGT GTG TGC TTG GAT
AAG GTG ATC GAA ACC ATG T ATGAG ACA GGT AAG GAC ATG AAT GCA AAG TAT
CGT GAA ACG TCT CGT G GA GGC CTG GCC ATG AAG ATC GTC GCG TGT GAC 33
tdcG L- ATG ATT AGT GCA TTC GAT ATT TTC AAG ATT GGA ATC GGC CCC TCG
TCA serine TCG CAC ACG GTG GGC CCA ATG AAC GCA GGT AAG TCC TTC ATT
GAT CG dehydratase C CTTGAG TCG AGT GGC TTA TTG ACA GCG ACA AGC CAC
ATT GTC GTG GA (Escherichia C CTG TAC GGG AGT CTG TCG TTG ACG GGC
AAA GGC CAT GCG ACC GAT G coli O157: H7 TT GCT ATTATC ATG GGA TTG
GCC GGG AAT TCA CCG CAG GAC GTA GTA A str. SS17) TC GAT GAA ATC CCG
GCC TTC ATT GAG CTG GTA ACT CGT TCG GGC CGT CTG CCA GTC GCAAGC GGA
GCT CAT ATC GTT GAC TTC CCA GTT GCC AAG AAC ATT ATT TTT CAC CCT GAA
ATG TTA CCT CGC CAT GAG AAC GGA ATG CGT ATC ACA GCA TGGAAA GCT CAG
GAA GAA TTA TTG AGT AAG ACG TAT TAC TCG GTT GGT GGC GGG TTC ATC GTC
GAG GAA GAG CAC TTC GGT TT A TCT CAT GAC GTA GAA ACACCA GTA CCA TAC
GAC TTC CAT TCA GCA GG T GAG TTG TTG AAA ATG TGC GAT TAC AAT GGC
CTT AGT ATT TCG GGA C TT ATG ATG CAT AAC GAA TTA GCGCTT CGT TCG AAG
GCC GAA ATT GAC G CC GGC TTC GCA CGT ATC TGG CAA GTT ATG CAT GAT
GGC ATC GAA CGT GGT ATG AAC ACC GAA GGT GTG TTA CCAGGA CCC TTG AAT
GTT CCG CGT CGT GCA GTC GCA CTG CGT CGT CAA CTT GTT AGT AGT GAC AAC
ATT TCC AAT GAT CCA ATG AAC GTG ATT GAC TGG ATCAAC ATG TAC GCG CTG
GCG GTC TCG GAG GAA AAC GCC GCT GGG GGT CGC GTG GTA ACA GCA CCT AC
G AAT GGG GCT TGC GGG ATC ATC CCT GCG GTA TTGGCC TAT TAC GAT AA G
TTT CGC CGT CCA GTC AAT GAG CGC TCA ATC GCT CGT TAC TTC CTG G CG
GCG GGG GCT ATC GGC GCT TTA TAC AAG ATG AAC GCCTCT ATT TCA G GG GCG
GAG GTC GGT TGT CAA GGA GAG ATT GGG GTC GCG TGC TCT ATG GCA GCT GCA
GGT TTG ACA GAA TTA TTA GGC GGC AGC CCA GCCCAA GTT TGC AAC GCG GCT
GAA ATC GCA ATG GAA CAT AAT CTT GGT CTG ACC TGT GAC CCT GTC GCA GGT
CAG GTA CAG ATT CCT TGC ATT GAG CGT AATGCA ATC AAC GCA GTA AAA GCT
GTT AAT GCG GCG CGT ATG GCT ATG CGT CG C ACA TCA GCC CCG CGT GTG
AGC CTG GAT AAG GTA ATC GAG ACC ATG T ACGAA ACC GGT AAA GAC ATG AAT
GAC AAA TAC CGC GAA ACC TCT CGC G GG GGT CTT GCA ATT AAA GTC GTG
TGT GGC 34 glyA ATG TTG AAA CGT GAG ATG AAT ATT GCC GAC TAT GAT GCA
GAA TTA TGG (Escherichia CAA GCT ATG GAA CAA GAG AAA GTC CGC CAG
GAA GAA CAT ATT GAA TT coli EPEC A ATCGCC TCT GAA AAT TAC ACT AGT
CCC CGC GTT ATG CAA GCC CAA GG C342-62) C AGC CAA TTA ACT AAC AAA
TAT GCC GAG GGA TAT CCT GGG AAA CGC T AC TAT GGAGGT TGC GAG TAT GTA
GAT ATT GTC GAA CAG TTA GCA ATC G AC CGC GCG AAA GAG CTT TTC GGC
GCA GAC TAT GCA AAC GTG CAG CCC CAT TCG GGT AGCCAA GCG AAT TTT GCG
GTC TAT ACC GCA CTG CTG GAA CCG GGA GAC ACG GTA CTG GGT ATG AAT TTA
GCT CAT GGT GGT CAC TTA ACG CAC GGG TCC CCCGTT AAT TTC TCT GGA AAA
CTG TAC AAC ATC GTC CCC TAT GGA ATC GAT GCT ACC GGC CAC ATT GAT TAC
GCG GAT CTT GA G AAG CAA GCT AAG GAA CATAAA CCA AAG ATG ATC ATT GGC
GGT TTT TC A GCT TAT AGT GGT GTC GTC GAC TGG GCT AAG ATG CGT GAA
ATT GCA G AC TCT ATT GGC GCG TAC CTT TTTGTC GAC ATG GCC CAC GTG GCT
GGC T TG GTG GCG GCA GGG GTC TAC CCG AAC CCC GTT CCC CAT GCG CAT
GTC GTG ACC ACC ACG ACA CAT AAG ACA CTGGCT GGG CCT CGT GGT GGC TTA
ATC TTG GCC AAG GGG GGG TCT GAG GAA TTA TAC AAA AAA CTT AAC TCA GCC
GTT TTT CCA GGC GGA CAG GGT GGT CCGTTG ATG CAC GTG ATT GCT GGA AAG
GCG GTC GCT CTT AAG GAA GCC ATG GAA CCT GAA TTC AAA AC G TAC CAA
CAG CAG GTT GCA AAA AAC GCC AAA GCGATG GTT GAG GTT TT C CTG GAA CGT
GGT TAC AAA GTC GTT AGT GGG GGT ACC GAT AAT CAT C TT TTC TTA GTT
GAC CTG GTA GAT AAA AAT TTG ACC GGAAAG GAG GCG G AC GCT GCC TTA GGC
CGT GCG AAT ATT ACC GTC AAT AAA AAC TCG GTG CCA AAT GAT CCC AAG TCG
CCT TTC GTG ACT TCA GGA ATC CGCGTA GGA ACT CCC GCA ATT ACA CGC CGC
GGG TTC AAG GAA GCT GAG GCG AAG GAG TTA GCA GGA TGG ATG TGT GAT GTT
TTA GAC TCG ATT AAC GAT GAGGCG GTG ATC GAA CGT ATC AAA GGT AAA GTA
TTA GAT ATT TGC GCC CGT TA T CCA GTT TAT GCC 35 SdaC serine ATG GAG
ACC ACG CAG ACT TCT ACA ATT GCG AGC AAA GAT AGC CGT TCT STP GCT TGG
CGC AAA ACT GAT ACT ATG TGG ATG TTG GGC CTG TAT GGA ACA transporter
GCTATT GGG GCC GGG GTA CTG TTT TTG CCA ATC AAT GCT GGA GTG GGG
(Escherichia GGT ATG ATC CCG CTG ATC ATT ATG GCG ATT CTT GCT TTC
CCA ATG ACA coli BL21 (DE3) TTT TTTGCA CAT CGC GGT CTT ACA CGC TTT
GTC CTT TCA GGA AAG AAT CCT GGG GAG GAC ATT ACG GAG GTT GTA GAA GAA
CAT TTT GGC ATT GGG GCT GGG AAACTT ATC ACA TTG CTG TAT TTT TTT GCA
ATC TAT CCC ATT TTG CTT GTC TAT AGC GTA GCA ATC ACG AAC ACC GTA GAA
TCA TTC ATG TCG CAC CAG TTAGGC ATG ACA CCT CCG CCA CGT GCG ATT CTG
TCA TTG ATC TTG ATC GTG GGA ATG ATG ACA ATT GTT CGT TTC GGA GAG CAA
ATG ATC GTG AAA GCC ATGTCA ATT TTG GTA TTT CCG TTC GTG GGA GTC TTA
ATG TTG CTG GCA TTG TAT TTA ATT CCC CAG TGG AAT GGT GCC GCT CTG GAG
ACC TTG TCG TTG GATACG GCG TCA GCG ACC GGT AAT GGT CTT TGG ATG ACG
CTT TGG TTG GCC ATT CCG GTC ATG GTT TTT TCA TTT AAC CAC TCA CCG ATC
ATT AGC TCG TTCGCT GTG GCG AAA CGC GAA GAA TAC GGT GAT ATG GCT GAA
CAA AAG TGC TCG AAG ATT TTG GCA TTC GCC CAC ATC ATG ATG GTA CTT ACG
GTC ATG TTCTTC GTG TTT TCT TGC GTC CTT AGT TTA ACC CCA GCG GAC CTG
GCG GCT GCA AAG GAA CAA AAT ATC AGC ATC
TTA AGC TAT TTG GCG AAT CAT TTC AACGCG CCT GTT ATC GCA TGG ATG GCA
CCC ATT ATC GCT ATC ATT GCA ATT ACC AAA TCT TTC TTA GGG CAC TAC TTG
GGT GCG CGC GAA GGA TTT AAC GGGATG GTT ATC AAG TCG CTT CGT GGG AAA
GGA AAG AGT ATC GAG ATC AAT AAA CTT AAT CGC ATC ACC GCC TTG TTC ATG
TTA GTA ACA ACG TGG ATC GTCGCT ACA CTT AAT CCC TCC ATT CTG GGG ATG
ATT GAA ACG CTT GGG GGT CCA ATC ATC GCA ATG ATC TTG TTT CTG ATG CCG
ATG TAC GCT ATC CAG AAGGTA CCC GCA ATG CGT AAA TAC TCT GGG CAT ATC
TCC AAC GTG TTT GTT GTT GTT ATG GGA TTA ATC GCT ATT TCT GCT ATC TTC
TAT AGT CTG TTC TCC 36 threonine ATG GCG TAT TCT GTC CAG TTC CTG
ATC CAA CTG TCC TTC TCG TAC CTT Serine GCC ACT GTG GCT TTT GCT ATC
TGC ATC AAC GTT CCA CGT CGT GCG TT Exporter A AATTTT GCC GGA TGG
GCC GGT GCC ATC GGG TGG ATC TGC TAC TGG CT (Lactobacillus G CTG AAC
ACA CAT GGC ACG GGC CGC ATG TTC GCT AAC CTG ATT GGC G saniviri CT
GTC GCAGTT GGG GTA TGT GGT ATC ATT TTC GCT CGC ATC AAG AAG A JCM
17471 = TG CCC GTG ATT ATT TTC AAT ATT CCG GGG CTG GTG CCA TTA GTG
CCT DSM 24301) GGA GCA ACC GCCTAC CAG GCA GTT CGC GCT CTT GCG TTG
GGA AAT ATG GAC CTT GCT ATC CAG CTT GGA GTT CGT GTT ATT ATG GTC GCA
GGG GCA ATC GCG GTG GGA TTCATG GTT AGT CAG CTT CTG TCA GAG TTG ACT
TAC CGC TTG CAC 37 glutaminase ATG CTG GAT GCT AAT AAG CTG CAG CAG
GCT GTC GAT CAG GCT TAT ACT YbaS CAA TTT CAT TCT TTG AAT GGT GGG
CAG AAT GCC GAT TAC ATT CCT TT (Escherichia C TTGGCT AAT GTC CCA
GGG CAA TTA GCA GCC GTA GCT ATT GTA ACA TC coli ST131) C GAT GGC
AAC GTG TAT TCT GCC GGG GAC TCG GAC TAC CGC TTC GCA C TT GAG TCTATC
AGT AAA GTC TGC ACT TTG GCA CTG GCG CTG GAG GAC G TT GGG CCT CAG
GCC GTG CAG GAC AAG GTT GGG GCT GAT CCT ACA GGG CTG CCA TTC AACTCA
GTA ATT GCT TTG GAA TTA CAC GGT GGA AAA CCA CTG TCA CCG CTG GTG AAC
GCG GGG GCA ATC GCT ACC ACG TCT TTG ATT AAT GCA GAA AAT ACGGAA CAG
CGT TGG CAA CGT ATT TTG CAT ATT CAG CAG CAG CTT GCT GGT GAG CAA GTC
GCA CTT TCT GAT GAA GTG AAC CA A AGT GAA CAA ACT ACT AATTTT CAC AAC
CGT GCA ATT GCT TGG TTA CT G TAC AGT GCT GGC TAC TTG TAC TGT GAC
GCA ATG GAA GCC TGT GAT G TT TAT ACA CGT CAG TGC AGT ACTTTG ATC AAC
ACA ATC GAA TTG GCA A CA TTG GGA GCT ACG TTA GCC GCT GGG GGC GTG
AAT CCG TTG ACA CAT AAA CGC GTT CTG CAA GCG GAC AAT GTGCCC TAT ATT
TTG GCT GAA ATG ATG ATG GAA GGG CTT TAT GGC CGC TCT GGG GAC TGG GCC
TAC CGT GTA GGC TTG CCA GGA AAG TCG GGG GTC GGA GGAGGG ATT CTG GCC
GTG GTG CCC GGC GTA ATG GGA ATT GCC GCG TTT TCG CCT CCC TTA GAC GAA
GA A GGT AAC AGC GTG CGC GGA CAA AAG ATG GTT GCGAGC GTT GCA AAG CA
G CTT GGG TAT AAC GTA TTT AAA GGG 38 Glutaminase ATG GCC GTC GCA
ATG GAT AAC GCC ATT TTA GAG AAT ATC CTG CGC CAA (Escherichia GTG
CGC CCA TTA ATC GGA CAA GGC AAG GTT GCG GAT TAC ATT CCG GC coli C
TTAGCT ACA GTG GAT GGG AGT CGC CTG GGA ATC GCT ATT TGC ACT GT o145:
H28 T GAC GGC CAA TTG TTT CAG GCA GGC GAC GCA CAA GAG CGC TTC TCC A
str. TC CAG AGCATT TCT AAA GTG TTG TCA TTG GTT GTT GCT ATG CGT CAC
T RM12581) AC TCT GAG GAG GAA ATT TGG CAG CGC GTG GGG AAG GAC CCG
TCC GGC AGT CCA TTT AATTCG TTG GTA CAG TTG GAG ATG GAA CAA GGA ATC
CCT CGT AAT CCC TTC ATC AAT GCA GGT GCT CTT GTA GTC TGC GAC ATG TTA
CAA GGT CGT TTA TCTGCC CCT CGC CAA CGC ATG TTG GAA GTT GTG CGT GGT
TTG TCT GGA GTT AGC GAT ATC AGC TAC GAC ACG GTC GTG GCT CG C AGT
GAA TTT GAA CAC TCAGCA CGC AAT GCA GCG ATT GCG TGG TTA AT G AAG TCG
TTT GGG AAT TTT CAT CAC GAT GTG ACG ACA GTC CTT CAA A AT TAT TTC
CAC TAC TGC GCA TTGAAG ATG TCG TGC GTA GAG CTT GCC C GT ACG TTC GTC
TTT CTT GCG AAC CAG GGC AAG GCC ATC CAT ATC GAC GAG CCC GTC GTA ACC
CCG ATG CAG GCGCGT CAA ATC AAT GCG CTG ATG GCG ACA TCG GGA ATG TAT
CAG AAT GCG GGG GAG TTC GCC TGG CGT GTC GGA TTA CCA GCT AAA TCC GGT
GTA GGC GGTGGA ATC GTT GCC ATT GTG CCC CAT GAA ATG GCT ATC GCT GTG
TGG TCC CCA GAA TTA GAT GAC GC A GGA AAT TCG TTA GCA GGT ATT GCG
GTT TTA GAACAA CTT ACG AAA CA A TTA GGA CGC TCG GTG TAT 39 ylaM ATG
GTG TGT CAG CAT AAT GAT GAA TTA GAG GCT CTT GTC AAG AAG GCA
(Bacillus AAA AAG GTT ACG GAT AAG GGG GAG GTG GCT AGT TAC ATT CCA
GCT CT subtilis G GCTAAG GCG GAC AAA CAC GAC TTA AGT GTC GCA ATC
TAC TAT AGC AA subsp. T AAT GTG TGC CTG TCC GCA GGG GAC GTT GAA AAG
ACG TTC ACT CTG C subtilis AA TCC ATCAGC AAA GTT CTG TCG TTA GCT
CTG GTA CTT ATG GAG TAT G str. 168) GG AAG GAT AAG GTA TTC AGT TAT
GTT GGG CAG GAA CCT ACA GGT GAT CCC TTT AAC AGCATC ATT AAA CTG GAG
ACA GTC AAC CCC TCT AAG CCA TTA AAT CCG ATG ATC AAT GCG GGC GCG TTA
GTA GTG ACC AGT CTT ATC CGC GGA CGT ACG GTGAAG GAG CGT CTT GAC TAT
CTT CTT AGC TTT ATC CGT CGT CTG ACT AAT AAT CAA GAA ATT ACA TAC TGC
CGC GAG GTA GC G GAA AGC GAA TAT TCT ACTTCA ATG ATT AAC CGT GCG ATG
TGC TAT TA T ATG AAA CAG TAT GGA ATT TTC GAA GAT GAC GTT GAA GCG
GTT ATG G AC CTT TAT ACA AAG CAA TGC GCTATT GAA ATG AAC TCA CTT GAT
TTG G CT AAG ATC GGT TCG GTT TTC GCC TTG AAC GGA CGC CAT CCT GAA
ACC GGG GAG CAA GTG ATT TCG AAG GAT GTAGCC CGT ATC TGT AAG ACG TTT
ATG GTG ACG TGT GGA ATG TAT AAT GCC TCT GGT GAA TTT GCG ATC AAA GTT
GGT ATC CCT GCG AAA TCG GGA GTG TCAGGT GGG ATT ATG GGT ATC TCC CCT
TAC GAT TTC GGA ATC GGG ATC TTT GGA CCC GCG CTG GAC GA G AAG GGG
AAT AGT ATT GCT GGT GTG AAG CTT TTAGAA ATC ATG AGC GA G ATG TAC CGT
CTT AGT ATC TTT 40 ybgJ (Bacillus ATG AAA GAG TTG ATT AAA GAG CAT
CAA AAG GAT ATC AAT CCT GCA TTA subtilis) CAA CTG CAT GAC TGG GTA
GAA TAC TAC CGT CCA TTT GCG GCA AAT GG C CAAAGT GCA AAC TAT ATC CCC
GCT TTA GGG AAG GTG AAC GAC AGC CA G TTA GGG ATC TGC GTA CTG GAA
CCG GAT GGC ACC ATG ATT CAC GCT G GG GAT TGGAAT GTG TCC TTT ACC ATG
CAG TCG ATT TCA AAA GTA ATT A GC TTC ATT GCT GCC TGC ATG TCG CGT
GGA ATC CCG TAT GTC TTG GAT CGT GTA GAC GTGGAA CCC ACA GGA GAT GCT
TTT AAT AGT ATC ATC CGT TTA GAG ATC AAC AAA CCA GGA AAG CCT TTC AAT
CCT ATG ATT AAT GCC GGA GCT TTG ACT ATCGCT AGC ATT CTT CCA GGA GAG
TCC GCT TAC GAA AAA CTT GAG TTT TTG TAT AGC GTG ATG GAG ACT TTA ATC
GGT AAA CG C CCC CGT ATT CAC GAA GAAGTA TTC CGT TCT GAA TGG GAG ACC
GCT CA T CGC AAT CGC GCC TTA GCC TAC TAT CTT AAA GAA ACA AAC TTC
TTA G AG GCC GAG GTC GAA GAG ACA CTGGAA GTA TAT TTG AAA CAA TGC GCG
A TG GAA TCG ACC ACG GAA GAC ATC GCC CTG ATC GGG TTG ATC CTG GCC
CAC GAT GGG TAT CAT CCT ATC CGT CATGAG CAG GTC ATT CCC AAG GAT GTT
GCC AAG TTG GCT AAA GCG TTA ATG TTG ACC TGT GGC ATG TAT AAC GCT TCT
GGA AAG TAT GCG GCT TTC GTT GGAGTA CCC GCA AAA TCT GGA GTT TCG GGT
GGT ATT ATG GCC TTG GTG CCT CCA AGT GCG CGT CGC GA A CAG CCG TTC
CAG AGC GGG TGC GGT ATC GGG ATTTAT GGA CCT GCA AT T GAT GAG TAC GGG
AAT AGC CTG ACG GGC GGC ATG CTT TTA AAA CAC A TG GCC CAA GAG TGG
GAA CTG AGT ATT TTC 41 Glutamine
CCATGGCAGAACGTGCAGTGCAGCTGGGCGGTGTAGCTCTGGGGACCACTCAAGTTATCAACA
permease
GCAAAACCCCGCTGAAAAGTTACCCGCTGGACATCCACAACGTTCAGGATCACCTGAAAGAAC
glnHPQ
TGGCTGACCGTTACGCAATCGTCGCTAATGACGTACGCAAAGCGATTGGCGAAGCGAAAGATG
operon
ACGACACCGCAGATATCCTGACCGCCGCGTCTCGCGACCTGGATAAATTCCTGTGGTTTATCG
(Escherichia
AGTCTAACATCGAATAAATCCATCGCTGATGGTGCAGAACTTTAGTACCCGATAAAAGCGGCT
coli)
TCCTGACAGGAGGCCGTTTTGTTTTGCAGCCCACCTCAACGCACTTATTTAGTGCATCCATCT
GenBank:
GCTATCTCCAGCTGATTAAGTAAATTTTTTGTATCCACATCATCACACAATCGTTACATAAAG
X14180.1
ATTGTTTTTTCATCAGGTTTTACGCTAAATAATCACTGTGTTGAGTGCACAATTTTAGCGCAC
CAGATTGGTGCCCCAGAATGGTGCATCTTCAGGGTATTGCCCTATAAATCGTGCATCACGTTT
TTGCCGCATCTCGAAAAATCAAGGAGTTGCAAAACTGGCACGATTTTTTCATATATGTGAATG
TCACGCAGGGGATCGTCCCGTGGATAGAAAAAAGGAAATGCTATGAAGTCTGTATTAAAAGTT
TCACTGGCTGCACTGACCCTGGCTTTTGCGGTTTCTTCTCATGCCGCGGATAAAAAATTAGTT
GTCGCGACGGATACCGCCTTCGTTCCGTTTGAATTTAAACAGGGCGATAAATATGTGGGCTTT
GACGTTGATCTGTGGGCTGCCATCGCTAAAGAGCTGAAGCTGGATTACGAACTGAAGCCGATG
GATTTCAGTGGGATCATTCCGGCACTGCAAACCAAAAACGTCGATCTGGCGCTGGCGGGCATT
ACCATCACCGACGAGCGTAAAAAAGCGATCGATTTCTCTGACGGCTACTACAAAAGCGGCCTG
TTAGTGATGGTGAAAGCTAACAATAACGATGTGAAAAGCGTGAAAGATCTCGACGGGAAAGTG
GTTGCTGTGAAGAGCGGTACTGGCTCCGTTGATTACGCGAAAGCAAACATCAAAACTAAAGAT
CTGCGTCAGTTCCCGAACATCGATAACGCCTATATGGAACTGGGCACCAACCGCGCAGACGCC
GTTCTGCACGATACGCCAAACATTCTGTACTTCATCAAAACCGCCGGTAACGGTCAGTTCAAA
GCGGTAGGTGACTCTCTGGAAGCGCAGCAATACGGTATTGCGTTCCCGAAAGGTAGCGACGAG
CTGCGTGACAAAGTCAACGGCGCGTTGAAAACCCTGCGCGAGAACGGAACTTACAACGAAATC
TACAAAAAATGGTTCGGTACTGAACCGAAATAATAACGCTACACCTGTAAAACGCACTGGCAG
TTCCCTCTCCCCTATGGGGAGAGGATTAGGGTGAGGGGCGCAAACCCGCTCCGGGGCCATTAA
TTACCCTGAATTTGATTATTTACACCACGGTAACAGGAACAACATATGCAGTTTGACTGGAGT
GCCATCTGGCCTGCCATTCCGCTTCTGATTGAAGGTGCCAAAATGACCCTGTGGATTTCGGTC
CTCGGTCTGGCAGGCGGTCTGGTAATCGGATTGCTGGCAGGTTTTGCACGCACCTTCGGAGGT
TGGATAGCCAACCACGTCGCGCTGGTCTTTATTGAAGTGATCCGCGGCACACCTATCGTCGTC
CAGGTGATGTTTATTTATTTCGCCCTGCCGATGGCGTTTAACGACTTACGCATCGACCCATTT
ACTGCGGCGGTGGTCACCATCATGATCAACTCCGGCGCGTATATTGCGGAAATCACGCGTGGT
GCGGTGCTGTCTATCCACAAAGGTTTTCGTGAAGCAGGACTGGCGCTCGGTCTTTCACGTTGG
GAAACCATTCGCTACGTCATTTTACCGCTGGCACTGCGTCGTATGCTGCCGCCGCTGGGTAAC
CAGTGGATCATCAGCATTAAAGACACCTCGCTGTTTATTGTGATCGGCGTGGCGGAACTGACC
CGTCAGGGGCAAGAAATTATTGCCGGTAACTTCCGCGCCCTTGAGATCTGGAGCGCCGTGGCG
GTGTTCTATCTGATTATTACCCTGGTGCTGAGCTTTATTCTGCGTCGTCTGGAAAGAAGGATG
AAAATCCTGTGATTGAATTTAAAAACGTCTCCAAGCACTTTGGCCCAACCCAGGTGCTGCACA
ATATCGATTTGAACATTGCCCAGGGCGAAGTCGTGGTGATTATCGGGCCGTCCGGTTCCGGTA
AATCGACCCTGCTGCGCTGCATCAACAAACTGGAAGAAATCACCTCCGGCGATCTGATTGTCG
ATGGCCTGAAGGTTAACGATCCGAAAGTTGACGAGCGCCTGATTCGCCAGGAAGCAGGTATGG
TGTTCCAGCAGTTTTACCTCTTCCCGCATCTGACAGCGCTGGAAAACGTCATGTTTGGCCCGC
TACGCGTGCGTGGCGCGAACAAAGAAGAGGCGGAAAAACTGGCACGTGAGCTGCTGGCGAAAG
TCGGTCTGGCAGAACGTGCACATCACTACCCTTCCGAACTTTCTGGTGGTCAACAGCAGCGTG
TGGCGATTGCCCGCGCGCTGGCGGTGAAGCCGAAAATGATGCTGTTTGATGAACCGACTTCCG
CTCTTGACCCGGAACTGCGCCATGAAGTGCTGAAGGTTATGCAGGATCTGGCTGAAGAAGGGA
TGACGATGGTGATCGTGACCCACGAAATCGGTTTTGCCGAGAAAGTAGCTTCGCGGCTGATCT
TTATCGACAAAGGCCGGATTGCGGAAGATGGCAATCCGCAGGTGTTGATCAAGAACCCGCCGA
GCCAGCGCTTGCAGGAATTTTTGCAGCACGTCTCTTAATAAGACACATTGCCTGATCGTACGC
TTATCAGGCCTACAGGATATCTGGCAACTTATTAAAATTGCATGAACTTGTAGGACGGATAAG
GCGTTCACGCGCATCCGGCAAAAAAGCCCGCACGTTGTCAGCAACCTGCTTAATATCCCTTCC
TCCCTTTCACCCGAAAGGGAGGCACACCAGATTCCTCTCATTTAAAATCGCCCCTCCTCCAGC
ATCTATACTTATCTTTTTGCTCTATTTTCTCACTGGAGGAGTCATGCGGTGGATCCTGTTCAT
CCTCTTCTGCCTGCTGGGCGCACCTGCCCACGCGGTATCCATACCCGGCGTTACAACCACAAC
GACAACGGACTCAACGACTGAACCGGCCCCGGAACCGGATATCGAACAAAAAAAAGCGGCCTA
TGCGCACTGGCGGATGTGCTGGATAATGACACCTCGCGTAAAGAGTTGATCGACCAGTTGCGC
ACCGTTGCCGCTACGCCCCTGCTGAACCGGTACC 42 Glutamine ATG AAA AGT GTA CTT
AAA GTG TCA TTG GCA GCA CTG ACA CTT GCA TTT permease H GCA GTC TCC
AGT CAT GCT GCG GAC AAA AAG TTA GTC GTA GCG ACT GA glnH C ACTGCG
TTT GTT CCT TTC GAA TTC AAG CAG GGG GAC AAG TAC GTC GG (Escherichia
C TTT GAC GTA GAC CTT TGG GCC GCC ATT GCA AAA GAG CTT AAG TTG G
coli EPEC AT TAC GAGTTA AAG CCT ATG GAC TTC AGT GGT ATC ATT CCC GCC
CTG C C342-62) AA ACG AAA AAC GTG GAT CTT GCG CTT GCA GGC ATT ACT
ATT ACC GAC GAA CGC AAG AAGGCG ATT GAC TTC AGC GAC GGC TAT TAT AAG
TCG GGT CTT TTA GTT ATG GTA AAA GCC AAC AAT AAT GAT GTG AAA AGC GTG
AAA GAT TTG GAC GGG AAAGTA GTG GCA GTT AAA TCA GGT ACA GGG AGT GTG
GAT TAC GCG AAA GCT AAT ATC AAA ACC AAA GAC TTA CGT CAA TTC CC G
AAT ATC GAC AAT GCG TATATG GAA CTG GGG ACG AAC CGT GCG GAT GC G GTG
CTG CAC GAT ACA CCC AAC ATC CTT TAT TTC ATT AAA ACA GCT G GT AAT
GGT CAA TTT AAA GCT GTAGGC GAC AGC CTG GAA GCC CAG CAA T AC GGG ATC
GCG TTC CCT AAG GGC TCT GAT GAG CTT CGT GAC AAG GTA AAC GGG GCG CTT
AAA ACG CTG CGT GAAAAC GGA ACG TAC AAT GAA ATC TAT AAG AAG TGG TTC
GGA ACC GAG CCC AAA 43 Glutamine ATG CAA TTC GAT TGG AGT GCG ATT
TGG CCT GCC ATT CCC CTT CTG ATT permease P GAG GGT GCA AAA ATG ACT
CTG TGG ATT TCA GTG CTG GGG TTA GCC GG glnP A GGTCTT GTT ATT GGG
TTA TTA GCA GGG TTT GCA CGC ACT TTC GGG GG (Escherichia A TGG ATT
GCA AAT CAT GTT GCG CTG GTC TTC ATC GAA GTC ATT CGT G coli B354) GC
ACC CCCATC GTG GTC CAA GTG ATG TTT ATT TAC TTC GCG TTG CCA A TG GCA
TTT AAC GAT CTT CGT ATT GAT CCA TTT ACT GCG GCA GTG GTG ACT ATC ATG
ATTAAT AGT GGG GCG TAC ATT GCG GAG ATT ACT CGC GGC GCT GTT CTT TCC
ATT CAC AAA GGT TTT CGT GAG GCC GGT TTA GCT CTT GGG CTT TCC CGC
TGGGAA ACA ATT CGT TAT GTT ATC TTG CCG CTT GCC TTG CGC CGT ATG TTG
CCG CCG CTG GGT AAC CAA TGG ATC ATT TCT AT C AAA GAT ACT TCG CTT
TTCATT GTT ATT GGA GTG GCT GAA TTA ACA CG C CAA GGT CAA GAA ATC ATC
GCG GGG AAT TTC CGT GCA TTA GAG ATC T GG AGT GCT GTC GCC GTT TTC
TACTTG ATC ATT ACG CTG GTG CTG TCC T TT ATT TTG CGC CGC TTG GAG CGT
CGC ATG AAG ATT CTT 44 Glutamine ATG ATT GAA TTT AAG AAT GTG TCG
AAG CAT TTC GGC CCC ACC CAA GTA Permease Q CTT CAC AAC ATT GAC CTT
AAC ATC GCC CAG GGC GAG GTT GTA GTA AT glnQ C ATCGGT CCA TCT GGT
AGT GGC AAG TCC ACC TTG CTG CGT TGT ATC AA (Escherichia T AAA CTT
GAG GAA ATC ACC AGC GGA GAC TTA ATT GTG GAC GGT CTT A coli EPEC AA
GTC AACGAT CCA AAA GTG GAC GAA CGC TTG ATT CGT CAG GAA GCG G
C342-62) GT ATG GTT TTC CAG CAG TTC TAC TTG TTT CCG CAC CTT ACG GCT
CTT GAG AAC GTC ATGTTC GGA CCG TTA CGC GTG CGC GGG GCC AAT AAG GAG
GAG GCG GAG AAG TTG GCA CGC GAG CTG TTA GCA AAA GTT GGC TTG GCT GAA
CGT GCA CAT CATTAC CCT TCT GAG CTG TCA GGT GGG CAA CAG CAA CGT GTC
GCC ATC GCA CGC GCG CTT GCT GTA AAA CCA AAG ATG ATG CT G TTC GAT
GAG CCA ACG TCGGCG CTT GAC CCG GAG TTG CGC CAT GAG GT C CTT AAG GTT
ATG CAA GAC TTA GCT GAA GAG GGA ATG ACG ATG GTA A TC GTG ACG CAC
GAG ATT GGA TTCGCA GAG AAG GTA GCA TCT CGT TTG A TC TTC ATC GAC AAA
GGT CGC ATT GCA GAA GAC GGC GAC CCA CAA GTT CTG ATT AAG AAC CCC CCT
TCA CAG CGCCTG CAA GAA TTT CTG CAA CAT GTC TCC Tryptophan 45
tryptophan ATG AGT TCC GCC ACA AGT CCG GCA CTG GAT TAT GCA TTG CTG
TTG TCT amino TCT TCT GCT CGT AAC CGT ATG CCT TCT GCA ATC CGT TCC
CTG TTC CC transferase G GCAGAA TTA ATT CCA GGC ATG GTC TCT CTT TTG
TCA GGT AAA CCG AA (transaminase) T TCG GAG ACC TTT CCC TTT CAG CGC
ATC AGT TTG GAA CTT AAA CCC T (Ustilago CC ATC CATCTG GAG GGA CAG
ACC GAG ACA GTG AGC ATC GAA GGT AGC G maydis 521) AT TTA GAC ATC
GCT CTT CAG TAT TCA GCA ACG AGT GGG TTG CCA AAG TTG GTA GAC TGGATC
ATT AAA TTT CAA TCT CGC GTT CAC GCT CGT AAG CAG GTC GAT GAG GGC AAT
AAG CCG GGT GAA GTA TGG CGC TGT AGC TTT GGC AAC GGA TCT CAAGAC CTG
CTG ACC AAG ACA TTT GAG GCT TTA GTT
GAC GCC GGT GAT TCA GTA GTC CTG GAA AGT CCG GCT TAC AGT GGA AT T
TTG CCG TCG TTG GTT GCGCAT AAA GCC AAC CTT TTC GAG GCA GAA AC T GAC
GCC GAG GGC GTT GAG CCC ACG GCT TTA GAC ACA TTG CTG ACT A AC TGG
AAG ACT GAC AGT GCA ACACGT GAC TCT CGT TTT CCC AAG TTT T TA TAT ACT
ACC CCG ACT GGT GCA AAT CCG TCC GGG ACA TCA GCC TCT GAT AAT CGC AAG
CGT GCG ATC CTT GATATT ATC CGC AAG CAC AAT TTA CTT CTG CTG GAG GAT
GAT CCT TAC TAT TTT TTG TCA TTC CAA GGG TTG GAA CCG GGG GCT GAC GCG
GTC AAA CGC ACTCGT GGG AAG AGC TAT TTT CAG TTG GAA GCT CAG GAC GAC
TAT GGC GTC GGC CGT GTT GTT CGC TT T GAT TCA TTT AGT AAG ATC TTG
TCT GCC GGA TTACGC CTG GGT TTC GT T ACA GGA CCC AAA GAG ATT CTG GAC
GCC ATC GAC CTG GAC ACT TCC T CC CGC AAT TTG CAG ACA AGT GGC ACT
TCC CAG GCA ATCGCC TAT GCT T TG TTG TCT AAG TGG GGA ATT GAC GGT TTT
TTA CAT CAT GCG GAC AAT GTC GCA CGT TTT TAC CAA AAT CGC TTA GAA CGC
TTT GAA GCCAGT GCC CAG GCA ATC TTA ACC GGA AGC CCT AGC ATC GCC TCG
TGG GTT CGT CCT TCG GCA GGG ATG TTC CTG TGG ATC AAG TTA AAG TTG CCT
CCG TCGCCC GAC TCG GCG GAG GGT GAT AGT TTT GAC CTG ATC TCT AAT AAA
GCT AA G GCA GCT GGG GTA TTG GCT TTA CCC GGT GTG GCC TTC AAA CCA
CCG A GCAGT TCA AGT ACG GGT GGC AAA CGT AAG ACA TCG GCA TAT GTC CGC
A CG TCA TTC TCC CAG GTG CCT CTG GAC CAA GTG GAT ACC GCA TTC ACA
CGC CTGCGT CAG GTG GTA GAG GAG GCC TGG CGT GAG GCT GGA CTT CAA ATC
CCC GCG 46 Mtr tryptophan ATG GCT ACC CTT ACT ACT ACT CAA ACT TCC
CCA TCG CTT CTT GGA GGA ArAAP GTC GTT ATC ATC GGT GGA ACT ATC ATC
GGA GCA GGG ATG TTT TCA CT transporter G CCGGTT GTG ATG TCG GGA GCA
TGG TTC TTT TGG TCA ATG GCG GCT CT (Escherichia T ATC TTC ACG TGG
TTC TGT ATG TTG CAT AGT GGC CTG ATG ATC CTG G coli BL21(DE3)) AA
GCA AATCTG AAC TAC CGT ATT GGG TCC TCT TTT GAT ACA ATT ACA A AG GAC
CTT CTG GGG AAA GGA TGG AAT GTA GTT AAT GGA ATT AGT ATC GCG TTC GTC
CTTTAC ATC TTG ACC TAC GCG TAT ATC TCT GCC TCA GGG AGC ATC TTG CAT
CAC ACT TTT GCC GAG ATG TCA TTG AAC GTG CCC GCA CGC GCT GCT GGC
TTTGGT TTT GCA CTG CTT GTG GCA TTC GTA GTC TGG TTA AGT ACG AAG GCT
GTG AGC CGT ATG ACC GCT ATC GTC CTT GGG GC T AAA GTA ATT ACC TTC
TTTTTA ACA TTC GGC TCG CTG TTA GGA CAC GT G CAG CCT GCC ACT TTG TTC
AAT GTG GCT GAA TCA AAC GCC TCG TAT G CC CCC TAT TTA CTT ATG ACT
TTGCCG TTT TGT CTG GCT TCC TTC GGT T AT CAC GGA AAC GTG CCA TCA CTG
ATG AAA TAT TAT GGT AAG GAT CCT AAA ACA ATT GTG AAG TGC TTG GTA
TACGGG ACC TTA ATG GCA CTT GCC CTT TAC ACG ATC TGG CTT CTT GCA ACG
ATG GGC AAT ATT CCT CGC CCT GAA TTT ATC GGG ATC GCA GAA AAA GGG
GGGAAT ATT GAC GTG CTG GTC CAG GCT TTA TCG GGT GTC TTG AAT AGC CGC
TCT TTG GAT CTT TTG TT A GTT GTC TTT TCC AAT TTT GCC GTG GCA TCG
AGTTTC TTA GGT GTG AC G CTG GGT CTT TTT GAT TAC CTG GCC GAT CTG TTC
GGA TTC GAC GAC A GC GCG GTG GGC CGT CTT AAA ACT GCT TTA TTA ACA
TTTGCG CCC CCT G TA GTG GGA GGT CTT CTG TTT CCT AAC GGA TTC TTA TAC
GCC ATC GGC TAC GCC GGA TTG GCG GCC ACG ATT TGG GCA GCT ATC GTC
CCGGCT TTA TTG GCA CGT GCC TCA CGC AAA CGC TTC GGG AGT CCT AAA TTC
CGT GTT TGG GGC GGG AAG CCT ATG ATT GCC CTT ATT TTA GTG TTT GGA
GTCGGT AAT GCA CTT GTG CAC ATC TTG TCA TCG TTC AAT CTG CTT CCC GTT
TA T CAA 47 tryptophan ATG ACC GAC CAA GCT GAA AAG AAG CAT TCG GCA
TTC TGG GGA GTA ATG permease Tna GTC ATT GCC GGT ACC GTG ATC GGC
GGT GGG ATG TTT GCT TTA CCT GT B G GACTTA GCA GGC GCG TGG TTT TTT
TGG GGG GCG TTC ATT CTG ATT AT (Escherichia T GCT TGG TTT TCC ATG
CTG CAT AGT GGC TTG CTG CTT CTT GAA GCG A coli str. K- AT CTT
AACTAT CCG GTG GGG TCA AGT TTC AAT ACC ATT ACA AAG GAC C 12 substr.
TG ATT GGT AAC ACA TGG AAT ATC ATT TCG GGG ATC ACG GTA GCA TTT
MC4100) GTA TTG TAT ATTCTT ACA TAT GCT TAT ATC AGT GCG AAT GGC GCA
ATC ATT TCC GAG ACG ATC TCC ATG AAC CTG GGG TAT CAC GCG AAT CCC CGT
ATT GTC GGC ATC TGCACA GCG ATT TTT GTT GCG AGC GTA TTA TGG CTG AGT
TCG TTG GCA GCT TCG CGT ATT ACT TCC CTT TTC CTT GGT TTG AA A ATC
ATC AGC TTC GTA ATTGTG TTT GGG AGT TTT TTT TTC CAG GTC GA C TAC TCC
ATT CTT CGC GAT GCA ACA AGT AGC ACA GCA GGC ACC AGT T AC TTC CCA
TAT ATC TTT ATG GCCTTA CCG GTT TGT TTA GCG TCT TTT G GT TTT CAT GGT
AAT ATC CCC TCA TTA ATT ATT TGC TAC GGC AAG CGC AAG GAC AAA TTA ATT
AAG TCT GTT GTTTTC GGC TCC TTG TTG GCG CTT GTA ATC TAT TTA TTT TGG
CTT TAT TGT ACG ATG GGG AAC ATC CCT CGC GAA TCC TTT AAG GCT ATT ATT
TCT TCA GGAGGC AAC GTA GAC AGT TTG GTA AAA AGT TTT TTG GGT ACG AAG
CAG CAT GGT ATC ATC GAG TTT TG T TTA CTT GTT TTC AGT AAT CTT GCC
GTT GCT TCCTCA TTC TTT GGC GT G ACT CTG GGG CTT TTT GAT TAT CTG GCA
GAT TTA TTC AAG ATC GAC A AC TCG CAT GGC GGG CGC TTC AAA ACG GTT
CTG CTT ACATTT CTT CCT C CA GCT TTA CTT TAC CTG ATC TTT CCG AAT GGT
TTT ATC TAT GGT ATT GGG GGG GCA GGC CTG TGC GCC ACT ATC TGG GCA GTT
ATC ATTCCT GCT GTA TTG GCT ATC AAG GCA CGC AAA AAG TTT CCC AAC CAG
ATG TTC ACC GTG TGG GGC GGC AAT TTG ATT CCG GCA ATC GTG ATC TTA TTT
GGTATC ACG GTT ATT CTT TGC TGG TTC GGC AAT GTG TTT AAC GTC CTG CCT
AA G TTT GGA 48 aroP ATG GAA GGG CAG CAG CAT GGC GAA CAG CTT AAG
CGT GGC CTG AAG AAT (Escherichia CGT CAT ATC CAG CTT ATC GCA TTA
GGC GGA GCT ATT GGG ACC GGC TT coli O104: H4 G TTCTTA GGC TCT GCT
TCA GTC ATT CAG TCT GCG GGG CCA GGC ATT AT str. C227- T TTA GGC TAC
GCG ATT GCG GGC TTC ATC GCC TTT TTA ATT ATG CGC C 11) AG CTT GGCGAG
ATG GTG GTG GAG GAA CCC GTG GCA GGC AGT TTC TCT C AC TTT GCA TAC
AAG TAT TGG GGA AGT TTT GCA GGC TTT GCG AGC GGT TGG AAC TAC TGGGTT
CTG TAC GTT CTG GTG GCC ATG GCG GAA CTG ACA GCA GTC GGT AAA TAT ATT
CAA TTC TGG TAC CCT GAA ATT CCC ACT TGG GTC TCT GCC GCT GTCTTC TTT
GTC GTC ATT AAT GCA ATC AAC TTG ACC AAC GTC AAA GTA TTC GGC GAG ATG
GAG TTT TGG TTC GCT ATT ATC AA A GTC ATT GCT GTT GTG GCCATG ATC ATT
TTC GGA GGC TGG CTG CTT TT C AGC GGC AAC GGA GGT CCC CAG GCA ACT
GTA TCG AAT CTT TGG GAC C AG GGT GGT TTC TTG CCA CAT GGGTTC ACG GGG
TTA GTT ATG ATG ATG G CC ATT ATT ATG TTC TCG TTT GGA GGG CTT GAA
TTG GTG GGC ATC ACT GCT GCT GAA GCT GAT AAC CCG GAG CAAAGC ATT CCT
AAG GCC ACA AAT CAA GTG ATC TAT CGC ATC CTT ATC TTT TAC ATT GGA TCG
TTG GCA GTA TTG CTG AGT TTG ATG CCC TGG ACC CGT GTCACC GCT GAT ACA
AGC CCT TTT GTT TTG ATT TTT CAT GAA TTA GGG GAT ACT TTT GTG GCA AAT
GC G TTA AAC ATC GTC GTA TTA ACT GCT GCC TTG TCAGTA TAT AAC TCC TG
C GTA TAC TGT AAT AGC CGT ATG CTG TTC GGC TTG GCT CAG CAG GGG A AC
GCT CCG AAA GCA CTG GCC AGT GTC GAC AAG CGT GGAGTA CCT GTG A AT ACG
ATT TTA GTT TCT GCT CTG GTC ACT GCA CTT TGT GTA TTG ATC AAC TAC CTG
GCG CCT GAG TCG GCG TTT GGC CTG CTG ATG GCGCTG GTG GTT AGC GCA TTG
GTC ATC AAT TGG GCG ATG ATC TCC TTG GCA CAC ATG AAA TTC CGC CGT GCT
AAA CAA GAA CAG GGT GTG GTT ACA CAC TTCCCA GCA TTA TTA TAC CCT CTG
GGC AAC TGG ATT TGC TTA CTT TTT ATG GC A GCG GTT CTG GTC ATC ATG
CTG ATG ACG CCT GGT ATG GCT ATT TCT G TTTAT CTG ATT CCG GTT TGG TTA
ATC GTA TTA GGG ATT GGC TAT TTA T TC AAG GAA AAA ACT GCA AAG GCT
GTC AAA GCG CAT 49 Aromatic ATG ACC CGC CAG AAG GCG ACT CTG ATC GGT
TTG ATT GCT ATC GTA TTA amino acid TGG TCC ACA ATG GTT GGT TTA ATT
CGT GGG GTT TCT GAG GGG CTT GG exporter Ydd C CCGGTG GGC GGA GCA
GCA GCT ATC TAC TCC CTG AGC GGT CTG TTA TT G G ATC TTT ACA GTT GGG
TTT CCG CGT ATC CGT CAA ATC CCC AAG GGA T (Escherichia AC TTA
TTGGCG GGG AGT TTA CTT TTT GTG AGC TAT GAA ATT TGC CTT G coli
TW10598) CC TTG TCT CTG GGC TAC GCA GCG ACA CGC CAT CAA GCA ATT GAG
GTA GGG ATG GTT AATTAC CTT TGG CCG TCA TTG ACG ATT CTT TTC GCA ATC
TTA TTT AAC GGT CAG AAG ACT AAT TGG TTG ATT GTA CCG GGT TTA TTA TTA
GCG TTG GTG GGAGTA TGC TGG GTG TTG GGA GGT GAC AAT GGT CTG CAT TAT
GAC GAG ATT ATT AAT AAT ATC ACA ACA TCG CCC TTA TCC TA C TTT CTG
GCT TTC ATT GGTGCC TTT ATC TGG GCC GCC TAT TGC ACC GT G ACG AAT AAG
TAC GCT CGT GGC TTC AAC GGA ATT ACA GTA TTT GTC T TG CTT ACT GGT
GCA TCT TTG TGGGTA TAT TAT TTC TTG ACC CCT CAA C CA GAG ATG ATC TTC
TCC ACC CCG GTT ATG ATC AAA TTA ATT TCA GCA GCT TTC ACT TTG GGA TTC
GCA TAC GCAGCT TGG AAT GTC GGC ATT CTT CAT GGG AAT GTG ACG ATT ATG
GCA GTC GGT TCC TAC TTC ACG CCC GTA CTT AGT TCC GCT TTA GCA GCG GTA
CTG CTGTCG GCG CCT TTG AGT TTT AGT TTC TGG CAG GGT GCC CTG ATG GTG
TGT GGG GGC TCC CTT TTG TG C TGG CTT GCT ACC CGC CGT GGT 50 S- ATG
GCA AAG CAC CTT TTC ACG TCG GAA TCT GTA TCT GAA GGG CAT CCC
adenosyl- GAC AAA ATT GCA GAT CAA ATC TCC GAC GCG GTA CTT GAT GCT
ATT CT methionine G GAACAA GAT CCC AAA GCC CGC GTC GCT TGC GAA ACT
TAT GTC AAG AC synthase A GGC ATG GTG TTA GTC GGC GGC GAG ATC ACT
ACC TCT GCG TGG GTG G (UniProtKB/ AT ATC GAGGAA ATC ACG CGC AAT ACG
GTG CGT GAG ATT GGC TAT GTA C Swiss-Prot: AC TCG GAC ATG GGG TTC
GAC GCC AAC AGT TGT GCG GTT TTA AGT GCC P0A817.2) ATT GGG AAA
CAGTCA CCT GAT ATT AAT CAG GGG GTG GAT CGT GCG GAC CCT CTT GAA CAA
GGT GCT GGT GAC CAA GGT CTG ATG TTC GGT TAT GCT ACG AAC GAA ACC
GATGTG TTG ATG CCC GCC CCG ATC ACA TAC GCC CAC CGT CTG GTC CAA CGC
CAG GCG GAG GTC CGT AAA AAC GGC ACG CTT CC T TGG CTT CGT CCA GAT
GCTAAG TCG CAG GTC ACT TTC CAA TAC GAC GA C GGG AAG ATT GTC GGA ATC
GAC GCC GTG GTC TTG TCA ACT CAG CAT T CA GAG GAG ATC GAT CAA AAG
AGCCTT CAG GAA GCC GTC ATG GAA GAG A TC ATC AAG CCG ATT CTG CCT GCA
GAA TGG TTA ACT TCC GCG ACC AAG TTC TTT ATT AAC CCC ACC GGG CGT
TTTGTC ATT GGC GGT CCT ATG GGC GAC TGT GGG TTG ACC GGC CGT AAA ATT
ATT GTC GAC ACT TAT GGC GGA ATG GCT CGT CAT GGC GGT GGG GCA TTC
AGTGGC AAG GAC CCG TCA AAG GTA GAT CGT TCA GCC GCC TAT GCC GCC CGT
TAC GTA GCC AAG AAC AT T GTT GCT GCA GGA CTT GCT GAC CGC TGT GAA
ATCCAA GTG AGC TAC GC G ATC GGC GTA GCA GAA CCC ACC TCC ATT ATG GTG
GAA ACT TTT GGC A CC GAA AAA GTC CCC AGT GAG CAA CTG ACC TTA TTG
GTTCGT GAG TTT T TT GAT TTG CGC CCT TAC GGA CTT ATC CAA ATG TTA GAC
CTT TTG CAC CCA ATC TAC AAA GAA ACT GCA GCA TAC GGT CAC TTT GGA
CGCGAG CAT TTT CCC TGG GAG AAG ACA GAC AAA GCA CAG CTG TTA CGT GAC
GCG GCC GGA TTG AAA 51 adenosylhomo ATG ACG GCT ACG ACG CCA CGC CTG
AAA CAT GAA GTG AAG GAC CTT GCG cysteinase CTT GCG CCT TTA GGT CGT
CAG CGT ATT GAG TGG GCG GGG CGC GAA AT (Anabaena G CCTGTT TTA AAG
CAA ATC CGC GAC CGC TTT GAA AAA GAA AAG CCC TT cylindrica C GCG GGC
CTG CGT ATC TCG GCT TGT GCG CAT GTT ACA ACA GAG ACG G PCC 7122) CT
CAT TTAGCA ATT GCC CTG AAG GCC GGG GGA GCT GAT GCC GTA TTG A
GenBank: TC GCA AGC AAC CCA CTG TCT ACG CAG GAT GAC GTA GCA GCC TCG
CTT AFZ60429.1 GTC GCT GAT CATGAG ATC TCT GTG TTT GCA CAA AAG GGC
GAA GAC GCC GCG ACG TAC TCG CGT CAC GTC CAA ATT GCG TTG GAC CAC CGC
CCC AAT ATC ATC GTT GAT GACGGT TCC GAC GTA GTA GCT GAA TTA GTA CAG
CAC CGT CAG AAT CAG ATC GCG GAT CTT ATT GGA TCC ACT GAA GAA ACT AC
A ACT GGG ATT GTT CGC CTTCGC GCT ATG TTC AAC GAG GGG GTT TTG AC G
TTT CCC GCG ATG AAT GTC AAC GAC GCA GAC ACA AAA CAT TTT TTT G AC
AAC CGC TAC GGT ACA GGA CAATCT ACC TTG GAC GGG ATC ATT CGT G CA ACC
AAC ATC TTG CTT GCC GGC AAA ACT ATC GTA GTT GTA GGC TAT GGC TGG TGC
GGA AAG GGG ACC GCA TTACGC GCC CGC GGG ATG GGA GCT AAT GTC ATT GTT
ACC GAG ATC GAT CAC ATT AAG GCA ATT GAG GCG GTG ATG GAT GGG TTT CGC
GTT CTG CCC ATG GCTGAA GCC GCA CCG CAT GGT GAT ATC TTT ATC ACT GTA
ACG GGT AAT AAA CAC GTA GTT CGT GGT GA A CAC TTT GAT GTC ATG AAA
GAC GGC GCC ATT GTTTGC AAC TCA GGT CA C TTC GAT TTG GAG TTG GAT TTA
AAA TAT TTA GCA GCA AAT GCC AAG G AA ATC AAA GAT GTG CGC CCA TTC
ACA CAA GAA TAT AAATTA ACC AAC G GC AAA AGC GTA GTG GTA TTA GGA GAG
GGG CGT TTG ATT AAT CTT GCA GCG GCA GAA GGT CAT CCG TCG GCA GTT ATG
GAC ATG TCT TTCGCC AAT CAA GCC TTA GCA GTC GAG TAT TTA GTG AAA AAT
AAA GGC TCC TTG GCG GCT GGA TTA CAT TCG ATC CCC CGC GAG GTT GAT GAG
GAA ATC GCTCGT TTA AAA TTG CAA GCG ATG GGG ATT TTT ATC GAT TCC CTG
ACA GCA GA T CAA ATC GAT TAT ATT AAT TCT TGG CAG TCA GGG ACG 52
Cystathionine- ATG GTA ATG TCG TTA TTC CAC AGT GTT AGC GAT TTA ATC
GGT CAC ACA beta- CCT TTA TTA CAA TTG CAT AAG CTT GAT ACA GGA CCC
TGT AGT TTG TT synthase C TTGAAA CTT GAG AAT CAA AAC CCA GGA GGG
TCA ATT AAA GAT CGT GT (Klebsiella A GCG CTT AGC ATG ATT AAC GAA
GCG GAA CGT CAG GGA AAA CTT GCG C quasipneumoniae CA GGA GGAACT ATC
ATC GAG GCT ACG GCG GGA AAT ACT GGG TTG GGG C subsp. TT GCT TTG ATC
GCA GCC CAG AAA AAC TAC CGT CTT ATC CTT GTA GTT Quasipneumoniae)
CCC GAC AAG ATGTCA CGT GAA AAA ATT TTC CAC TTG CGT GCC TTA GGC
GenBank: GCA ACC GTG CTT TTG ACC CGT TCA GAC GTG AAC AAG GGG CAC
CCG GCA CDQ16225.1 TAT TAT CAG GAC TATGCT CGC CGC TTG GCA GAT GAG
ACT CCA GGG GCG TTC TAC ATT GAC CAA TTC AAT AAT GAT GCC AAT CCT TTA
GCA CAT GC A ACA AGC ACG GCC CCT GAGCTG TTC CAA CAA TTA GAA GGG GAC
ATC GA T GCC ATT GTG GTT GGT GTT GGG TCG GGT GGA ACG TTG GGC GGC
TTG C AG GCC TGG TTC GCA GAA CAC TCTCCC AAA ACA GAG TTC ATC TTG GCT
G AT CCA GCT GGG TCG ATT CTT GCC GAC CAG GTA GAC ACA GGC CGC TAC
GGG GAA ACG GGA AGC TGG CTT GTA GAGGGT ATT GGC GAG GAT TTT ATC CCA
CCA CTT GCT CGC CTG GAA GGA GTT CAT ACC GCA TAT CGT GTA TCT GAT CGC
GAA GCC TTT CTT ACA GCC CGT CAACTG CTT CAG GTA GAG GGT GTA TTA GCG
GGC TCG TCA ACG GGA ACA TTG TTA TCT GCG GCC TTG CG C TAT TGC CGT
GCC CAG TCT CGC CCA AAG CGT GTGGTT ACC TTC GCA TG T GAC TCT GGA AAT
AAG TAC TTG AGT AAG ATG TTC AAT GAC GAC TGG A TG CGC CAA CAG GGA
CTT ATT GCG CGC CCG GAA CAG GGAGAT CTG AGT G AT TTC ATC GCC TTA CGT
CAC GAC GAG GGG GCC ACG GTC ACC GCC GCG CCC GAC GAC ACA CTG GCG GCT
GTA TTT ACT CGC ATG CGC TTGTAC GAT ATC TCC CAG CTT CCG GTC TTG GAA
GAC GGT CGT GTC GTT GGC ATT GTG GAC GAA TGG GAT TTA ATT CGC CAT GTA
CGT GGC GAC CGT CAA CGCTTT TCC CTG CCA GTC AGC GAG GCT ATG TCC CGT
CAC GTA GAA ACG TTA GA C AAA CGC GCC CCC GAA TCC GAA TTG CAA GCT
ATC TTA GAC CGT GGA C TGGTA GCA GTC ATT GCA GAC AAT GCG CGC TTT CTG
GGA CTG GTT ACA C GT TCA GAT GTC TTA ACG GCA TGG CGC AAT CGT GTG
GCG CAA 53 cystathionine- ATG TCG TCT ATT CAC ACC CTG TCT GTT CAT
AGT GGC ACC TTC ACG GAC gamma- TCA CAT GGC GCG GTG ATG CCC CCA ATC
TAT GCC ACC TCC ACG TTC GC lyase G CAACCT GCG CCC GGA CAG CAC ACC
GGA TAT GAA TAC TCG CGC AGT GG (Klebsiella A AAT CCT ACT CGT CAT
GCC TTA GAG ACT GCG ATC GCA GAC CTG GAG A pneumoniae AT GGA ACGCGC
GGG TAC GCA TTT GCC TCG GGC TTG GCA GCG ATC TCG
A subsp. CT GTC CTT GAA TTG TTG GAT AAG GAC AGC CAT TTA GTT GCA GTG
GAT pneumoniae GAT GTC TAT GGTGGG ACC TAC CGT TTA CTT GAA AAC GTT
CGT CGT CGT HS11286) TCT GCT GGG CTG CAA GTG TCG TGG GTC AAG CCA
GAC GAT TTA GCG GGG NCBI ATT GAG GCG GCT ATCCGT CCT GAC ACC CGT ATG
ATC TGG GTC GAA ACA Reference CCT ACT AAT CCT TTG CTG AAA TTA GCC
GAT TTG AGC GCC ATC GCA GC Sequence: T ATC GCA CGC CGT CAC AATCTT
ATT TCA GTT GCG GAT AAC ACG TTC GC YP_005228837.1 T TCA CCA GCC ATC
CAC CGT CCT CTT GAA CAC GGT TTC GAC ATT GTG G TG CAT TCT GCG ACA
AAA TAC TTAAAT GGA CAT TCC GAT GTG GTT GCG G GG TTA GCT GTC GTC GGA
GAT AAC TCC GGC TTA GCC GAG AAA TTA GGT TAT TTA CAA AAT GCA GTT GGC
GGG GTATTA GAC CCC TTT TCC TCG TTC CTT ACA TTG CGC GGC ATC CGC ACT
CTG GCA CTG CGT ATG GAA CGT CAT AGC GCG AAT GCA CTG CAG TTA GCC GAA
TGGTTG GAA CAA CAG CCC GAA GTA GAG CGT GTA TGG TTT CCT TGG CTG GCC
TCC CAT CCT CAT CAT CA A TTG GCA CGT CAG CAG ATG GCA TTA CCT GGC
GGGATG ATT AGC GTA GT A GTC AAA GGA GAT GAG GGA TAT GCT GAG CGC ATC
ATC AGT AAA CTG C GT TGG TTC ACT CTT GCC GAG TCT TTA GGC GGC GTC
GAGTCG TTA GTT T CC CAG CCG TTC TCA ATG ACA CAT GCT TCG ATC CCA CTT
GAA AAG CGT CTT GCG AAC GGC ATT ACG CCC CAG CTT ATT CGC CTT AGT
GTGGGG ATC GAA GAC CCA CAT GAT CTT ATC GCG GAT TGG CAA CAA GCC CTG
CGT GCC GAA 54 cysteine ATG GAG TTA TAC GAG TGC ATC CAG GAC ATC TTC
TCG GGG TTG AAA AAC dioxygenase CCT TCC GTG AAA GAT CTG GCA ACA TCC
CTG AAA CAA ATC CCG AAT GC (Bacillus A GCTAAA TTA TCT CAG CCT TAC
ATT AAA GAG CCT GAC CAG TAT GCA TA subtilis sub C GGT CGC AAT GCC
ATC TAC CGT AAC AAC GAG TTG GAG ATT ATT GTT A sp. subtilis TC AAC
ATTCCT CCC AAC AAA GAG ACA ACC GTA CAC GAT CAC GGA CAA T str.
BAB-1) CC ATT GGA TGC GCA ATG GTT CTG GAA GGT AAA TTA CTT AAT AGC
ATT GenBank: TAT CGT TCT GCTGGT GAG CAC GCC GAG CTG TCC AAC TCT TAC
TTT GTT AGI30235.1 CAC GAG GGG GAA TGC CTT ATC TCG ACT AAA GGC TTG
ATT CAC AAA ATG AGC AAC CCC ACA AGCGAG CGC ATG GTA TCG TTG CAT GTT
TAT TCG CCA CCG CTT GAG GAC ATG ACA GTA TTT GAG GAA CAG AAA GAG GTG
TTA AA G AAC TCT 55 Glutamate ATG AGC GTT AGT AAA AAA CTG TTC TCT
ACG GCT GTG CGT GGT AAG AGC Oxaloacetate TGG TGG TCA CAC GTC GAG
ATG GGC CCT CCT GAT GCG ATT TTG GGG GT Transaminase G ACTGAA GCT
TTC AAA GCT GAT TCT AAC CCC AAG AAG ATC AAT TTG GG (Caenorhabditis
C GTG GGA GCG TAC CGT GAT GAC CAA GGA AAA CCG TTC GTA CTT CCT A
elegans) GC GTC AAGGAA GCC GAA CGT CAA GTT ATT GCA GCA AAT CTT GAC
AAG G NCBI AG TAC GCC GGG ATC GTT GGC CTG CCT GAA TTC ACG AAA CTT
AGT GCT Reference CAG TTA GCA TTAGGG GAA AAC AGT GAC GTA ATC AAA
AAC AAG CGT ATT Sequence: TTT ACG ACG CAA AGT ATT TCT GGG ACT GGT
GCG CTG CGT ATT GGA AGT NP_741811.1 GAG TTC CTG AGT AAATAT GCA AAG
ACT AAG GTT ATC TAT CAA CCC ACG CCT ACA TGG GGA AAC CAC GTG CCT ATC
TTC AAG TTC GCG GGC GTG GA T GTG AAA CAG TAT CGT TATTAT GAC AAG TCT
ACA TGT GGA TTT GAT GA G ACG GGG GCA TTG GCT GAT ATT GCG CAA ATC
CCC GAA GGT AGC ACT A TT TTG CTG CAC GCG TGC GCA CATAAC CCA ACG GGG
GTC GAC CCT AGT C GT GAC CAA TGG AAA AAG ATT TCA GAT ATT GTT AAG
AAA CGC AAT TTG TTC GTG TTT TTT GAC ATG GTG AAT GAGTCA GTC CTG AGT
CCG TTA CTG CCT CGC ACG CTT ATG CGC CTG CTT GTG TTG TTA CTG AAA TCC
CGC AGT CTT TTC GCC CAC TCA ACA CCC ACC CAT CAGTCG ATG GAA TTA GCT
CTT TTG CCG GCC TCG TCG CGT ATC CAA CTT TCT ACC TCC AAT GGG TCA GA
A ATG TCC AGC TCT TGG CTT ATC GTC AGC AGC CCT 56 methionine ATG AAA
CGC GAC CAA CAT TTT GAA ACA CGC GCG ATC CAT ACT GGT TAC gamma lyase
AAG CCG AAC GAG CAT TTT GAT AGC TTG ACT CCC CCT ATT TAC CAA AC
(Bacillus C AGCACG TTC ACA TTT GCA TCA ATG GAG CAA GGT GGC AAC CGT
TTC GC halodurans A GGC GAG GAA GCA GGA TAT GTT TAT TCA CGC CTG GGG
AAC CCC ACC G C-125) TG CAA ATTTTG GAA CAA CGC ATT GCT GAG TTG GAG
GGT GGG GAG GCA G GenBank: CT CTT GCC TTT GGA TCT GGC ATG GCT GCT
GTC AGT GCG ATT TTG GTG BAB04518.1 GGG CTT ACG AAGGCC AAC GAC CAC
ATC TTA GTG AGC AAT GGA GTG TAT GGT TGT ACG TTT GGG TTG TTA ACG ATG
TTA AAG GAA AAA TAC AAC ATC GAC GCC ACT TTC AGTCCG ATG GAC AGC GTA
GAG GAA ATC CTG GCA AAC ATC CAG GAT AAT ACC ACG TGC ATT TAT GTG GAA
ACA CCT ATC AAC CC C ACC ATG CAG TTA ATC GATTTG GAA CTG GTT GTG CGC
GTA GCG AAG GA A AAG GGT ATT AAG GTA ATC GTT GAT AAC ACG TTT GCC
ACA CCA TAC T TA CAA CAA CCG ATT GCT CTG GGATGT GAC TTC GTT GTC CAT
TCG GCC A CG AAA TAC ATC GGG GGT CAT GGG GAC GTG GTC GCC GGA GTG
CTG ATT GGA GAC AAG GAA ACA ATT CAG TTG ATCCGT AAG ACC ACC CAG AAG
GAT ATG GGG GGC GTA ATT TCT CCA TTT GAT GCG TGG CTG CTG TTG CGC GGA
TTG AAA ACA CTT GCA GTA CGT ATG GAT CGCCAT TGC GAG AAT GCT GAA AAA
TTG GCC GAG AAA CTG AAA GAG CAT CCA AAA GTA AGT ACG GTT CT G TAC
CCG GGA GAC TTT GAG CAT CCC GAT CAC TCCATC GTC GCC AAA CA G ATG AAA
AAG GGA GGC GGT TTA TTA AGC TTT GAG ATC AAG GGG ACT G AG GCG GAC
ATC GCC AAA GTT GTA AAT CAG TTA AAA CTGATT CGT ATT G CT GTT AGT TTG
GGT GAC GCA GAG ACC TTG ATT CAG CAT CCT GCA ACC ATG ACC CAT GCA GTA
GTA CCC GAA AAG CGC CGC ACT CAA ATGGGT ATT AGT AAA AAG TTG TTA CGC
ATG TCG GCC GGG TTA GAG GCC TGG CAA GAT GTC TGG GCT GAC TTA GAG CAG
GCG TTA AAT CAA CTG 57 Methionine ATG ACC GCG ATT CCG GCC TTG GCA
GAC CTG CAG GCT CGT TAT GCC GAC aminotransferase TTA CAA GGG CGT
GGT CTG AAG TTA GAT ATG ACG CGC GGT AAA CCG GC (Methylobacterium G
CCAGAG CAG TTG GAT TTA TCG GAC GAT CTT TTC ACT TTA CCA GGT AA
aquaticum) C CGC GAT CAC CGC ACA GAG AGC GGA GAA GAC GCG CGT AAT
TAC GGC G GenBank: GA GTA CAGGGC CTG GCT GAG GTC CGT GCC TTA TTC
GCC CCT GTG CTT G BAQ48233.1 GT GCG TCA CCC GAT CGC ATT GCC GTA GGT
AAT AAC TCA TCG TTG GCA TTG ATG CAT GACTGC ATT GCC TAT GCA TTG CTT
AAG GGT GTA CCC GGC GGC GCT CGT CCT TGG GCA AAG GAA GAG GAG ATT CGT
TTT TTA TGC CCA GTC CCA GGG TAC GACCGT CAC TTC GCT CTG TGC GAG ACC
TAC GGG ATT GGA ATG ATT CCA GTC CCT ATG ACC GCT GAC GGG CCT GAT ATG
GAA AT G GTT GAA CGT GAG GTA CGCGAT CCA CGC GTC AAA GGT ATG TGG GCG
GT G CCG CAG TAT AGT AAC CCA GGC GGT GAG ACA TAC TCC GAC GCG ACT G
TT GAG CGC CTG GCT CGT ATG GAAACC GGT GCC CCT GAC TTC CGT CTT T TT
TGG GAC AAC GCG TAT GCA CTT CAC CAT TTG ACC GAA CGT CGC CCA ACC CTT
CGT AAT GTG TTA GAT GCC TGTGCG GAA GCC GGG TCA CCG GAT CGT GCT ATT
GTG TTT GCT AGT ACG TCG AAA GTT ACA CTG GCG GGG GCA GGC CTT GCG ATG
CTT GCG TCC AGC GAG GGCAAT ATT CGC TGG TAT TTA GCT AAC GCC GGC AAA
CGC TCA ATT GGT CCA GAT AAG CTT AAC CAG TT G CGC CAT GTT CGC TTT
CTG CGT GAC CAG GGC GGACTT GAT GCA TTA AT G GAC GGC CAC CGC CGT CTT
TTA GCT CCT AAG TTC CGC GCT GTA ACG G AA ACC CTT GCT CGT CAT CTG
GGC GGG ACT GGA GTA GCGCGC TGG AGC G AG CCG GAA GGG GGG TAC TTT ATC
CTG CTG GAA GTC CCT GAG GGC TGT GCG ACA CGC GTA GTT AAG CTT GCT GCT
GCT TGC GGA CTG GCTCTG ACG CCC GCA GGG GCG ACG CAC CCA TAC GGG CGT
GAC CCT CAA GAT AAG CTG TTA CGT CTT GCC CCG TCA TAC CCG AAA CCA GCG
GAG GTC GAG GCAGCC GCT GAG GTA GTC GCT GTG TGC GTT TTA CTT GCG GCA
GCT GAA AGC CG C GAA GCT GGC GGT TCG GGG CAG GTT GCT GCA 58 Aro10p
ATG GCA CCC GTC ACT ATT GAG AAA TTC GTG AAT CAA GAA GAG CGT CAT
decarboxylase TTA GTG AGC AAT CGT TCC GCC ACG ATC CCT TTT GGA GAA
TAT ATT TT (Saccharomyces C AAGCGC CTT CTT TCC ATT GAC ACC AAA AGC
GTC TTC GGG GTT CCC GG cerevisiae C GAC TTC AAT TTA TCT TTA TTG GAA
TAT TTA TAC TCG CCC TCC GTG G YJM1615) AA TCT GCGGGT CTT CGT TGG
GTT GGC ACC TGT AAC GAG TTA AAT GCA G GenBank: CC TAC GCT GCA GAT
GGA TAT TCC CGC TAC TCT AAT AAA ATT GGA TGC AJV21157.1 TTA ATC ACC
ACATAC GGC GTA GGA GAA CTG AGT GCG CTT AAT GGA ATC GCG GGG TCA TTC
GCT GAA AAT GTA AAG GTT CTG CAT ATC GTA GGG GTC GCC AAG TCC ATT
GATTCC CGT TCG TCT AAC TTC TCG GAT CGT AAC TTA CAT CAC TTG GTC CCG
CAG TTA CAT GAT TCG AAC TTT AAA GGA CCC AA C CAT AAG GTC TAT CAC
GACATG GTT AAA GAT CGT GTC GCA TGT TCC GT C GCC TAC CTG GAG GAT ATT
GAG ACG GCC TGT GAC CAA GTT GAT AAC G TG ATC CGT GAC ATT TAT AAG
TATTCA AAA CCT GGT TAC ATT TTC GTC C CA GCC GAC TTT GCC GAC ATG TCC
GTA ACC TGC GAC AAC TTG GTC AAT GTA CCG CGT ATC AGC CAA CAA GAT
TGTATT GTC TAC CCC AGC GAG AAC CAA CTG TCA GAC ATC ATT AAT AAA ATC
ACT AGC TGG ATC TAC TCG TCT AAG ACT CCA GCA ATC CTT GGA GAC GTC
TTAACT GAT CGT TAT GGG GTA TCA AAC TTT CTG AAC AAA CTG ATC TGC AAA
ACC GGT ATC TGG AAC TT C TCC ACC GTG ATG GGA AAA TCA GTC ATT GAC
GAGAGT AAC CCA ACT TA T ATG GGT CAA TAC AAC GGC AAA GAA GGT CTT AAA
CAG GTC TAT GAA C AT TTC GAG CTG TGT GAT TTG GTT TTA CAC TTC GGA
GTAGAT ATT AAC G AG ATC AAT AAT GGT CAC TAC ACG TTC ACT TAC AAG CCA
AAT GCG AAA ATT ATT CAA TTC CAC CCT AAT TAT ATT CGT TTA GTA GAC
ACTCGT CAG GGG AAT GAA CAA ATG TTC AAA GGC ATC AAT TTT GCG CCA ATC
TTG AAA GAG TTG TAT AAG CGT ATC GAC GTC TCT AAA TTA TCG TTG CAA
TACGAT TCC AAT GTA ACA CAA TAC ACC AAT GAG ACT ATG CGT CTG GAG GAC
CC A ACG AAT GGT CAA TCG AGC ATC ATT ACC CAA GTA CAC CTG CAA AAG A
CCATG CCG AAA TTT TTG AAT CCC GGC GAC GTC GTC GTG TGT GAG ACT G GT
AGT TTC CAA TTC AGT GTA CGC GAC TTC GCA TTC CCC AGT CAG TTG AAA
TATATC AGC CAG GGT TTC TTT TTA TCC ATT GGT ATG GCC TTG CCT GCC GCG
TTG GGG GTT GGG ATC GCA ATG CAG GAT CAT TCC AAC GCG CAT ATT AAC
GGAGGG AAC GTC AAA GAA GAC TAC AAG CCC CGC TTA ATT TTG TTT GAA GGT
GAC GGC GCC GCG CAG ATG ACC ATC CAG GAG CTT AGC AC G ATC CTT AAA
TGCAAT ATC CCT TTG GAG GTC ATT ATC TGG AAT AAC AA T GGA TAC ACT ATC
GAG CGT GCC ATC ATG GGT CCA ACA CGT TCA TAT A AC GAT GTG ATG TCG
TGGAAA TGG ACA AAG TTG TTC GAA GCC TTT GGG G AT TTC GAT GGT AAG TAT
ACG AAT TCG ACT TTA ATT CAG TGT CCT AGC AAA TTA GCG TTA AAA CTT
GAAGAA TTG AAG AAT TCT AAT AAG CGT TCG GGG ATC GAA CTG TTA GAA GTG
AAG CTG GGT GAG CTT GAC TTC CCA GAG CAA TTG AAG TGT ATG GTA GAG
GCCGCA GCT CTT AAA CGT AAT AAG 59 Methionine ATG TTT GAG AAG TAT
TTT CCA AAT GTT GAC TTG ACC GAG TTA TGG AAT import GCC ACA TAT GAA
ACT CTG TAT ATG ACA TTG ATT TCC TTA CTG TTT GC system C TTCGTA ATC
GGC GTC ATC CTG GGA TTG CTG TTA TTC TTA ACA TCT AA permease G GGG
TCT CTT TGG CAA AAT AAA GCA GTA AAT TCC GTT ATC GCA GCC G protein
MetP TT GTC AACATC TTT CGT TCA ATT CCC TTC CTT ATT TTA ATC ATC CTG
C (Bacillus TT CTT GGT TTC ACT AAA TTC TTA GTG GGA ACA ATT TTG GGA
CCA AAT subtilis) GCG GCT CTT CCCGCG TTA GTC ATC GGT AGT GCT CCC
TTT TAT GCT CGT GenBank: CTG GTC GAA ATC GCA CTT CGT GAA GTG GAC
AAA GGA GTG ATT GAG GCG KIX81758.1 GCG AAA TCG ATG GGGGCT AAG ACG
AGC ACT ATT ATT TTT AAG GTT CTT ATC CCC GAG TCC ATG CCC GCG CTG ATT
TCC GGA ATT ACA GTG ACT GC G ATT GCA TTG ATC GGG TCAACC GCC ATC GCA
GGA GCT ATT GGT TCT GG T GGA TTG GGA AAC TTA GCA TAC GTT GAA GGC
TAT CAA TCG AAT AAT G CG GAT GTG ACC TTC GTG GCC ACAGTT TTC ATC CTG
ATT ATT GTT TTC A TC ATT CAG ATC ATT GGT GAC CTT ATT ACC AAC ATC
ATC GAT AAA CGC 60 DL- ATG ATT AAA CTG AGC AAC ATT ACT AAG GTG TTC
CAC CAA GGT ACA CGT methionine ACG ATC CAG GCT CTT AAT AAT GTG TCA
CTG CAC GTT CCT GCT GGT CA transporter G ATTTAT GGG GTT ATC GGT GCC
AGT GGG GCT GGG AAG AGC ACT CTG AT subunit MetN C CGC TGC GTC AAT
CTG TTA GAG CGC CCT ACA GAG GGC TCG GTA CTG G (Escherichia TG GAC
GGTCAA GAG TTG ACT ACT CTG TCG GAG TCC GAG TTG ACA AAA G coli
K-12]) CA CGC CGC CAG ATT GGC ATG ATT TTC CAA CAT TTC AAT TTG TTA
TCG GenBank: AGC CGT ACA GTTTTC GGG AAC GTG GCC TTA CCA CTG GAG TTG
GAC AAT CQR79802.1 ACT CCC AAA GAC GAA GTC AAA CGT CGT GTG ACC GAA
TTA TTG TCC TTG GTG GGT CTT GGT GACAAA CAC GAC AGT TAT CCC AGT AAT
TTG AGT GGC GGG CAA AAA CAG CGT GTT GCC ATC GCA CGC GCA TTA GCT TCG
AAT CC C AAG GTG CTG TTA TGT GATGAA GCG ACC AGC GCC CTT GAC CCA GCC
AC A ACT CGT AGC ATC CTG GAG CTT TTG AAA GAT ATC AAT CGT CGC CTG G
GT TTG ACC ATC TTA TTG ATT ACGCAC GAG ATG GAC GTT GTA AAG CGT A TC
TGT GAC TGT GTA GCG GTG ATC TCC AAC GGT GAA TTA ATC GAA CAG GAC ACC
GTA TCG GAG GTC TTC TCA CATCCT AAG ACA CCC CTT GCA CAA AAA TTC ATC
CAA AGC ACG CTG CAT TTA GAT ATT CCT GAA GAT TAT CAG GAA CGC CTG CAG
GCT GAA CCG TTT ACT GATTGC GTT CCA ATG CTT CGC TTA GAG TTC ACA GGG
CAA TCG GTT GAC GCT CCC TTA TTG AGT GAA AC C GCC CGC CGT TTC AAT
GTT AAT AAC AAC ATC ATTTCC GCG CAA ATG GA C TAC GCG GGG GGT GTT AAA
TTT GGA ATC ATG TTA ACC GAA ATG CAC G GC ACA CAG CAG GAT ACA CAG
GCG GCG ATC GCA TGG CTGCAG GAA CAT C AT GTT AAA GTA GAA GTC CTT GGG
TAT GTG 61 metI
ATGTCTGAGCCGATGATGTGGCTGCTGGTTCGTGGCGTATGGGAAACGCTGGCAATGACCTTC
(Escherichia
GTATCCGGTTTTTTTGGCTTTGTGATTGGTCTGCCGGTTGGCGTTCTGCTTTATGTCACGCGT
coli)
CCGGGGCAAATTATTGCTAACGCGAAGCTGTATCGTACCGTTTCTGCGATTGTGAACATTTTC
CGTTCCATCCCGTTCATTATCTTGCTTGTATGGATGATTCCGTTTACCCGCGTTATTGTCGGT
ACATCGATTGGTTTGCAGGCAGCGATTGTTCCGTTAACCGTTGGTGCAGCACCGTTTATTGCC
CGTATGGTCGAGAACGCTCTGCTGGAGATCCCAACCGGGTTAATTGAAGCTTCCCGCGCAATG
GGTGCCACGCCGATGCAGATCGTCCGTAAGGTGCTGTTACCGGAAGCGCTGCCGGGTCTGGTG
AATGCGGCAACTATCACCCTGATTACCCTGGTCGGTTATTCCGCGATGGGTGGTGCAGTCGGT
GCCGGTGGTTTAGGTCAGATTGGCTATCAGTATGGCTACATCGGCTATAACGCGACGGTGATG
AATACGGTACTGGTATTGCTGGTCATTCTGGTTTATTTAATTCAGTTCGCAGGCGACCGCATC
GTCCGGGCTGTCACTCGCAAGTAA 62 metQ
ATGGCGTTCAAATTCAAAACCTTTGCGGCAGTGGGAGCCCTGATCGGATCACTGGCACTGGTA
(Escherichia
GGCTGCGGTCAGGATGAAAAAGATCCAAACCACATTAAAGTCGGCGTGATTGTTGGTGCCGAA
coli)
CAGCAGGTTGCAGAAGTCGCGCAGAAAGTTGCGAAAGACAAATATGGCCTGGACGTTGAGCTG
GTAACCTTCAACGACTATGTTCTGCCAAACGAAGCATTGAGCAAAGGCGATATCGACGCCAAC
GCCTTCCAGCATAAACCGTACCTTGATCAGCAACTGAAAGATCGTGGCTACAAACTGGTCGCA
GTAGGCAACACTTTTGTTTATCCGATTGCTGGTTACTCCAAGAAAATCAAATCACTGGATGAA
CTGCAGGATGGTTCGCAGGTTGCCGTGCCAAACGACCCAACTAACCTTGGTCGTTCACTGCTG
CTGCTGCAAAAAGTGGGCTTGATCAAACTGAAAGATGGCGTTGGCCTGCTGCCGACCGTTCTT
GATGTTGTTGAGAACCCCAAAAATCTGAAAATTGTTGAACTGGAAGCACCGCAACTGCCGCGT
TCTCTGGACGACGCGCAAATCGCTCTGGCAGTTATCAATACCACCTATGCCAGCCAGATTGGC
CTGACTCCGGCGAAAGACGGTATCTTTGTTGAAGATAAAGAGTCCCCGTACGTAAACCTGATC
GTGACGCGTGAAGATAACAAAGACGCCGAGAACGTGAAGAAATTCGTCCAGGCTTATCAGTCT
GACGAAGTTTACGAAGCAGCAAACAAAGTGTTTAACGGCGGAGCTGTTAAAGGCTGGTAA 63
MetE ATG ACG ACT ATC AAA ACA TCA AAT CTG GGC TTC CCT CGC ATT GGA
CTT (Bacillus AAT CGC GAA TGG AAA AAA TCA CTG GAA GCG TTT TGG AAA
GGT AAC AG atrophaeus C GACAAA GAT ACA TTT CTT AAG CAG ATG GAT GAG
TTA TTT CTT ACT GC UCMB-5137) C GTA AAA ACC CAG ATT GAT CAA AAA ATC
GAC ATC GTG CCC GTG AGC G GenBank: AC TTC ACTCAC TAC GAC CAC GTT
CTT GAC ACA GCT ATC TCT TTT AAT T AKL84080.1 GG ATT CCA GAA CGC TTT
AAA CAC ATT ACG GAT GCG ACT GAT ACA TAT TTC GCG CTG GCACGT GGC ATT
AAG GAT GCT GTT AGT TCG GAA ATG ACT AAG TGG TTT AAT ACC AAT TAC CAC
TAT ATC GTT CCG GAA TAC AAT AAA GAC ATC GAA TTC CGTTTA ACC CGC AAC
AAG CAG TTA GAG GAC TAC CGC CGC GTC AAA CAA GCG TTT GGC GTC GAA ACT
AAA CCC GTC ATT GTC GG T CCT TAC ACA TTC GTG ACGCTT GCC AAG GGC TAC
GAA CAA AGT GAG GC C AAA GAA ATC CAA AAG CGT TTA GTC CCA TTG TAT
GTG CAA TTA TTG A AA GAA TTG GAA CAA GAG GGC GTGCAG TGG GTA CAA ATC
GAT GAG CCA G CA CTT GTG ACA GCC TCA TCC GAG GAT GTT AGC GCG GCC
AAG GAG TTA TAC CAG GCC ATT ACG AAT GAG TTA TCCGGC TTG AAT GTC CTT
TTG CAG ACT TAC TTC GAT TCT GTT GAT GCT TAT GAG GAG TTA ATC AGC TAC
CCG GTA CAG GGT ATC GGC TTG GAT TTT GTA CACGAT AAA GGG CGC AAC TTG
GAG CAA TTA AAA GCG CAT GGA TTT CCG AAG GAT AAG GTA TTA GCA GC T
GGT GTT ATT GAT GGT CGT AAC ATT TGG AAG ACGGAT TTA GAT GAG CG C TTG
GAC GCC ATC CTT GCG CTG TTA TCT TCG ACG GAC ATT GAC GAA T TA TGG
ATT CAA CCA AGC AAT TCG CTT CTT CAT GTA CCAGTA GCA AAG C AC CCA GAC
GAG CAC CTG GAG AAG GAT CTG TTG AAT GGC TTG AGT TAC GCA AAA GAA AAG
CTG GCA GAA CTG TCC GCT TTA AAA GAG GGTTTG TTA TCG GGT AAA GCG GCA
ATC TCG GCC GAC ATT CAG CAG GCC AAA GCG GAT TTA CAG GCC CTG AAG CAA
TTC GCC ACC GGG GCT AAC AGT GAG CAGAAA GAG GAA TTA AAT CAG TTG ACC
GAG AAA GAC TTT AAG CGC CCG ATC CC C TTC GAA GAG CGC CTG AAA ATC
CAG AAT GAA TCC TTG GGG CTT CCC C TGCTT CCT ACT ACG ACT ATT GGT TCT
TTT CCT CAA AGC GCC GAG GTG C GT TCG GCG CGC CAA AAG TGG CGC AAA
AGT GAG TGG AGC GAC GAG CAA TAT CAAGAA TTT ATC AAC GCG GAA ACG AAG
CGC TGG ATC GAC ATT CAG GAA GAG CTT GAT CTT GAC GTT TTA GTA CAT GGA
GAG TTC GAG CGC ACC GAC ATG GTCGAA TAT TTC GGT GAG AAA CTG GCT GGA
TTC GCG TTT ACT AAA TAC GCA TGG GTC CAG AGC TAC GGA TCC CGC TGT GTA
CGC CCT CC C GTC ATC TAT GGGGAC GTG GAG TTT ATT GAA CCT ATG ACT GTC
AAG GA C ACA GTG TAC GCT CAA TCT TTA ACG AGT AAG CAG GTT AAA GGG
ATG T TG ACT GGC CCG GTC ACAATC TTG AAT TGG AGC TTC CCG CGT AAC GAC
A TT AGC CGT AAG GAG ATC GCC TTC CAA ATC GGG TTA GCT CTT CGC AAA
GAG GTC AAG GCG TTG GAA GATGCT GGT ATT CAA ATC ATC CAA GTT GAC GAA
CCG GCC CTG CGT GAA GGG CTG CCT CTG AAA GAA AAC GAT TGG GAA GAG TAT
TTA ACG TGG GCC GCG GAGGCG TTC CGC TTA ACT ACT TCG GCT GTG AAA AAC
GAC ACT CAG ATT CAT ACA CAC ATG TGT TAT TCC AAT TT T GAG GAC ATT
GTC GAC ACA ATT AAT GACTTG GAT GCG GAC GTC ATT AC A ATC GAA CAC TCC
CGC AGT CAC GGT GGG TTC TTG GAC TAC TTG CGC G AT CAT CCG TAT CTT
AAA GGT TTA GGT CTT GGCGTG TAC GAT ATT CAC A GC CCT CGT GTA CCC CCG
ACA GAG GAA ATT TAT AAG ATC ATT GAC GAA GCC CTG ACC GTA TGT CCT ACT
GAC CGC TTC TGG GTAAAC CCA GAC TGC GGG CTG AAG ACC CGT CAC CAG GAG
GAA ACG ATT GCC GCG TTG AAG AAC ATG GTC GAG GCT GCT AAA CAG GCT CGT
GCC AAA CAG AGTCAA CTT GTC 64 BrnF ATG CAG AAA ACA CAG GAG ATT CAC
AGC TCG TTA GAG GTT AGC CCC AGT (Corynebacterium AAA GCT GCT CTG
GAG CCC GAC GAT AAG GGG TAT CGT CGT TAC GAA AT glutamicum) C GCACAA
GGC CTG AAG ACC TCT CTT GCT GCA GGC CTG GGA ATG TAT CC GenBank: T
ATC GGA ATT GCA TTC GGC TTA CTG GTG ATT CAA TAT GGT TAT GAA T
AAM46686.1 GG TGG GCCGCT CCA CTG TTC TCC GGC CTG ATT TTT GCG GGG
TCT ACG G AG ATG CTT GTA ATT GCA CTT GTG GTC GGC GCT GCT CCG CTG
GGT GCC ATT GCC CTT ACGACC TTA CTT GTT AAT TTC CGT CAT GTT TTC TAT
GCC TTT TCC TTT CCC TTG CAC GTT GTT AAA AAC CCT ATT GCG CGC TTC TAT
TCT GTA TTC GCT CTTATT GAT GAA GCA TAC GCT GTT ACA GCC GCT CGT CCC
GCC GGT TGG AGT GCA TGG CGT CTG ATT TCA ATG CAG ATT GCG TT C CAC
TCC TAC TGG GTA TTTGGA GGC TTG ACC GGT GTA GCA ATC GCA GA G TTA ATT
CCT TTC GAG ATC AAA GGC CTG GAG TTC GCA CTT TGT TCG T TA TTT GTA
ACT CTT ACT TTA GACAGT TGT CGC ACT AAG AAA CAA ATT C CG AGT TTG TTA
TTG GCT GGA CTG AGC TTT ACT ATC GCG TTA GTA GTG ATC CCC GGC CAA GCT
CTG TTC GCT GCGTTA CTT ATC TTT CTG GGG CTT CTG ACA ATC CGT TAT TTT
TTC TTA GGG AAG GCA GCC AAA 65 BrnE ATG ACG ACT GAT TTC TCC TGC ATC
CTG TTG GTG GTC GCG GTA TGT GCA (Corynebacterium GTC ATT ACA TTT
GCG CTT CGT GCC GTA CCT TTT CTG ATC TTG AAA CC glutamicum) C TTGCGT
GAA TCG CAA TTT GTG GGA AAA ATG GCC ATG TGG ATG CCT GC GenBank: G
GGC ATT CTG GCA ATC CTG ACG GCT TCT ACC TTC CGT TCA AAC GCC A
AAM46685.1 TC GAT TTAAAG ACG TTG ACG TTC GGT CTG ATT GCC GTG GCA
ATC ACA G TC GTA GCC CAC TTA TTA GGA GGC CGT CGC ACC TTA TTA TCT
GTT GGC GCT GGA ACA ATTGTG TTT GTA GGT CTT GTT AAT TTG TTT
Threonine 66 threonine 3- ATG AAG GCC CTG AGC AAA TTG AAA GCC GAG
GAG GGG ATC TGG ATG ACC dehydrogenase GAT GTT CCT GAA CCA GAA GTG
GGG CAC AAC GAC CTT TTA ATC AAA AT (Salmonella T CGCAAG ACT GCA ATC
TGC GGG ACA GAC GTA CAT ATC TAT AAC TGG GA enterica C GAG TGG AGT
CAA AAA ACT ATT CCC GTC CCT ATG GTG GTC GGG CAC G subsp. AG TAT
GTCGGA GAG GTT GTA GGA ATC GGA CAA GAA GTC AAA GGA TTT A enterica
AA ATC GGG GAT CGT GTG AGT GGG GAG GGT CAC ATT ACC TGT GGG CAT
serovar TGC CGC AAT TGCCGT GGA GGA CGC ACA CAT TTG TGC CGT AAC ACT
ACA Typhistr. GGC GTA GGC GTG AAT CGT CCC GGA TGT TTC GCG GAA TAC
CTT GTC ATT CT18) CCA GCG TTT AAC GCCTTT AAG ATC CCT GAC AAC ATT
TCA GAT GAT TTA GenBank: GCA TCC ATT TTT GAC CCA TTC GGT AAC GCG
GTC CAT ACT GCG TTG AG CAD03286.1 C TTC GAC TTA GTT GGA GAAGAT GTA
TTA GTT TCC GGC GCC GGA CCG AT T GGC GTC ATG GCA GCT GCC GTT GCG
AAG CAC GTG GGC GCA CGT CAT G TG GTA ATT ACG GAC GTA AAT GAGTAT CGT
CTG GAG CTG GCA CGT AAA A TG GGG GTT ACA CGT GCC GTA AAC GTT GCG
AAA GAG TCT TTA AAC GAT GTC ATG GCT GAA CTG GGC ATG ACG GAAGGG TTT
GAT GTC GGA CTG GAA ATG TCC GGT GCC CCG CCA GCC TTC CGT ACC ATG TTG
GAC ACC ATG AAC CAT GGG GGC CGT ATC GCA ATG TTG GGA ATTCCC CCG AGC
GAC ATG TCT ATC GAC TGG ACA AAG GTA ATT TTT AAA GGC CTG TTC ATT AAG
GGG AT T TAC GGT CGT GAG ATG TTT GAG ACG TGG TAC AAGATG GCT GCC TTG
AT T CAA TCG GGG TTG GAT CTG AGC CCT ATC ATC ACA CAC CGT TTT TCA G
TG GAT GAC TTT CAA AAA GGG TTT GAC GCC ATG TGC AGCGGT CAA TCA G GG
AAA GTA ATT CTT TCT TGG GAC 67 threonine ATG ATT GAC CTT CGT TCG
GAC ACC GTA ACC CGC CCA TCT CAC GCA ATG aldolase TTG GAA GCT ATG
ATG GCC GCG CCT GTG GGG GAT GAC GTT TAT GGG GA (Escherichia T
GACCCG ACC GTC AAC GCT TTA CAA GAT TAC GCT GCT GAA TTG TCG GG coli
O26: H11 C AAA GAA GCA GCA ATC TTC TTA CCT ACA GGT ACA CAA GCT AAT
CTT G str. TC GCC CTGCTT AGT CAC TGT GAG CGT GGC GAA GAA TAC ATT
GTT GGT C CVM10026) AA GCA GCG CAT AAT TAC CTG TTC GAA GCT GGA GGG
GCT GCT GTT CTT GenBank: GGT AGC ATT CAGCCC CAA CCC ATT GAT GCT GCT
GCC GAT GGT ACT CTT EIL35157.1 CCT CTG GAT AAA GTC GCT ATG AAA ATT
AAG CCA GAC GAC ATT CAC TTC GCA CGC ACA AAG CTGCTG TCG CTT GAG AAT
ACA CAC AAT GGA AAA GTC CTG CCC CGT GAG TAC CTG AAA GAG GCT TGG GAA
TTT ACA CGC GAA CG C AAC CTG GCT CTG CAC GTAGAC GGT GCT CGC ATC TTC
AAC GCC GTT GT C GCC TAC GGT TGC GAA TTG AAA GAG ATT ACG CAA TAC
TGT GAC TCC T TC ACG ATT TGC TTG TCC AAA GGCTTA GGC ACC CCG GTG GGT
TCA TTG T TG GTA GGA AAC CGT GAC TAT ATT AAG CGC GCC ATC CGC TGG
CGT AAA ATG GCA GGG GGT GGA ATG CGT CAA TCAGGG ATT CTT GCG GCA GCT
GGC ATG TAC GCG CTG AAA AAT AAT GTG GCT CGC CTT CAA GAG GAT CAC GAT
AAT GCT GCG TGG ATG GCT GAG CAA TTA CGTGAG GCG GGT GCA GAC GTA ATG
CGC CAA GAT ACC AAT ATG CTG TTC GTA CGT GTT GGG GAA GAA AA C GCT
GCG GCC TTA GGA GAA TAC ATG AAG GCG CGTAAC GTG TTG ATC AA C GCA TCC
CCT ATT GTT CGC CTT GTA ACT CAC CTT GAT GTT TCA CGT G AA CAA TTG
GCG GAA GTT GCC GCC CAC TGG CGT GCC TTTCTT GCT CGC 68 serine ATG
CTT AAA CGT GAG ATG AAT ATC GCC GAC TAC GAC GCC GAA TTA TGG
hydroxymethyl- CAG GCG ATG GAG CAG GAG AAA GTC CGC CAA GAG GAA CAC
ATT GAG CT transferase T ATTGCG TCG GAG AAC TAT ACA TCC CCT CGC GTT
ATG CAG GCG CAA GG (Escherichia C TCA CAG TTG ACG AAC AAA TAC GCT
GAG GGA TAT CCG GGA AAG CGT T coli) AT TAT GGCGGT TGC GAG TAC GTT
GAC ATT GTT GAA CAG TTA GCG ATT G GenBank: AT CGT GCT AAG GAG TTA
TTT GGA GCG GAT TAT GCC AAT GTT CAA CCT AAA23912.1 CAC TCG GGC
AGCCAG GCT AAC TTT GCT GTA TAC ACC GCA CTT TTA GAA CCT GGT GAC ACG
GTC CTG GGT ATG AAT TTG GCC CAT GGA GGC CAC TTA ACT CAT GGA AGC
CCTGTG AAT TTT AGT GGG AAG TTG TAT AAC ATC GTG CCC TAC GGG ATC GAC
GCC ACA GGA CAC ATT GAT TAC GCA GAT TTG GA G AAA CAA GCC AAG GAA
CATAAG CCT AAA ATG ATC ATC GGC GGA TTT TC A GCA TAT AGC GGA GTG GTA
GAC TGG GCC AAA ATG CGC GAG ATT GCT G AT TCG ATT GGT GCT TAC CTG
TTTGTC GAT ATG GCG CAT GTC GCT GGT C TG GTC GCT GCG GGA GTT TAT CCT
AAC CCC GTG CCT CAC GCT CAC GTC GTG ACG ACT ACT ACA CAT AAG ACT
TTAGCG GGT CCT CGT GGG GGT TTG ATT CTT GCG AAG GGG GGC TCA GAG GAA
CTT TAT AAG AAG CTT AAC TCT GCC GTA TTT CCC GGC GGT CAG GGG GGC
CCTCTT ATG CAC GTC ATC GCA GGA AAG GCG GTG GCT CTG AAG GAA GCG ATG
GAA CCC GAA TTC AAG AC T TAC CAA CAG CAA GTA GCC AAA AAC GCC AAA
GCCATG GTG GAG GTA TT C CTG GAG CGC GGC TAC AAG GTA GTT AGC GGG GGG
ACG GAC AAC CAT T TG TTC TTA GTC GAT TTA GTG GAC AAA AAC CTT ACT
GGTAAG GAG GCT G AT GCT GCT CTT GGG CGT GCA AAT ATC ACA GTC AAT AAG
AAT AGC GTG CCC AAT GAC CCA AAG TCG CCA TTT GTG ACT TCT GGC ATC
CGCGTT GGG ACT CCG GCA ATC ACC CGT CGT GGC TTT AAG GAG GCA GAG GCC
AAG GAG CTG GCA GGG TGG ATG TGT GAC GTA CTG GAC TCT ATT AAT GAT
GAGGCA GTT ATC GAA CGT ATT AAA GGC AAA GTG CTT GAC ATT TGT GCG CGC
TA C CCC GTG TAT GCC 69 tdcC (Escherichia ATG TCT ACT TCG GAC TCT
ATT GTT TCA TCG CAA ACA AAA CAG TCA TCC coli) TGG CGT AAA TCA GAT
ACC ACC TGG ACT TTG GGT CTG TTT GGT ACC GC GenBank: G ATCGGG GCT
GGT GTA TTG TTT TTC CCG ATC CGC GCT GGA TTT GGT GG AAA24662.1 T TTA
ATT CCT ATC CTG CTG ATG CTT GTA CTG GCA TAT CCT ATT GCT T TT TAT
TGTCAT CGC GCA GCG CGC TTG TGT TTA AGC GGA AGC AAC CCC T CG GGT AAT
ATC ACA GAG ACG GTG GAG GAG CAT TTC GGG AAA ACA GGA GGG GTC GTA
ATCACA TTT CTG TAC TTT TTT GCT ATT TGT CCC CTG TTG TGG ATT TAT GGG
GTT ACG ATC ACC AAT ACT TTT ATG ACG TTT TGG GAG AAT CAA CTG GGC
TTTGCA CCG CTT AAC CGC GGA TTC GTG GCG CTG TTC CTT TTA CTG TTG ATG
GCG TTT GTC ATC TGG TTC GGT AAA GAC TTA AT G GTG AAA GTC ATG TCT
TATTTG GTA TGG CCT TTC ATT GCT TCA CTT GT C TTA ATT AGT CTG TCA TTA
ATC CCT TAT TGG AAC TCG GCA GTA ATC G AT CAA GTA GAT CTG GGT AGC
CTGTCT TTG ACC GGA CAT GAT GGG ATC T TA ATT ACC GTA TGG CTG GGC ATT
TCT ATT ATG GTC TTT AGT TTT AAC TTT TCA CCT ATC GTG TCC TCC TTT
GTGGTG TCC AAG CGC GAG GAA TAT GAG AAG GAT TTT GGT CGT GAT TTT ACG
GAA CGT AAG TGC TCA CAA ATT ATT AGC CGC GCG TCT ATG CTT ATG GTG
GCTGTC GTT ATG TTC TTT GCT TTC TCC TGC TTA TTT ACC TTG TCA CCG GCG
AAC ATG GCG GAA GCG AA G GCG CAA AAC ATT CCA GTT TTA TCA TAT CTT
GCTAAT CAT TTC GCT TC T ATG ACA GGG ACC AAA ACT ACT TTT GCC ATC ACA
TTG GAG TAT GCG G CG TCT ATC ATT GCA TTA GTG GCC ATT TTT AAG TCG
TTCTTT GGC CAT T AT TTA GGT ACT TTA GAA GGG TTG AAT GGC TTA GTC TTG
AAA TTC GGA TAC AAG GGG GAC AAA ACT AAA GTT TCC TTG GGT AAG TTG
AACACA ATC TCG ATG ATC TTT ATT ATG GGG AGT ACA TGG GTC GTT GCG TAT
GCA AAT CCA AAC ATT CTG GAT TTA ATT GAG GCG ATG GGA GCA CCG ATT
ATCGCG TCA TTG TTG TGC CTT TTG CCG ATG TAC GCC ATC CGT AAG GCG CCT
TC A CTG GCC AAA TAT CGT GGG CGC TTG GAT AAC GTG TTC GTA ACC GTC A
TCGTT TGC 70 Threonine/ ATG CCT GGT TCC TTG CGT AAA ATG CCG GTT TGG
TTG CCG ATT GTT ATT homoserine CTT CTG GTT GCA ATG GCT AGC ATC CAA
GGA GGC GCT AGT TTA GCA AA exporter Rht A AGTCTG TTT CCT TTG GTG
GGG GCA CCG GGT GTG ACC GCG CTG CGT TT A G GCT TTG GGC ACT TTA ATT
TTG ATT GCC TTC TTT AAG CCC TGG CGC C UniProtKB/ TT CGT TTTGCT AAA
GAA CAA CGT TTG CCG CTT TTG TTC TAC GGC GTC T Swiss-Prot: CA CTT
GGT GGC ATG AAC TAT CTT TTT TAT TTA AGC ATC CAA ACC GTA P0AA67.1
CCC CTG GGT ATTGCG GTG GCT TTG GAG TTC ACG GGT CCA TTG GCA GTT GCC
CTT TTC AGC TCG CGT CGC CCA GTC GAT TTC GTC TGG GTA GTG CTT GCG GTA
CTT GGA CTGTGG TTC TTA CTG CCC TTA GGC CAA GAC GTG AGT CAC GTA GAC
CTT ACC GGG TGT GCG CTG GCT TTG GGA GCC GGT GCT TG T TGG GCA ATT
TAC ATC CTGTCG GGA CAG CGT GCG GGA GCA GAG CAC GG G CCT GCG ACA GTA
GCG ATT GGG TCG CTG ATC GCA GCC CTG ATT TTC G TC CCC ATT GGT GCC
TTA CAG GCAGGA GAG GCG TTG TGG CAC TGG TCA G TG ATT CCC TTA GGT TTG
GCG GTA GCA ATC CTG TCT ACC GCA CTT CCT TAT TCT TTA GAG ATG ATT GCC
TTA ACCCGT CTG CCG ACA CGT ACG TTT GGC ACC TTA ATG TCG ATG GAA CCG
GCA TTG GCT GCC GTT TCA GGT ATG ATC TTC CTG GGA GAG ACG TTA ACT CCC
ATTCAG TTG TTA GCT CTT GGG GCA ATC ATC GCT GCG AGT ATG GGA TCG ACC
CTT ACG GTT CGT AAA GA G TCG AAG ATT AAA GAA TTG GAC ATC AAT 71 htB
r ATG ACG CTG GAG TGG TGG TTC GCA TAC TTG CTG ACA TCC ATC ATC CTG
(Escherichia AGT TTA AGC CCC GGA TCT GGT GCA ATC AAC ACG ATG ACT
ACG TCT TT coli FVEC130 G AATCAC GGC TAT CGT GGT GCT GTT GCA TCC
ATT GCC GGC TTG CAG AC 2) G GGA TTA GCC ATC CAT ATT GTT TTA GTG GGT
GTA GGA CTT GGA ACA T GenBank: TA TTC AGTCGC TCG GTT ATC GCC TTT
GAG GTC TTA AAG TGG GCT GGT G EFI17945.1 CC GCT TAT TTG ATT TGG CTG
GGA ATT CAG CAA TGG CGT GCA GCC GGT GCG ATT GAC TTGAAG AGC CTT GCG
TCC ACA CAG AGC CGC CGT CAC TTG TTT CAA CGT GCA GTA TTC GTC AAT TTG
ACC AAC CCC AAA AGT ATC GTC TTT CTG GCG GCA CTGTTT CCC CAG TTC ATT
ATG CCT CAA CAG CCG CAG TTG ATG CAG TAC ATC GTC TTG GGC GTC ACC ACC
ATC GTA GTG GAC AT T ATT GTA ATG ATT GGA TACGCC ACT CTG GCC CAA CGT
ATT GCG CTG TG G ATC AAG GGC CCG AAA CAG ATG AAG GCA CTG AAC AAA
ATT TTT GGT T CT TTG TTT ATG TTG GTT GGG GCACTT CTT GCC AGT GCA CGT
CAC GCG 72 RhtC threonine ATG CTG ATG CTT TTT TTA ACA GTA GCA ATG
GTG CAT ATC GTC GCA TTG Rht ATG TCA CCG GGA CCT GAC TTT TTT TTT GTT
TCA CAA ACA GCA GTA TC Transporter A CGCTCA CGT AAG GAG GCA ATG ATG
GGT GTC TTA GGG ATC ACT TGC GG
(Escherichia C GTA ATG GTA TGG GCC GGT ATT GCA CTT CTG GGA CTG CAT
TTA ATT A coli BL21(DE3)) TT GAG AAGATG GCC TGG CTT CAC ACA TTA ATC
ATG GTA GGC GGT GGG C GenBank: TT TAT TTA TGT TGG ATG GGC TAT CAA
ATG CTG CGT GGA GCT CTT AAG CAQ34168.1 AAA GAA GCC GTGTCC GCA CCG
GCT CCC CAA GTG GAA CTT GCG AAA TCA GGT CGC TCC TTC TTG AAG GGG TTG
TTG ACT AAT CTT GCG AAC CCT AAG GCC ATC ATT TAT TTCGGT TCT GTG TTT
AGT TTG TTC GTT GGG GAT AAT GTG GGA ACC ACG GAA CGC TGG GGA ATC TTC
GCA TTA ATC ATT ATC GA G ACG TTA GCT TGG TTC ACCGTC GTG GCC TCC CTT
TTT GCT CTG CCG CA A ATG CGC CGT GGT TAC CAA CGT TTA GCA AAG TGG
ATC GAC GGT TTT G CT GGA GCT TTA TTT GCG GGT TTCGGC ATT CAT CTG ATT
ATT AGC CGT 73 cysteine ATG CCC CTG CAC AAC TTA ACA CGT TTT CCA CGC
CTG GAA TTC ATT GGT desulfhydrase GCA CCG ACT CCC TTG GAA TAT CTG
CCT CGC TTT TCG GAC TAC TTA GG (Escherichia C CGCGAG ATT TTC ATT
AAG CGC GAT GAT GTT ACA CCG ATG GCT ATG GG coli) G GGT AAC AAA TTG
CGT AAA TTG GAA TTT CTT GCA GCG GAT GCA CTG C GenBank: GT GAA
GGCGCG GAC ACT TTA ATT ACC GCT GGT GCA ATT CAG TCA AAT C ALI49110.1
AC GTA CGC CAA ACT GCG GCA GTT GCT GCG AAG TTA GGT CTT CAT TGT GTC
GCC CTT TTGGAA AAT CCA ATT GGC ACA ACG GCA GAA AAT TAC CTT ACC AAC
GGG AAC CGT TTG TTG CTT GAC CTT TTT AAC ACA CAG ATC GAA ATG TGC GAC
GCT TTAACT GAT CCC AAC GCT CAA TTG GAG GAG CTT GCG ACT CGC GTG GAA
GCT CAA GGC TTC CGT CCG TAT GTT ATT CCG GTC GG C GGC AGC AAT GCT
CTT GGGGCA TTA GGG TAT GTA GAG TCC GCT CTG GA G ATC GCG CAA CAA TGT
GAG GGC GCG GTT AAC ATT TCG AGT GTA GTT G TG GCC TCT GGA AGT GCG
GGC ACCCAC GCC GGG CTG GCT GTG GGT CTT G AG CAC TTA ATG CCT GAA TCT
GAA CTG ATC GGG GTC ACA GTC TCG CGT TCC GTC GCA GAT CAG TTA CCT AAG
GTAGTA AAC TTA CAG CAA GCC ATT GCG AAA GAA TTA GAA TTA ACC GCT AGT
GCA GAA ATC TTA TTA TGG GAT GAT TAC TTT GCG CCT GGG TAC GGT GTC
CCCAAT GAT GAA GGT ATG GAA GCA GTC AAG CTT TTA GCT CGT TTG GAG GGG
ATC TTG CTG GAC CCT GT T TAC ACC GGC AAA GCA ATG GCA GGC TTA ATT
GACGGT ATC AGT CAG AA A CGC TTC AAA GAC GAG GGA CCA ATT CTG TTC ATC
CAT ACC GGC GGC G CT CCT GCC CTT TTT GCC TAC CAC CCT CAC GTT 74
tnaA ATG GAG AAT TTC AAG CAT TTG CCC GAG CCG TTC CGC ATT CGT GTC
ATT (Escherichia GAG CCT GTC AAG CGT ACT ACT CGC GCG TAT CGC GAA
GAG GCG ATT AT coli DH1) C AAATCG GGT ATG AAT CCA TTT TTA CTT GAT
TCA GAA GAT GTG TTC AT GenBank: C GAT TTA CTT ACA GAT TCT GGG ACA
GGC GCG GTA ACG CAA TCG ATG C BAJ45452.1 AA GCA GCGATG ATG CGC GGT
GAC GAA GCC TAT TCT GGC TCG CGC TCC T AT TAT GCT CTG GCC GAA TCA
GTC AAA AAC ATT TTT GGT TAC CAA TAT ACG ATT CCC ACGCAT CAG GGA CGC
GGA GCA GAG CAA ATC TAT ATC CCA GTC TTA ATC AAA AAG CGC GAG CAA GAA
AAG GGA TTG GAC CGC TCG AAA ATG GTA GCC TTC TCAAAT TAC TTC TTC GAC
ACT ACT CAG GGG CAC TCG CAA ATC AAC GGC TGC ACT GTT CGC AAT GTG TAT
ATC AAG GAA GCC TT T GAT ACA GGC GTA CGT TACGAT TTC AAG GGG AAC TTT
GAC CTG GAA GG T CTT GAA CGT GGC ATT GAA GAA GTA GGA CCC AAC AAC
GTA CCC TAT A TC GTC GCC ACG ATC ACA TCT AATAGC GCA GGA GGT CAG CCT
GTG TCT T TG GCG AAT CTG AAA GCG ATG TAT TCG ATC GCC AAA AAG TAT
GAT ATC CCC GTC GTA ATG GAT TCT GCA CGT TTTGCA GAG AAC GCC TAC TTC
ATT AAA CAG CGT GAA GCG GAG TAC AAA GAT TGG ACC ATC GAA CAG ATC ACT
CGT GAG ACT TAT AAA TAT GCT GAC ATG CTGGCT ATG TCG GCT AAG AAG GAC
GCT ATG GTC CCA ATG GGA GGC CTT TTA TGC ATG AAG GAC GAT AG T TTT
TTT GAC GTT TAT ACG GAA TGT CGC ACC CTTTGT GTA GTG CAG GA A GGA TTC
CCC ACT TAT GGC GGC CTT GAA GGT GGA GCG ATG GAA CGT T TA GCT GTT
GGA CTG TAT GAT GGT ATG AAT CTG GAT TGGCTG GCA TAT C GT ATT GCG CAG
GTG CAG TAC CTG GTA GAC GGG TTA GAG GAG ATC GGG GTT GTG TGC CAG CAG
GCC GGG GGC CAT GCG GCG TTC GTG GACGCA GGA AAA CTG CTT CCC CAC ATT
CCC GCC GAT CAG TTC CCT GCG CAG GCA CTT GCT TGC GAG TTA TAC AAG GTG
GCC GGT ATC CGT GCG GTA GAG ATCGGC TCG TTT CTT TTG GGG CGC GAC CCT
AAA ACA GGA AAA CAA TTG CCC TG C CCT GCC GAA CTT CTT CGC CTT ACT
ATC CCT CGT GCG ACC TAC ACT C AAACC CAC ATG GAC TTT ATT ATC GAG GCC
TTC AAA CAT GTG AAG GAG A AT GCT GCT AAT ATC AAG GGC CTG ACC TTT
ACC TAC GAG CCA AAG GTT TTG CGCCAC TTT ACA GCA AAA CTT AAA GAA GTT
75 cysK ATG TCA AAA ATT TTC GAG GAT AAC TCG TTA ACG ATC GGC CAC ACT
CCC (Escherichia TTG GTT CGT CTG AAT CGT ATC GGT AAC GGG CGC ATT
CTG GCA AAG GT coli O104: H4 T GAATCA CGC AAT CCG TCC TTC TCA GTT
AAG TGC CGT ATT GGA GCG AA str. C227- T ATG ATT TGG GAT GCT GAG AAG
CGC GGA GTC CTG AAG CCT GGG GTG G 11) AG TTG GTGGAG CCA ACC TCT GGG
AAT ACA GGT ATC GCG CTG GCT TAT G GenBank: TA GCT GCA GCG CGT GGC
TAC AAA TTA ACA CTT ACC ATG CCC GAG ACC EGT66151.1 ATG TCA ATC
GAACGT CGT AAG TTG TTG AAG GCA TTA GGA GCG AAT CTG GTA CTG ACC GAA
GGA GCT AAG GGA ATG AAG GGC GCT ATT CAA AAA GCG GAA GAA ATT GTC
GCAAGT AAC CCC GAA AAG TAT CTT TTA CTG CAA CAG TTT TCT AAC CCT GCA
AAT CCT GAG ATC CAC GAA AAA ACA ACA GGT CC C GAA ATC TGG GAA GAC
ACCGAC GGT CAA GTT GAC GTA TTT ATC GCC GG G GTA GGA ACT GGA GGA ACC
TTA ACG GGG GTC AGT CGT TAT ATT AAG G GT ACG AAG GGA AAG ACT GAT
TTGATT AGC GTA GCA GTG GAG CCA ACG G AT AGT CCT GTT ATT GCC CAA GCC
CTG GCG GGG GAG GAA ATC AAA CCG GGA CCT CAC AAA ATC CAA GGG ATT
GGTGCG GGT TTT ATC CCA GCC AAT CTG GAT CTG AAA CTT GTC GAC AAG GTC
ATT GGA ATT ACT AAT GAA GAG GCG ATC TCC ACT GCG CGC CGT TTG ATG
GAGGAA GAA GGG ATT TTG GCA GGG ATT TCA AGC GGT GCG GCG GTG GCA GCA
GCT TTG AAA TTG CAA GA A GAC GAG TCA TTC ACT AAT AAG AAT ATT GTT
GTTATT TTA CCA AGC AG C GGT GAG CGC TAC TTA TCA ACC GCT TTG TTC GCT
GAT TTA TTT ACG G AA AAA GAG TTA CAA CAA 76 cysm ATG TCA ACA TTA
GAA CAG ACA ATT GGT AAT ACC CCC CTG GTC AAA TTG (Escherichia CAG
CGC ATG GGG CCA AAC AAT GGA AGC GAG GTT TGG CTG AAA TTG GA coli
FVEC1412) A GGCAAC AAC CCG GCG GGA TCT GTG AAA GAC CGT GCC GCA CTG
TCC AT GenBank: G ATC GTA GAA GCT GAG AAA CGT GGC GAG ATT AAA CCT
GGG GAT GTT T EFF01099.1 TA ATC GAGGCT ACA AGT GGG AAC ACT GGA ATC
GCC CTT GCC ATG ATT G CG GCT TTA AAG GGT TAT CGT ATG AAG TTA CTT
ATG CCC GAT AAC ATG AGC CAG GAG CGCCGT GCC GCT ATG CGT GCC TAT GGT
GCT GAA CTT ATC TTA GTT ACC AAG GAG CAA GGC ATG GAA GGT GCG CGT GAC
TTG GCA TTA GAA ATG GCG AAT CGTGGC GAA GGG AAG CTG CTT GAC CAA TTT
AAT AAT CCA GAT AAC CCT TAT GCA CAC TAT ACC ACG ACC GGC CCG GAA ATC
TG G CAA CAA ACC GGC GGG CGCATC ACC CAC TTT GTA TCA TCC ATG GGC AC
A ACT GGT ACA ATT ACG GGC GTT TCT CGT TTC ATG CGC GAG CAG AGT A AA
CCT GTT ACA ATC GTG GGA CTTCAA CCT GAG GAG GGA TCT TCG ATC C CA GGC
ATT CGT CGT TGG CCT GCT GAG TAC TTA CCT GGC ATT TTC AAC GCA TCC TTA
GTG GAT GAA GTT CTT GACATT CAT CAG CGC GAA GCA GAG AAT ACC ATG CGC
GAG TTG GCA GTA CGT GAG GGC ATT TTC TGC GGG GTT TCT TCT GGG GGG GCC
GTG GCG GGT GCT TTACGT GTC GCC AAA GCA AAC CCC GGA GCA GTA GTT GTT
GCC ATT ATT TGT GAT CGT GGT GAC CGC TA C TTA TCT ACG GGA GTC TTC
GGA GAG GAA CAC TTTTCA CAA GGG GCC GG A ATT 77 ATG TTC GAT TTT TCG
AAA GTC GTC GAT CGT CAT GGG ACC TGG TGC ACT malY CAA TGG GAC TAC
GTG GCG GAC CGC TTT GGG ACA GCA GAT TTG TTA CC (Escherichia G
TTCACT ATT AGC GAC ATG GAT TTT GCC ACA GCA CCT TGC ATT ATC GA coli)
G GCA CTG AAT CAG CGC TTA ATG CAT GGG GTT TTC GGT TAT AGC CGT T
GenBank: GG AAG AACGAT GAG TTC CTT GCA GCA ATT GCA CAT TGG TTC AGT
ACC C AAA24099.1 AA CAT TAT ACC GCT ATC GAT TCC CAG ACG GTT GTG TAC
GGC CCC AGC GTT ATT TAC ATGGTG AGC GAA TTG ATC CGT CAG TGG TCT GAA
ACA GGA GAA GGT GTA GTA ATC CAT ACT CCC GCC TAT GAC GCG TTC TAC AAA
GCC ATT GAG GGG AAT CAACGT ACA GTA ATG CCC GTT GCC TTA GAA AAA CAG
GCA GAC GGA TGG TTT TGC GAT ATG GGA AAA TTA GAG GCG GTA CTT GC A
AAA CCC GAG TGC AAA ATCATG CTT TTA TGC AGT CCG CAA AAC CCA AC A GGC
AAG GTC TGG ACC TGT GAT GAA TTA GAG ATT ATG GCG GAT TTG T GC GAG
CGT CAC GGA GTC CGT GTCATC TCT GAC GAG ATT CAC ATG GAC A TG GTC TGG
GGG GAA CAG CCG CAC ATT CCT TGG TCT AAT GTC GCA CGT GGT GAT TGG GCC
CTT TTG ACA TCG GGTTCG AAA AGC TTT AAC ATT CCA GCC CTG ACC GGG GCA
TAT GGA ATT ATC GAA AAC TCG TCG AGC CGT GAC GCG TAT TTA TCT GCC CTT
AAG GGA CGT GATGGA CTT TCG AGC CCG TCG GTT CTT GCC TTG ACG GCA CAC
ATT GCT GCT TAC CAA CAG GGA GCG CC G TGG CTG GAC GCT CTT CGC ATT
TAC CTG AAG GATAAC CTT ACT TAC AT T GCG GAT AAG ATG AAT GCG GCC TTC
CCA GAA CTT AAC TGG CAG ATT C CC CAG TCA ACG TAT TTA GCC TGG CTT
GAC CTT CGT CCCTTA AAC ATT G AT GAC AAC GCA CTG CAA AAG GCA CTG ATC
GAA CAG GAA AAG GTA GCC ATC ATG CCT GGC TAT ACC TAC GGC GAG GAG GGC
CGT GGG TTCGTC CGC CTG AAC GCA GGA TGT CCC CGC TCG AAA CTT GAA AAA
GGG GTA GCT GGT CTT ATT AAT GCT ATT CGC GCT GTG CGC 78 MetC ATG GCC
GAC AAG AAG TTG GAT ACT CAA CTG GTG AAC GCC GGG CGT TCC
(Escherichia AAA AAA TAT ACC TTG GGA GCT GTT AAT AGC GTT ATC CAA
CGT GCA TC coli) A AGTTTA GTT TTC GAT AGT GTC GAA GCA AAG AAG CAT
GCG ACA CGC AA GenBank: T CGC GCA AAT GGG GAA TTA TTT TAT GGA CGC
CGC GGG ACC TTG ACC C ADK47401.1 AC TTC TCTTTA CAG CAG GCC ATG TGT
GAG CTG GAA GGG GGA GCC GGT T GT GTA TTG TTC CCC TGC GGA GCC GCG
GCG GTG GCT AAC AGT ATC CTG GCG TTC GTG GAGCAG GGT GAT CAC GTC CTG
ATG ACG AAC ACC GCG TAC GAA CCC TCG CAA GAC TTC TGC AGT AAA ATC TTA
TCC AAA TTA GGT GTG ACT ACC TCG TGG TTTGAC CCG TTG ATC GGG GCG GAC
ATT GTG AAA CAT CTG CAG CCC AAC ACG AAA ATT GTT TTT TTG GAG TCT CCC
GGT TCG AT T ACT ATG GAG GTA CAC GACGTG CCA GCT ATC GTT GCA GCA GTT
CGT TC C GTG GCG CCC GAC GCA ATT ATC ATG ATC GAC AAT ACA TGG GCC
GCA G GC GTC CTT TTT AAA GCC TTA GATTTT GGC ATT GAT GTA AGT ATC CAA
G CG GCT ACC AAG TAC TTG GTC GGA CAT TCC GAT GCG ATG ATT GGT ACA
GCA GTA TGC AAT GCA CGC TGC TGG GAGCAA TTG CGT GAA AAC GCT TAC CTG
ATG GGG CAA ATG GTA GAC GCA GAT ACC GCT TAT ATT ACC AGT CGT GGG TTG
CGT ACA TTA GGA GTG CGT TTG CGTCAA CAC CAC GAG TCA TCC CTG AAA GTG
GCT GAA TGG CTG GCT GAA CAT CCC CAG GTT GCT CGC GT A AAC CAC CCC
GCA CTT CCG GGA TCA AAG GGC CATGAA TTT TGG AAG CG C GAC TTC ACG GGC
TCC AGT GGA TTG TTT TCT TTC GTA CTT AAG AAA A AG TTG TCT AAT GAA
GAA TTG GCG AAT TAC CTT GAT AACTTT AGC TTG T TT AGT ATG GCA TAT AGT
TGG GGG GGA TAT GAA TCA CTG ATT TTG GCA AAT CAA CCA GAA CAT ATT GCT
GCG ATT CGT CCT CAA GGC GAAATT GAT TTT AGC GGA ACG TTA ATT CGT CTG
CAC ATC GGG CTT GAG GAT GTG GAC GAT TTA ATT GCA GAT TTG GAT GCG GGA
TTT GCA CGT ATT GTG 79 cystathione ATG TCA GGT GCC CAG CAC TTG TTC
GCA GAT TTC AGC GAA GGA TCA GGA gamma lyase TCG TGG CAA CCC CAG GCC
CAA GGG TTT GAG ACG CTT CTG GTA CAT GG (Trypanosoma T GGCGTA AAG
CCA GAT CCC GTC ACG GGG GCA ATC CTG ACC CCC GTC TA grayi) C CAG TCT
ACG ACG TTC GTG CAA GAG AGT ATC GAA CGT TAT CAA GCA A NCBI AG GGC
TATAGC TAT ACC CGT TCA GCC AAT CCT ACC GTA TCT GCA TTG G Reference
AA GAG AAA TTG TGC GCA ATC GAG CAC GGC GAA TAT GCC ACT GTG TAT
Sequence: AGC ACC GGC ATGTCC GCT ACG ACA ACG GCC ATC AGT AGT TTT
ATG TCT XP_009313447.1 GCT GGC GAC CAC GCT ATT GTG ACC GAA TGT AGC
TAT GGC GGA ACC AAT CGT GCC TGC CGT GTCTTC TTC ACG CGC TTA GGT ATG
TCT TTT ACA TTC GTA GAT ATG CGC GAC GTT AAA AAT GTA GAG GCT GCC ATC
AAA CCC AA T ACC AAG CTG GTT ATC TCAGAA TCG CCA GCA AAC CCT ACA CTG
ACG CT T ACT GAT ATT GAC GCA CTT AGC TCG CTT TGC AAG GCT AAG GGT
ATT A TT CAC ATG TGT GAC AAC ACT TTCGCA ACC GCT TTC ATT ATG CGT CCG
C TT GAT CAC GGA GCA GAC GTG ACC CTG ATC TCC ACG ACT AAG TTT GTT
GAT GGC CAC AAT ATG ACC GTC GGA GGGGCC TTG GTC ACT AAA TCC AAG GAA
TTA GAC GGA AAG GTA CGT TTA ACG CAA AAT ATC TTA GGT AAC TGT ATG AGT
CCA TTT GTT GCG TTC CTT CAA TTACAA ACG GTG AAG ACG ATG AGC CTT CGC
ATT TCT CGT CAA TCA GAA AAC GCC CAG AAA GTA GCG GA A TTT CTT GAG
ACC CAC CCC GCA GTG GAA CGC GTAATG TAT CCA GGT CT T AAA TCT TTC CCA
CAG AAG GCC TTA GCG GAT CGT CAG CAC GCA AAC A AT TTA CAT GGC GGT
ATG TTA TGG TTT GAA GTG CGC GGAGGA ACA GCG G CA GGG CGT CGC TTG ATG
GAC ACC GTT CAG CGC CCG TGG AGC TTA TGC GAG AAT CTG GGT GCG ACG GAA
TCC ATC ATT ACT TGC CCG AGTGTC ATG ACC CAC GCG AAC ATG ACT ACT GAG
GAC CGT ATG AAG GTC GGT ATC ACC GAC GGA TTT GTA CGT GTC AGC TGC GGG
ATC GAA GAT GCA GCC GATCTT ATC TCA GCT TTG AAG GCC GCA CTG GAT GCC
TTG GGC AAG 80 Cystathione ATG ATC TTA ACA GCA ATG CAA GAT GCA ATC
GGG CGT ACA CCT ATC TTC beta- AAG TTT ACA CGT AAA GAT TAC CCA ATT
CCA TTG AAG TCG GCA ATT TA synthase C GCGAAA TTG GAA CAC TTA AAC
CCG GGG GGA TCC GTG AAA GAT CGC CT (Helicobacter T GGG CAG TAT CTT
ATT AAG GAG GCC TTC CGT ACA CAC AAG ATT ACC T pylori CT ACT ACCACT
ATC ATC GAA CCT ACT GCT GGG AAT ACT GGC ATC GCC C 2017) TT GCC CTT
GTA GCT ATC AAA CAT CAT CTT AAA ACG ATC TTT GTT GTT GenBank: CCC
GAA AAA TTTTCG GTT GAG AAA CAA CAG ATC ATG CGT GCT CTT GGT
ADZ49193.1 GCC TTA GTA ATC AAT ACG CCT ACC TCA GAG GGT ATC TCA GGG
GCC ATT AAA AAA AGC AAA GAGTTA GCC GAG TCT ATC CCG GAC AGC TAC TTG
CCT CTT CAA TTT GAG AAT CCC GAC AAT CCG GCT GCT TAT TAC CAC ACT CT
T GCT CCT GAA ATT GTA AAGGAA CTG GGG ACG AAT TTT ACC TCT TTT GT A
GCG GGC ATC GGT TCT GGA GGA ACT TTC GCA GGC ACC GCC AAG TAC C TT
AAA GAA CGT ATC CCG AAC ATCCGC TTG ATT GGA GTT GAA CCA GAA G GT TCT
ATT TTA AAT GGG GGT GAA CCG GGG CCC CAC GAA ATC GAA GGA ATT GGA GTA
GAG TTC ATC CCA CCA TTCTTC GCT AAT TTG GAT ATT GAT GGG TTT GAG ACG
ATT TCA GAC GAA GAG GGC TTC AGT TAT ACG CGC AAA TTA GCC AAA AAG AAC
GGA TTA TTA GTG GGTAGT TCG TCC GGA GCA GCG TTC GCC GCG GCT CTT AAG
GAA GTA CAA CGT CTG CCC GAA GGG TCA CA A GTG TTG ACG ATT TTC CCA
GAT ATG GCT GAT CGCTAC CTT AGT AAA GG C ATT TAT TCC 81 putative
amino ATG CAA GCT TTC TTG AAC CGT TCG TTC GCG CCC CTT TTA AAC CCA
AAT transferase GAG AAC CTG CTG GAT CAA GTT AAG AGT TCG ATT ATT TTG
AAG AAA GG (Helicobacter T GTTAGC TAC TTT GAC TGG GGT GCT AGT GGG
CTG GCC AGT GCA TTG GT pylori C GAG AAA CGT GTT AAG TCC CTG CTT CCA
TAT TAT GCC AAT GCC CAC A 2017) GC GTA GCAAGT AAA CAT GCC ATC TTA
ATG GGC ATG TTA CTT AAA GAA T GenBank: GC CAA GAG AAG CTG AAA CGC
TCG TTA AAC CTT AGT ACT AAC CAT TGC ADZ50111.1 GTG CTT AGC GCCGGG
TAT GGC GCG AGC TCA GCG ATC AAG AAA TTC CAA GAG ATC CTG GGA GTT TGC
ATC CCC TCT AAA ACC AAA AAG AAT CTG GAA CCT TAT TTA AAA GACATG GCG
CTG AAA CGC GTA ATC GTA GGT CCT TAT GAA CAT CAC TCT AAC GAG GTC TCT
TGG CGC GAG TCT CTT TGT GAG GT G GTG CGC ATT CCA CTT AACGAA CAT GGA
CTG CTG GAT TTG GAG ATT TT A GAG CAG ATC TTA AAG AAA TCC CCC AAT
TCT CTG GTC TCC GTC TCG G
CC GCA AGT AAT GTA ACG GGG ATTCTG ACA CCC CTG AAA GAA ATT AGC T CA
CTG TGC AAG GAG TAT CGC GCG ATC CTG GCG CTT GAT CTG GCC AAC TTT TCC
GCA CAC GCG AAC CCG AAA GACTGC GAG TAC CAG ACG GGG TTC TAT GCA CCA
CAC AAG TTG TTG GGT GGT ATT GGG GGA TGC GGG CTT CTT GGA ATC TCC AAA
GAC TTG ATC GAT ACA CAGATC CCA CCT AGT TTT TCA GCC GGA GGA GTC ATT
AAG TAC GCA AAC CGC ACG CGT CAC GAA TTT AT T GAT GAG CTG CCG TTG
CGT GAG GAG TTC GGA ACTCCG GGA CTG CTG CA A TTT TAT CGC TCA GTG TTA
GCC TAC CAG TTA CGT GAC GAA TGC GGT T TG GAT TTC ATT CAT AAG AAG
GAG AAT AAT CTG CTT CGTGTG TTA ATG C AT GGC TTG AAA GAT CTG CCA GCT
ATC AAC ATT TAC GGC AAT TTA ACC GCA AGC CGC GTA GGA GTA GTC GCG TTT
AAC ATC GGA GGC ATTAGT CCA TAC GAT CTT GCC CGT GTC CTG AGT TAC GAA
TAT GCT ATT GAG ACT CGC GCA GGG TGC TCT TGT GCC GGC CCG TAT GGA CAT
GAC TTA CTG AATTTG AAT GCA CAA AAG TCT TCC GAT TTC AAT GCA AAA CCT
GGA TGG TTG CG C GTC TCA CTT CAT TTT ACA CAC AGT ATT AAT GAC ATT
GAC TAT CTG T TGGAC TCT CTG AAG AAA GCT GTT AAG AAA CTG CGT 82 YdeD
ATG AAC GGT GAA CAC GCC GCG TTG GCC CAC TCC CGC ACA AAA GGG ATT
(Bacillus GCT TTG GTT TTA ACG GGC AGT ATC TTA TGG GGC GTT TCA GGG
ACA GT atrophaeus T GCGCAG TAC TTA TTC CAA CAA CAA CAT TTT AAC GTA
GAG TGG TTG AC UCMB-5137) C GTC GTT CGC TTG TTG CTG TCT GGT ATC TTG
CTG CTT GGC CTT GCC T GenBank: AT CGT AAGGAA AAG CAA CGC ATC TGG
GCT GTC TGG AAA GAC AAG ACA G AKL87093.1 AT GGT CTG AAT CTG GTT CTG
TTC GGG ATT TTG GGG ATG TTG TCC GTC CAG TAC ACA TACTTT GCG GCT ATC
CAG CAT GGT AAT GCG GCG ACG GCA ACT GTA CTT CAG TAT CTG GCC CCG GCA
CTT ATT ACC TGC TAC GTA GCC ATT CGC TCT AAG CGTCTT CCA ACC GTC AAA
GAG TTG ATC GCA GTT TTC CTG GCT ATT ATT GGA ACG TTT TTT TTA GTC ACC
CAT GGG GAC ATC CA C AGT CTT AGT ATC TCA GGGTGG GCT TTA TTC TGG GGA
TTA AGT TCG GC G TTT GCC CTG GCG TTT TAC ACT TTG CAC CCT CAT AAA
CTT CTG GCC A AG TGG GGG GCG GCT ATC GTT GTTGGC TGG GGT ATG CTT ATC
GGA GGG C TT GGT CTT TCC TTA ATC CAT CCT CCA TGG AAA TTT GAG GGA
CAG TGG TCG GTC TCG GCT TAT GCC GCC GTT ATTTTC ATT GTC CTG TTT GGG
ACC CTG ACT GCC TTC TAC TGC TAC CTG GAA TCT TTA AAG TAC TTA ACT GCC
AGC GAA ACT TCA TTA ATC GCC TGC GCG GAGCCC TTA AGT GCT GCG TTC TTA
AGC GTG ATT TGG TTG CAT GTG ACT TTT GGT ATC AGC GAG TGG CT T GGT
ACT TGT TGT ATT TTA TCT ACG ATT ATG ATCTTA TCG ATT AAG GA G AAG AAG
CTG AAG 83 protein YfiK ATG ACA CCC ACG TTG CTT AGC GCC TTC TGG ACG
TAC ACC CTT ATT ACA (Escherichia GCC ATG ACG CCT GGG CCA AAT AAT
ATC CTT GCC TTA TCA TCC GCA AC coli) G TCGCAT GGG TTC CGC CAG TCC
ACC CGT GTG CTT GCA GGT ATG TCT CT GenBank: T GGC TTT TTA ATC GTT
ATG CTG TTG TGC GCG GGA ATC AGT TTC TCC T AJE57139.1 TG GCG GTAATC
GAC CCC GCC GCC GTA CAT TTA TTG TCT TGG GCT GGT G CC GCG TAT ATT
GTT TGG CTG GCT TGG AAA ATT GCC ACG TCT CCG ACT AAG GAA GAT GGTTTA
CAA GCA AAA CCC ATC TCG TTT TGG GCT TCA TTT GCA CTT CAG TTC GTG AAT
GTC AAG ATT ATT CTT TAC GGG GTA ACA GCC CTG TCC ACT TTC GTTTTA CCC
CAG ACG CAG GCG TTG TCA TGG GTA GTC GGA GTG TCC GTC TTA TTA GCC ATG
ATC GGT ACG TTT GGG AAT GTG TG C TGG GCG CTG GCG GGC CACTTG TTT CAA
CAA TTA TTC CGT CAG TAC GG T CGC CAG TTA AAT ATC GTT CTT GCT TTA
TTA CTG GTG TAT TGT GCA G TC CGC ATC TTC TAT 84 multidrug ATG ACG
ACC CGC CAG CAT AGC TCG TTC GCA ATC GTA TTT ATT CTT GGA efflux TTG
CTT GCT ATG TTG ATG CCA TTA TCA ATC GAC ATG TAC TTA CCA GC
transporter. C CTGCCT GTT ATT TCG GCC CAA TTT GGA GTA CCC GCT GGG
TCA ACC CA Bcr (Escherichia A ATG ACA TTA TCA ACA TAC ATT CTG GGG
TTC GCT TTA GGA CAG TTG A coli) TT TAT GGTCCA ATG GCT GAC TCG TTT
GGG CGC AAA CCA GTG GTC TTG G GenBank: GC GGG ACA CTG GTC TTT GCG
GCC GCA GCC GTT GCG TGT GCC TTG GCT CDZ21005.1 AAC ACG ATC GACCAG
CTT ATT GTA ATG CGT TTC TTC CAT GGC TTA GCT GCG GCG GCT GCC AGT GTA
GTG ATT AAT GCG CTT ATG CGT GAC ATC TAT CCG AAG GAG GAA TTCAGC CGC
ATG ATG AGC TTC GTA ATG TTG GTA ACG ACC ATC GCT CCA TTA ATG GCC CCT
ATT GTT GGG GGT TGG GTC TTA GT C TGG CTT TCA TGG CAT TACATT TTT TGG
ATC CTT GCC CTG GCG GCT AT T CTG GCC TCA GCG ATG ATT TTC TTC CTG
ATT AAA GAA ACC CTT CCT C CG GAG CGC CGT CAG CCT TTC CATATT CGC ACT
ACT ATC GGT AAT TTT G CG GCC TTG TTT CGC CAT AAA CGC GTG CTG TCA
TAC ATG TTG GCA AGC GGC TTT TCT TTC GCG GGT ATG TTC TCGTTT TTA AGT
GCT GGT CCC TTC GTG TAT ATC GAA ATC AAT CAC GTA GCC CCG GAG AAC TTC
GGC TAT TAC TTC GCA TTA AAT ATC GTG TTT CTT TTC GTCATG ACC ATC TTC
AAC TCT CGC TTC GTC CGT CGT ATC GGT GCC TTA AAT ATG TTT CGT TCG GGG
CT G TGG ATC CAA TTT ATC ATG GCT GCG TGG ATG GTGATC TCC GCA CTG TT
G GGG CTT GGG TTT TGG TCG CTT GTG GTG GGC GTG GCT GCA TTC GTT G GA
TGT GTC AGC ATG GTA TCT TCT AAC GCG ATG GCT GTAATT TTG GAT G AG TTC
CCA CAT ATG GCA GGG ACT GCT TCC TCT CTG GCT GGC ACA TTT CGC TTC GGA
ATT GGT GCA ATC GTA GGC GCG TTG CTG AGC TTAGCG ACA TTC AAT TCG GCG
TGG CCC ATG ATT TGG TCC ATT GCG TTT TGT GCG ACC AGC AGC ATC CTG TTC
TGC CTT TAT GCT TCC CGT CCA AAG AAG CGT 85 TolC ATG AAC AAA CTT AGT
ATG CTG GGA GCT GCC TTC GCG TTG TTG GCA GGG (Pseudomonas AAC TCA
GCA TTG GCA GCA ATG GGG CCT TTC GAA ATC TAC GAA CAG GC fluorescens
T CTTCGC AAT GAC CCA GTT TTC TTA GGG GCC ATT AAG GAG CGT GAC GC
R124) C GGA TTG GAA AAC CGC ATC ATC GGC CGC GCA GGA TTG TTA CCA CGC
T GenBank: TG GGG TACAAC TAC AAT CGT GGC CAT AAC ACC TCT AAA GCG
ACC CAG T EJZ58348.1 TG ACA AAT CGT GGC TCT CTG ACT GAA GAC CGT AAC
TAT AAT TCG TAT GGT TCA ACT CTTACA TTA CAG CAA CCC TTA TTA GAC TAT
GAG GCC TAT GCC GCC TAC CGT AAG GGA GTA GCG CAA AGC TTG TTC GCC GAT
GAA GCC TTT CGC GGT AAG TCACAG GAA TTA TTG GTT CGC GTC TTA GAT AAT
TAC ACG AAA GCG TTG TTC GCA CAA GAC CAA ATC GAT ATC GCA CAG GCG AA
A AAA AAA GCT TAT GAA CAACAA TTT CAG CAG AAC GAA CAT ATG TTC AA A
CAA GGC GAG GGG ACG CGC ACT GAC ATT TTG GAA GCT GAA AGT CGT T AT
GAA CTT GCC ACG GCA GAA GAAATC GAG GCG CGT AAC GAA CAG GAT G CC GCT
CTT CGC GAG CTT GGT GCG CTT GTC GGT GTC CCA ACT GTC GAC ATT TCT GAA
CTT GCA CCC TTA GAC CAGAAT TTT CAA ACG TTC GCG CTG ATG CCT GCT AAC
TAT GAT ACG TGG CAC GAG TTA GCA ATT TCT AAT AAT CCG AAC CTG GCA TCA
CAG CGT CAG GCC GTGGAA GTA GCA AAA TAC GAA GTT GAA CGT AAC CGT GCA
GGA CAT TTA CCC AAG GTC TCA GCA TAT GC C AGC ATT CGT CAG ACT GAG
TCT GAC AGT GGT AATACC TAC AAT CAA CG T TAT GAT ACG AAC ACC ATT GGC
TTT GAG GTA AAC GTC CCT CTG TAT G CA GGA GGA GGA GTC TCA GCA AGT
ACA CGC CAA GCA TCACGC ACG ATG G AG CAG GCG GAG TAT GAA TTA GAT GGA
AAG ACG CGT GAG ACG TTA ATT GAA TTA CGT CGT CAG TTC AGC GCG TGC CTT
AGT GGA GTT AATAAG TTA CGC GCC TAT CAG AAA GCC CTG GCC TCG GCC GAA
GCA CTG GTG GTC TCA ACC AAG CAG AGC ATT CTT GGC GGC GAA CGC ACC AAC
TTG GAC GCGCTT AAC GCG GAA CAG CAG CTG TTC ACC ACG CGT CGC GAC CTT
GCA CAG GC C CGC TAT GAC TAC TTG ATG GCG TGG ACG AAA CTG CAT TAT
TAC GCA G GAACC CTG AAC GAA CAA GAT TTA GCG CGT GTG GAC GAG GCA TTT
GGC C AA GGG CCC AAA TCA AAT CCT CGC 86 Tyrosine ATG TTC GAT GCG
CTG GCG CGT CAA GCG GAT GAT CCG CTT TTG GCG CTG transaminase ATC
GGA CTG TTT CGC AAA GAC GAG CGC CCC GGT AAA GTG GAC TTA GG
(Sinorhizobium T GTGGGA GTT TAC CGC GAC GAA ACT GGC CGC ACT CCG ATC
TTT CGC GC meliloti G GTT AAA GCA GCC GAA AAA CGC TTG CTT GAG ACT
CAG GAC TCG AAG G AK83) CC TAC ATCGGC CCG GAA GGA GAC CTG GTT TTT
CTT GAC CGT TTG TGG G GenBank: AA CTT GTT GGG GGG GAT ACC ATT GAA
CGT TCT CAC GTA GCT GGT GTA AEG55340.1 CAA ACA CCT GGCGGG AGC GGC
GCA CTT CGT TTG GCG GCA GAT TTA ATC GCC CGC ATG GGC GGT CGC GGG ATT
TGG TTG GGG TTG CCA TCC TGG CCG AAT CAC GCT CCC ATTTTC AAA GCG GCT
GGA CTG GAT ATC GCG ACT TAC GAT TTC TTT GAT ATC CCG AGT CAA TCC GTT
ATT TTT GAT AAC CTG GT G TCT GCC CTG GAA GGT GCAGCA TCT GGC GAT GCC
GTC TTA TTG CAT GC T AGC TGC CAC AAT CCA ACT GGA GGG GTA TTA TCC
GAG GCA CAG TGG A TG GAA ATT GCC GCG CTG GTC GCCGAA CGC GGA CTG TTA
CCA CTT GTT G AT CTT GCG TAT CAA GGG TTC GGA CGT GGG CTG GAT CAA
GAC GTC GCG GGC TTA CGC CAT TTA TTA GGT GTA GTTCCC GAA GCC CTT GTC
GCC GTT AGC TGC TCT AAA TCG TTC GGC TTG TAC CGC GAA CGC GCT GGA GCC
ATC TTC GCC CGT ACA TCA TCT ACC GCT TCA GCCGAC CGC GTC CGC AGT AAC
TTA GCT GGC CTT GCT CGC ACA TCG TAT AGT ATG CCC CCC GAT CAC GG G
GCC GCG GTT GTC CGT ACG ATC TTA GAC GAC CCAGAG CTG CGT CGT GA C TGG
ACC GAG GAA TTA GAG ACA ATG CGC TTG CGT ATG ACG GGT CTT C GC CGC
TCT CTT GCA GAG GGC TTG CGC ACC CGT TGG CAGTCT CTT GGC G CC GTA GCT
GAC CAA GAA GGG ATG TTC TCG ATG CTG CCG TTG TCC GAA GCA GAG GTT ATG
CGC CTT CGC ACT GAG CAT GGA ATT TAC ATGCCC GCA TCA GGA CGC ATT AAC
ATT GCG GGG TTA AAA ACG GCG GAG GCT GCC GAA ATT GCA GGT AAA TTT ACG
AGT TTG 87 tyrosine ATG AAG AAC CGC ACT CTT GGA TCA GTA TTC ATT GTT
GCG GGG ACC ACC transporter ATC GGT GCA GGT ATG CTT GCC ATG CCC CTG
GCT GCA GCT GGC GTC GG TyrP (Escherichia G TTCAGC GTT ACC CTG ATT
TTA CTG ATT GGT CTG TGG GCT CTG ATG TG coli W) T TAC ACG GCA TTG
CTT TTG CTT GAA GTG TAC CAG CAT GTA CCC GCA G GenBank: AC ACC
GGTCTT GGC ACT CTG GCG AAA CGT TAT TTA GGA CGT TAT GGT C AFH11702.1
AA TGG CTG ACC GGT TTC TCC ATG ATG TTT CTG ATG TAT GCG CTG ACG GCC
GCA TAC ATTAGT GGT GCA GGT GAA CTG CTG GCA AGT TCA ATT TCT GAC TGG
ACG GGC ATC TCT ATG AGC GCG ACT GCT GGG GTT TTA TTG TTT ACA TTT GTG
GCT GGCGGT GTA GTG TGT GTA GGG ACG TCA TTA GTT GAT CTG TTT AAC CGC
TTC CTT TTC AGT GCA AAA ATC ATT TTC CTT GTA GT A ATG CTT GTC TTA
TTA TTACCA CAT ATT CAT AAG GTA AAT CTT TTG AC A TTA CCA TTG CAG CAG
GGA TTG GCG TTA TCA GCC ATC CCT GTA ATC T TC ACA TCC TTC GGA TTC
CAC GGGTCC GTC CCA TCC ATC GTG TCC TAC A TG GAC GGC AAT GTA CGC AAG
TTA CGT TGG GTC TTT ATC ACA GGG AGC GCC ATT CCC CTT GTA GCG TAT ATT
TTTTGG CAA GTT GCT ACT CTG GGG TCA ATC GAC TCT ACC ACC TTC ATG GGT
TTA CTT GCG AAC CAC GCG GGG TTG AAC GGA CTG TTA CAG GCT TTG CGT
GAAATG GTT GCC TCG CCA CAT GTT GAG TTG GCG GTT CAT CTT TTT GCT GAC
TTA GCC TTA GCT ACC TC T TTC CTT GGG GTT GCG CTG GGA TTA TTC GAC
TATCTG GCT GAT CTT TT T CAA CGC TCC AAC ACC GTA GGT GGA CGT TTA CAG
ACT GGA GCC ATT A CT TTC TTG CCC CCT TTA GCC TTT GCG CTG TTT TAT
CCACGT GGG TTT G TT ATG GCC TTG GGG TAT GCT GGA GTC GCC TTA GCT GTA
CTT GCT CTT ATT ATT CCA TCG TTA TTA ACG TGG CAA TCG CGT AAA CAC
AACCCC CAA GCA GGG TAC CGC GTG AAG GGA GGA CGC CCC GCG CTG GTG GTT
GTT TTT CTG TGC GGG ATT GCC GTC ATC GGC GTG CAA TTT TTG ATT GCA
GCAGGT TTG TTG CCG GAG GTG GGG Phenylalanine 88 Beta- ATG ACT CAT
GCT GCA ATT GAC CAG GCG TTG GCA GAC GCC TAT CGT CGT phenylalanine
TTT ACT GAC GCA AAC CCT GCC AGC CAG CGT CAG TTT GAA GCG CAA GCC
transaminase CGCTAT ATG CCC GGG GCT AAC TCT CGC TCT GTT TTG TTT TAT
GCA CCC (Aromatic TTT CCA TTG ACG ATC GCA CGT GGG GAA GGC GCC GCT
CTT TGG GAT GCG beta-amino GAC GGCCAC CGT TAC GCT GAC TTT ATC GCG
GAA TAC ACA GCT GGG GTG acid TAT GGA CAC AGT GCC CCA GAG ATT CGT
GAC GCA GTA ATC GAA GCT ATG aminotransferase; CAG GGT GGGATT AAT
TTG ACG GGT CAT AAT TTG TTG GAA GGC CGC TTA Beta- GCC CGC CTT ATT
TGT GAG CGT TTC CCA CAG ATC GAA CAG TTG CGT TTC phenylalanine- ACG
AAT AGC GGAACA GAG GCC AAT CTG ATG GCC CTT ACC GCG GCG CTT
aminotransferase; CAT TTT ACT GGT CGC CGC AAA ATC GTC GTA TTT AGT
GGA GGT TAT CAT VpAT) GGG GGG GTT CTT GGGTTC GGT GCC CGT CCT AGC
CCT ACC ACA GTA CCA UniProtKB/ TTT GAC TTC CTT GTG CTG CCT TAC AAC
GAT GCT CAG ACG GCT CGT GCT Swiss-Prot: CAG ATC GAG CGC CAC GGCCCG
GAG ATC GCG GTC GTG TTA GTC GAG CCC H8WR05.1 ATG CAA GGT GCT TCT
GGC TGC ATC CCA GGT CAG CCC GAC TTT CTG CAA GCC CTG CGC GAA TCC GCT
ACTCAG GTA GGG GCG CTG TTA GTT TTT GAC GAA GTG ATG ACT AGT CGC TTA
GCG CCA CAT GGT TTA GCT AAC AAA TTG GGG ATC CGT TCG GAT TTG ACA
ACCCTG GGT AAG TAC ATT GGC GGC GGT ATG TCA TTT GGG GCC TTT GGC GGT
CGT GCT GAT GTC ATG GCC CTG TTC GAC CCT CGC ACT GGA CCT TTG GCT
CATTCC GGT ACG TTT AAC AAC AAT GTG ATG ACG ATG GCT GCC GGT TAT GCT
GGC TTA ACG AAA TTA TTC ACT CCG GAA GCG GCA GGG GCA TTG GCA GAG
CGTGGA GAA GCG CTT CGC GCA CGT CTT AAC GCC CTG TGT GCT AAC GAA GGA
GTA GCA ATG CAG TTC ACT GGC ATC GGC TCG CTG ATG AAT GCC CAC TTC
GTCCAG GGA GAC GTT CGT AGC TCT GAG GAT CTG GCC GCA GTT GAT GGG CGT
TTA CGT CAG TTG TTG TTC TTT CAT TTA TTG AAT GAA GAT ATT TAC TCT
TCACCG CGT GGG TTT GTT GTA TTA TCG TTG CCA TTG ACT GAC GCT GAT ATT
GAC CGC TAC GTT GCT GCG ATC GGT TCA TTT ATT GGC GGT CAT GGG GCG
TTGTTA CCG CGC GCT AAC 89 gadA
ATGGACCAGAAGCTGTTAACGGATTTCCGCTCAGAACTACTCGATTCACGTTTTGGCGCAAAG
glutamate
GCCATTTCTACTATCGCGGAGTCAAAACGATTTCCGCTGCACGAAATGCGCGATGATGTCGCA
decarboxylase
TTTCAGATTATCAATGATGAATTATATCTTGATGGCAACGCTCGTCAGAACCTGGCCACTTTC
(Escherichia
TGCCAGACCTGGGACGACGAAAACGTCCATAAATTGATGGATTTGTCGATCAATAAAAACTGG
coli)
ATCGACAAAGAAGAATATCCGCAATCCGCAGCCATCGACCTGCGTTGCGTAAATATGGTTGCC
GATCTGTGGCATGCGCCTGCGCCGAAAAATGGTCAGGCCGTTGGCACCAACACCATTGGTTCT
TCCGAGGCCTGTATGCTCGGCGGGATGGCGATGAAATGGCGTTGGCGCAAGCGTATGGAAGCT
GCAGGCAAACCAACGGATAAACCAAACCTGGTGTGCGGTCCGGTACAAATCTGCTGGCATAAA
TTCGCCCGCTACTGGGATGTGGAGCTGCGTGAGATCCCTATGCGCCCCGGTCAGTTGTTTATG
GACCCGAAACGCATGATTGAAGCCTGTGACGAAAACACCATCGGCGTGGTGCCGACTTTCGGC
GTGACCTACACCGGTAACTATGAGTTCCCACAACCGCTGCACGATGCGCTGGATAAATTCCAG
GCCGACACCGGTATCGACATCGACATGCACATCGACGCTGCCAGCGGTGGCTTCCTGGCACCG
TTCGTCGCCCCGGATATCGTCTGGGACTTCCGCCTGCCGCGTGTGAAATCGATCAGTGCTTCA
GGCCATAAATTCGGTCTGGCTCCGCTGGGCTGCGGCTGGGTTATCTGGCGTGACGAAGAAGCG
CTGCCGCAGGAACTGGTGTTCAACGTTGACTACCTGGGTGGTCAAATTGGTACTTTTGCCATC
AACTTCTCCCGCCCGGCGGGTCAGGTAATTGCACAGTACTATGAATTCCTGCGCCTCGGTCGT
GAAGGCTATACCAAAGTACAGAACGCCTCTTACCAGGTTGCCGCTTATCTGGCGGATGAAATC
GCCAAACTGGGGCCGTATGAGTTCATCTGTACGGGTCGCCCGGACGAAGGCATCCCGGCGGTT
TGCTTCAAACTGAAAGATGGTGAAGATCCGGGATACACCCTGTACGACCTCTCTGAACGTCTG
CGTCTGCGCGGCTGGCAGGTTCCGGCCTTCACTCTCGGCGGTGAAGCCACCGACATCGTGGTG
ATGCGCATTATGTGTCGTCGCGGCTTCGAAATGGACTTTGCTGAACTGTTGCTGGAAGACTAC
AAAGCCTCCCTGAAATATCTCAGCGATCACCCGAAACTGCAGGGTATTGCCCAGCAGAACAGC
TTTAAACACACCTGA 90 glutamate ATG GAT AAA AAG CAA GTG ACG GAC CTG
CGC TCT GAA CTT CTT GAC AGT decarboxylase CGT TTT GGG GCA AAG AGT
ATT AGT ACC ATT GCT GAG TCA AAG CGT
TT (Escherichia T CCTTTG CAT GAG ATG CGC GAT GAC GTC GCA TTC CAG
ATT ATC AAC GA coli KO11FL) C GAG CTG TAT TTG GAC GGC AAT GCC CGC
CAA AAC TTG GCC ACG TTT T GenBank: GT CAG ACTTGG GAT GAC GAG AAT
GTT CAT AAA CTT ATG GAC CTT TCA A ADX50933.1 TT AAC AAA AAT TGG ATT
GAC AAA GAA GAG TAC CCC CAA TCT GCC GCA ATT GAT TTA CGTTGT GTT AAT
ATG GTG GCC GAC TTA TGG CAT GCA CCA GCC CCT AAA AAC GGC CAA GCG GTG
GGA ACC AAC ACG ATC GGG TCT AGT GAG GCA TGT ATG TTAGGC GGG ATG GCC
ATG AAG TGG CGT TGG CGT AAA CGC ATG GAG GCA GCA GGG AAA CCA ACC GAT
AAA CCT AAT TTA GTC TG C GGA CCG GTT CAG ATC TGTTGG CAT AAA TTT GCG
CGC TAC TGG GAT GT G GAA TTA CGC GAA ATT CCG ATG CGT CCG GGC CAA
CTG TTC ATG GAT C CC AAA CGT ATG ATC GAA GCA TGTGAC GAA AAC ACG ATT
GGG GTG GTA C CC ACC TTT GGG GTC ACA TAT ACA GGT AAC TAC GAG TTT
CCA CAA CCG TTG CAT GAT GCT CTG GAC AAG TTT CAAGCT GAC ACC GGG ATC
GAC ATT GAT ATG CAC ATT GAC GCT GCC TCC GGC GGA TTC TTG GCC CCA TTT
GTA GCC CCT GAC ATT GTC TGG GAC TTT CGT CTTCCC CGT GTG AAA TCC ATC
AGC GCA TCC GGT CAC AAG TTT GGG CTT GCC CCA TTA GGG TGT GGA TG G
GTC ATC TGG CGT GAT GAG GAA GCA TTA CCC CAAGAA CTT GTC TTC AA T GTA
GAT TAC CTT GGG GGA CAG ATT GGC ACT TTT GCC ATC AAC TTT T CT CGC
CCA GCG GGT CAA GTG ATC GCC CAG TAT TAC GAGTTT CTG CGC C TG GGA CGT
GAG GGA TAT ACA AAA GTG CAG AAC GCA TCG TAC CAG GTA GCG GCT TAC CTT
GCG GAC GAA ATT GCA AAG CTG GGA CCA TACGAG TTT ATC TGT ACC GGG CGT
CCA GAT GAA GGT ATT CCG GCT GTG TGT TTT AAG CTG AAA GAC GGG GAA GAT
CCC GGA TAT ACG CTG TAT GAT CTG TCTGAA CGT TTA CGT TTG CGC GGT TGG
CAA GTT CCA GCC TTC ACG TTG GGT GG C GAA GCC ACT GAT ATT GTA GTC
ATG CGT ATC ATG TGT CGT CGC GGC T TTGAA ATG GAT TTC GCA GAG TTA CTT
CTG GAA GAC TAC AAA GCG AGC T TA AAA TAT TTG TCT GAC CAT CCC AAG
TTG CAA GGG ATC GCA CAG CAA AAT TCGTTT AAA CAC ACT 91 GltT ATG AAG
AAA TTA CGC TTC GGA CTG GCG ACT CAA ATC TTT GTG GGG CTG (Bacillus
ATT CTT GGG GTA GTA GTG GGC GTT ATC TGG TAC GGT AAT CCG GCG GT
atrophaeus G GTAACT TAT TTG CAG CCA GTT GGG GAC CTT TTT TTA CGT TTG
ATT AA UCMB-5137) A ATG ATC GTT ATT CCT ATC GTG GTG TCT TCT TTG ATC
ATT GGC GTC G GenBank: CG GGA GCTGGG TCC GGA AAA CAG GTC GGA AAG
CTG GGC TTT CGT ACT A AKL83763.1 TT CTG TAC TTC GAG ATC ATC ACT ACC
TTT GCC ATC ATT CTG GGA CTT GCT CTG GCG AATCTT TTC CAG CCT GGT ACA
GGA GTA AAT ATC GAG AGC GCG CAG AAA AGT GAC ATT TCC CAG TAC GTG GAG
ACT GAA AAA GAG CAA TCC ACC AAA TCC GTAGCT GAG ACT TTC CTG CAT ATC
GTG CCC ACC AAT TTC TTT CAA TCA CTT GCG GAA GGT GAT CTT CTT GCT ATT
ATC TGC TT T ACC GTA CTT TTC GCC CTTGGC ATT TCG GCT ATC GGT GAA CGT
GGC AA A CCG GTG CTT GCT TTC TTT GAC GGA GTA TCC CAC GCG ATG TTT
CAT G TA GTG AAC CTT GTG ATG AAG GTTGCT CCG TTC GGC GTA TTT GCT CTG
A TT GGA GTA ACA GTA AGC AAA TTT GGA CTG GGT TCT TTA CTG AGC CTG
GGT AAA CTT GTG GGG CTG GTA TAT GTTGCT CTG GCA TTT TTT CTT ATT GTA
ATC TTT GGT ATT GTT GGA AAG CTG GCT GGC GTG AAT ATC TTC AAG TTT TTA
GCT TAC ATG AAG GAT GAA ATC TTATTA GCG TTC TCG ACC TCA TCG TCC GAG
ACT GTG TTG CCC CGC ATC ATG GAG AAA ATG GAG AAG AT C GGG TGT CCA
AAG GGA ATT GTA AGC TTT GTA GTCCCC ATC GGT TAC AC A TTC AAT CTT GAC
GGC TCG GTC TTA TAC CAA TCT ATT GCT GCG CTG T TC TTG GCA CAG GTT
TAC GGA ATC GAC CTG ACT ATT TGGCAT CAG ATT A CT CTG GTG TTA GTT CTG
ATG GTC ACT AGC AAA GGC ATG GCA GCC GTT CCT GGA ACT AGC TTT GTA GTC
CTG CTG GCA ACC TTA GGT ACCATT GGT GTT CCA GCG GAA GGG CTT GCA TTC
ATT GCG GGG GTT GAC CGC ATT ATG GAC ATG GCT CGC ACT GTG GTC AAT TTA
ACA GGC AAT GCT CTT GCGAGT GTC GTA ATG AGC AAG TGG GAG GGT CAG TAC
GAC CCG GTG AAA GGT GC A GAG ATT ATG AGC CGC AGC AAG ACG GAA CAG
GAC GCT ACT ATC TCC G GA 92 mechanosensitive ATG GAG GAC TTG AAC
GTA GTA GAT AGC ATT AAT GGA GCG GGC TCA TGG channel Msc TTA GTA GCC
AAC CAA GCC CTG TTG TTA TCG TAT GCT GTA AAT ATC GT S (Escherichia C
GCAGCC TTA GCC ATC ATT ATC GTT GGG TTA ATC ATC GCC CGT ATG AT coli)
T TCT AAT GCG GTG AAT CGC TTA ATG ATC TCG CGC AAG ATC GAC GCC A
GenBank: CT GTC GCGGAT TTC TTG TCC GCC CTG GTG CGT TAC GGT ATC ATC
GCG T CTX26261.1 TC ACA TTG ATT GCG GCA TTA GGG CGC GTA GGA GTC CAG
ACA GCT TCT GTG ATT GCG GTATTA GGT GCA GCA GGA TTA GCT GTG GGA TTG
GCG TTA CAG GGG TCT CTT TCC AAT CTG GCG GCC GGC GTA CTT CTG GTT ATG
TTT CGC CCC TTT CGC GCCGGA GAG TAT GTG GAT TTG GGA GGA GTG GCC GGA
ACA GTG CTG TCA GTG CAA ATC TTT TCT ACC ACG ATG CGT ACA GCA GA T
GGA AAA ATC ATC GTG ATCCCC AAT GGC AAG ATC ATC GCG GGT AAC AT T ATC
AAC TTC TCC CGC GAA CCT GTT CGC CGC AAC GAA TTT ATC ATC G GT GTT
GCC TAT GAT TCA GAC ATCGAT CAG GTC AAA CAA ATT CTT ACG A AC ATC ATT
CAG TCA GAG GAC CGT ATT CTG AAA GAC CGC GAA ATG ACG GTG CGT TTG AAT
GAG TTA GGG GCT TCAAGT ATC AAC TTC GTA GTC CGC GTG TGG AGC AAT TCC
GGT GAT TTG CAA AAC GTG TAT TGG GAC GTC CTT GAG CGC ATT AAG CGT GAA
TTC GAT GCT GCCGGG ATC TCC TTT CCG TAT CCT CAG ATG GAT GTG AAT TTC
AAG CGT GTA AAG GAA GAT AAG GCT GC C 93 HutH ATG ATG GTC ACC TTG
GAT GGG TCT TCA TTA ACG ACG GCT GAT GCA CAA (Bacillus CGT GTA CTT
TTC GAT TTT GAA GAG GTA CAG GCA TCG GCT GAA TCG AT
amyloliquefaciens G GAGCGC GTA AAA AAG AGC CGT GCC GCC GTG GAA CGC
ATT GTA CAA GA subsp. A GAA AAA ACT ATC TAC GGA ATC ACT ACG GGG TTT
GGT AAG TTT TCC G plantarum AT GTG CTGATC CAA AAA GAG GAC GCT GCG
GAT TTA CAA TTG AAT TTG A str. FZB42) TC TTG TCA CAT GCA TGT GGA
GTC GGC GAT CCT TTC CCA GAG TCA GTC GenBank: TCC CGC GCC ATGCTG CTT
CTG CGT GCA AAC GCA TTG TTA AAA GGC TTC ABS75970.1 TCC GGT GTT CGT
ACG GAA TTA ATT GAC CAG CTT TTA GCG TAC TTA AAC CAC CGT ATC CAC
CCTGTT ATC CCC CAA CAA GGT TCG CTG GGG GCC TCC GGC GAT TTG GCC CCT
CTT AGC CAC CTT GCG TTG GCA CTG ATC GGA CA A GGG GAA GTG TTC TAC
GAAGGA GCA CGT ATG CCC ACT GCT CAT GCC CT T GAA CAA ACC AAT CTG CAG
CCC GCA GTC CTG ACA TCG AAG GAA GGG C TG GCG TTG ATC AAT GGG ACT
CAGGCT ATG ACC GCA ATG GGC TTA ATC G CA TAC CTT GAA GCC GAA AAG TTG
GCA TAT CAG AGC GAG CGC ATC GCT TCA TTG ACT ATC GAA GGA TTG CAA
GGTATT ATT GAC GCG TTT GAC GAA GAT ATT CAT GCC GCT CGT GGA TAC CAG
GAA CAA ATG GAT GTC GCT GAG CGC ATT CGC TAT TAT CTT TCG GAT TCG
AAGCTG ACA ACC GTA CAA GGC GAG CTG CGT GTG CAA GAT GCT TAC TCC ATT
CGC TGC ATC CCT CAA GT C CAC GGA GCT TCT TGG CAG ACC CTG GCG TAT
GTGAAG GAG AAG TTA GA A ATT GAG ATG AAC GCT GCT ACT GAT AAC CCT TTA
ATT TTT GAA GAC G GG GCC AAA ATT ATC TCG GGG GGG AAC TTT CAC GGG
CAACCG ATC GCG T TT GCA ATG GAC TTC TTG AAA GTA GCT GCT GCT GAG TTG
GCT AAT ATC AGC GAG CGC CGT ATT GAG CGT CTT GTC AAT CCA CAG CTG
AATGAC CTT CCT CCT TTT CTT TCG CCG CAA CCG GGT TTA CAG TCT GGT GCC
ATG ATT ATG CAG TAC GCC GCT GCC TCC TTG GTC TCG GAA AAC AAA ACA
CTTGCG CAT CCC GCC TCA GTC GAC TCA ATC CCC TCC TCG GCT AAC CAG GAG
GA T CAC GTC TCC ATG GGG ACG ATC GCT TCA CGT CAT GCT TAC CAG ATT A
TTGCA AAC ACT CGT CGC GTA TTA GCC GTC GAG GCC ATT TGC GCT TTA C AA
GCT GTA GAG TAC CGT GGG GAA GAG CAC TGC GCT AGC TAC ACG AAA CAA
CTTTAC CAT GAG ATG CGT AAC ATC GTG CCA TCG ATT CAG GAG GAC CGT GTT
TTC TCG TAC GAC ATC GAG CAC TTA TCC GAC TGG CTT AAA AAG GAA TCC
TTCTTA CCT AAT GAA CAC CAC CAA AAG TTA ATG ACT AAT GAG GGC GGG TTA
ACT CGC 94 Histidine ATG AAG AAA CTT GTC CTT TCA TTG TCT CTG GTA
TTA GCG TTC AGT TCA ABC GCA ACT GCA GCA TTC GCT GCT ATT CCG CAA AAT
ATC CGC ATC GGG AC transporter, G GATCCC ACG TAT GCG CCA TTC GAG
TCA AAG AAT TCA CAA GGT GAA TT histidine- G GTC GGG TTC GAT ATT GAC
CTG GCG AAA GAA TTG TGT AAA CGT ATC A binding AT ACC CAATGC ACG TTC
GTG GAA AAT CCC TTG GAT GCA TTA ATT CCG T periplasmic CT TTG AAA
GCG AAA AAA ATC GAT GCC ATC ATG TCA TCC CTT TCT ATC protein ACA GAA
AAG CGCCAG CAG GAG ATT GCC TTC ACA GAC AAG TTG TAC GCT precursor
HisJ GCA GAC AGC CGC CTG GTC GTT GCA AAG AAT TCT GAC ATT CAA CCT
ACC (Escherichia GTG GAA TCG CTG AAGGGC AAG CGC GTA GGG GTC TTG CAG
GGC ACT ACT coli O145: H28 CAG GAA ACA TTT GGG AAC GAA CAT TGG GCG
CCT AAG GGA ATT GAG AT str. C GTG TCT TAT CAG GGT CAGGAT AAC ATC
TAC AGT GAT CTG ACA GCC GG RM12581]) A CGT ATT GAC GCC GCT TTT CAG
GAC GAG GTG GCG GCA TCT GAA GGG T GenBank: TC TTA AAG CAG CCA GTC
GGC AAAGAC TAC AAA TTT GGT GGG CCG AGC G AHY71563.1 TG AAG GAC GAG
AAA TTG TTT GGG GTA GGA ACA GGG ATG GGC TTG CGT AAG GAG GAC AAT GAA
TTA CGT GAA GCTCTT AAT AAA GCC TTT GCT GAG ATG CGT GCG GAC GGG ACT
TAC GAA AAA CTT GCA AAA AAG TAT TTC GAC TTT GAC GTC TAC GGC GGT 95
Histidine ATG CTG TAT GGA TTC AGT GGC GTT ATC TTG CAG GGG GCT CTT
GTC ACT ABC TTA GAG TTA GCT ATC TCG TCC GTT GTG TTA GCT GTC ATT ATT
GGA CT transporter, T ATCGGG GCT GGT GGC AAA TTG AGT CAG AAC CGT
TTG AGC GGC CTT AT permease T TTT GAA GGG TAC ACA ACC TTA ATT CGC
GGA GTC CCA GAC TTA GTG C protein HisQ TG ATG TTGCTT ATT TTC TAT
GGT TTA CAG ATC GCT TTG AAT ACG GTT A (Escherichia CC GAG GCA ATG
GGG GTC GGC CAA ATC GAT ATC GAT CCT ATG GTG GCT coli O145: H28 GGA
ATC ATT ACTTTG GGC TTC ATT TAC GGG GCA TAT TTC ACG GAG ACG str. TTC
CGC GGA GCT TTC ATG GCC GTC CCG AAG GGC CAC ATT GAA GCG GCA
RM12581) ACA GCT TTT GGA TTCACT CGT GGG CAA GTT TTC CGT CGC ATC ATG
TTT GenBank: CCA GCG ATG ATG CGC TAT GCG CTT CCT GGG ATC GGG AAT
AAC TGG CA AHY71562.1 G GTA ATC TTA AAA TCG ACGGCT TTA GTC AGT TTA
TTG GGG TTG GAA GA T GTC GTA AAA GCG ACC CAG TTG GCT GGG AAA TCG
ACT TGG GAG CCC T TT TAC TTC GCT ATT GTG TGT GGCGTT ATT TAC TTA GTT
TTC ACT ACA G TA TCA AAC GGT GTG TTA TTG TTT TTG GAA CGT CGC TAC
AGC GTG GGT GTA AAG CGT GCT GAT TTG 96 hisP ATG TCC GAG AAC AAA TTA
AAT GTT ATC GAT TTG CAT AAG CGT TAT GGA (Escherichia GAG CAT GAA
GTG TTG AAA GGA GTG TCT CTT CAA GCA AAC GCG GGG GA coli EPEC C
GTAATT TCT ATC ATC GGA TCG TCT GGT TCT GGT AAG TCA ACC TTC CT
C342-62) G CGT TGT ATT AAC TTC TTA GAG AAG CCG TCT GAG GGT TCT ATT
GTA G GenBank: TT AAT GGGCAG ACC ATC AAT CTT GTG CGC GAT AAG GAC
GGC CAG TTG A EIQ70323.1 AA GTG GCA GAC AAA AAC CAA CTT CGT TTG CTT
CGC ACC CGT CTT ACC ATG GTA TTC CAACAC TTC AAC CTG TGG TCG CAC ATG
ACG GTA CTT GAG AAC GTG ATG GAA GCG CCA ATT CAG GTA CTT GGA TTG AGC
AAA CAA GAA GCC CGC GAA CGT GCGGTG AAA TAT TTG GCC AAG GTG GGT ATC
GAC GAG CGT GCG CAG GGC AAA TAC CCC GTT CAC TTG TCC GGG GGT CAA CAA
CA G CGT GTC AGT ATT GCC CGCGCT CTG GCT ATG GAA CCA GAG GTG CTT CT
G TTT GAC GAG CCG ACG TCA GCT TTG GAC CCG GAA TTA GTG GGC GAA G TA
TTG CGC ATC ATG CAG CAG TTAGCA GAA GAA GGC AAG ACC ATG GTT G TT GTC
ACA CAC GAA ATG GGG TTT GCG CGT CAT GTC TCG ACT CAT GTA ATC TTC TTG
CAT CAA GGT AAA ATC GAGGAA GAA GGA GCG CCG GAA CAG TTA TTC GGG AAT
CCT CAA TCC CCC CGT CTG CAG CAG TTT CTT AAA GGG TCC TTA AAG 97
proline ATG TCA ATG TCC GCT GAG CAC GCT GAG GAA TTA AAA AAT GAA CCT
GCG reductase GTC GTT TGT TGT CGC ACT GAG GAG GGG ACC ATC TTG TCA
GCC GAT AA (Clostridium T TTGGAA GAC CCA AAC ATT TTT CCA GAT ATG
GTG GAT AGC GGT TTA CT botulinum) G AAC ATT CCT GGG GAC TGC TTA AAA
GTT GGG GAA GTA ATC GGG GCC A NCBI AA CTG CTTAAG ACG ATT GAC TCT
TTG ACC CCT CTT GCC AAG GAC ATC A Reference TT GAG GGG GCC AAA TCC
TTA GAC GGA GAC GTA CGC AGT AAA TCA GAG Sequence: ATT CAG ATC
GAATCA CCA GAG GAG AAG GCG ATC CTT AAA AAC AAT TTG WP_024933653.1
AAG GCG GGA GAT ATT ATC AAG GTT GAG GAC CTG GAG AAC CCT ATG CAC TTC
GCC AAG TTA CAAGAT TCG CTT CTT ATC AAG CTG GAT GAG AAA GTG CTT ACG
CGC CGC GAA GTT GTA GAC GCG AAA CTT ACG GAA GAT GCA CC G GCG ATT
TCA GGG GTC ACTGCA TCA ATG TTG GAA GGC TTC GAG GAA AA G GCC CTG GAG
ATT ACC CAA GAT AGC AAG GAT GTG GAC TTC AAT TCA G TA ATT CCA CTG
AAC GGC AAT CGTGAA TTC CTT CGT TTG AAA ATC GAG G AA GGC ACA GGC ATT
TAT ATC GAA ATT CCC TTT ACC CAA GTC 98 Proline porter ATG TCA GAA
AAA CTT CCG GCA CCT CGC GAG GGT TTA TCC GGT AAA GCT II ATG CGT CGT
GTT GTC ATG GGT AGC TTT GCC GGT GCA TTA ATG GAA TG (Escherichia G
TATGAT TTC TTC ATC TTT GGG ACG GCG GCG GGT CTT GTT TTT GCA CC coli
PMV- G CTG TTT TAT CCT GAC AGT GAT CCG TTT ATT GGG TTG ATC GCG TCG
T 1) TC GCT ACATTT GGA GTT GGT TTT TTG ACC CGC CCG TTA GGA GGT ATC
G GenBank: TG TTC GGT CAT TTT GGT GAC AAG ATC GGG CGT AAG ATT ACC
TTA ATC CDH67546.1 TGG ACA TTG GCGATT GTG GGG TGT TCT ACA TTC TTA
ATC GGT TTC ATT CCA ACG TAC CAA GAA ATC GGC ATT TGG GCC CCT TTG GTC
CTT ATG GTT TTG CGC CTG ATT CAGGGT TTT GGC TTG GGA GGA GAA TAC GGA
GGG GCG GCG TTA ATG ACC ATC GAA AGT GCC CCC GAA AGC CGC CGT GGT TTT
CT T GGG TCA TTG CCA CAG ACGGCC GCC AGC GTC GGC ATC ATG CTT GCA AC
G GGT ATT TTC GCG CTT TGT AAT CAT TTC CTT ACT TCT GAA CAG TTC T TA
TCA TGG GGC TGG CGT ATT CCCTTC TGG TTG TCC GCG GTT ATG TTA A TC GTC
GGA CTT TTT ATC CGT CTG CAT ACT GAA GAG ACG CTT GAC TTT CAG AAG CAA
AAA ACG ACT AAC AAT AAAGAA AAG TCG GTT CCC CCC CTT ATC GAG CTT TTT
AAG AAA CAT CCA CGC AAT ATT TTG TTG GCC TTG GGG GCC CGC CTT GCG GAG
TCA GTA AGC AGC AACATC ATT AAT GCA TTC GGC ATC GTC TAT ATC AGC AGT
CAA CTT GCA CTG AGC CGT GAC ATC CCC TT G ACT GGG ATG CTG ATT GCA
AGC GCC ATC GGA ATTTTT AGT TGC CCA TT G GTA GGC TGG CTT TCG GAC CGT
ATC GGA CAG AAA TCA TTA TAT TTG T CA GGC GCT GGA TTT TGT GTC CTG
TTT GCC TTC CCG TTTTTT CTG CTG T TG GAC TCG AAG AGT ACA CTG ATT ATC
TGG TGC TCA ATG ATT TTG GGC TAT AAC TTG GGT CCA ACT ATG ATG TTT GCT
GTA CAA CCA ACATTG TTT ACT CGT ATG TTC GGC ACC AAG GTC CGC TAC ACA
GGC TTA TCA TTT GCT TAC CAG TTC TCG GCT ATC TTA GGC GGC CTG TCC CCA
CTG ATT GCATCC TCA CTT CTT GCG TTG GGG GGC GGC AAA CCC TGG TAT GTC
GCC TTG TT C CTT TTC GCT GTG TCC GTG TTA TCT TTC GTC TGT GTA TGG
TTA ATC G AGCCC ACA GAC GAA CAA GAG ACG GCT TCA TAC CGC TAC ATC CGC
GAA C AA AGT CAT GAG AAC 99 Escherichia
ATGAAAAACGCGTCAACCGTATCGGAAGATACTGCGTCGAATCAAGAGCCGACGCTTCATCGC
coli PheP
GGATTACATAACCGTCATATTCAACTGATTGCGTTGGGTGGCGCAATTGGTACTGGTCTGTTT
CTTGGCATTGGCCCGGCGATTCAGATGGCGGGTCCGGCTGTATTGCTGGGCTACGGCGTCGCC
GGGATCATCGCTTTCCTGATTATGCGCCAGCTTGGCGAAATGGTGGTTGAGGAGCCGGTATCC
GGTTCATTTGCCCACTTTGCCTATAAATACTGGGGACCGTTTGCGGGCTTCCTCTCTGGCTGG
AACTACTGGGTAATGTTCGTGCTGGTGGGAATGGCAGAGCTGACCGCTGCGGGCATCTATATG
CAGTACTGGTTCCCGGATGTTCCAACGTGGATTTGGGCTGCCGCCTTCTTTATTATCATCAAC
GCCGTTAACCTGGTGAACGTGCGCTTATATGGCGAAACCGAGTTCTGGTTTGCGTTGATTAAA
GTGCTGGCAATCATCGGTATGATCGGCTTTGGCCTGTGGCTGCTGTTTTCTGGTCACGGCGGC
GAGAAAGCCAGTATCGACAACCTCTGGCGCTACGGTGGTTTCTTCGCCACCGGCTGGAATGGG
CTGATTTTGTCGCTGGCGGTAATTATGTTCTCCTTCGGCGGTCTGGAGCTGATTGGGATTACT
GCCGCTGAAGCGCGCGATCCGGAAAAAAGCATTCCAAAAGCGGTAAATCAGGTGGTGTATCGC
ATCCTGCTGTTTTACATCGGTTCACTGGTGGTTTTACTGGCGCTCTATCCGTGGGTGGAAGTG
AAATCCAACAGTAGCCCGTTTGTGATGATTTTCCATAATCTCGACAGCAACGTGGTAGCTTCT
GCGCTGAACTTCGTCATTCTGGTAGCATCGCTGTCAGTGTATAACAGCGGGGTTTACTCTAAC
AGCCGCATGCTGTTTGGCCTTTCTGTGCAGGGTAATGCGCCGAAGTTTTTGACTCGCGTCAGC
CGTCGCGGTGTGCCGATTAACTCGCTGATGCTTTCCGGAGCGATCACTTCGCTGGTGGTGTTA
ATCAACTATCTGCTGCCGCAAAAAGCGTTTGGTCTGCTGATGGCGCTGGTGGTAGCAACGCTG
CTGTTGAACTGGATTATGATCTGTCTGGCGCATCTGCGTTTTCGTGCAGCGATGCGACGTCAG
GGGCGTGAAACACAGTTTAAGGCGCTGCTCTATCCGTTCGGCAACTATCTCTGCATTGCCTTC
CTCGGCATGATTTTGCTGCTGATGTGCACGATGGATGATATGCGCTTGTCAGCGATCCTGCTG
CCGGTGTGGATTGTATTCCTGTTTATGGCATTTAAAACGCTGCGTCGGAAATAA 100 Anabaena
ATGAAAACACTATCACAGGCCCAATCTAAAACTTCTTCACAGCAATTCAGCTTTACCGGGAAC
variabilis
TCGTCTGCGAATGTAATTATCGGCAATCAAAAGCTGACCATTAATGATGTAGCTCGCGTTGCC
PAL1
CGGAATGGCACTTTGGTGTCACTGACGAACAATACCGACATTCTGCAAGGTATTCAAGCTAGC
TGCGATTATATCAATAACGCCGTTGAATCTGGCGAGCCAATCTACGGGGTAACAAGCGGTTTT
GGTGGGATGGCGAACGTTGCCATTAGCCGTGAACAGGCGAGCGAACTTCAGACCAACCTCGTT
TGGTTCCTAAAGACAGGAGCTGGTAATAAGTTACCTCTGGCTGACGTAAGAGCCGCGATGCTG
CTTCGCGCTAATAGTCACATGCGCGGCGCCAGTGGTATCCGTCTTGAGCTTATCAAGAGGATG
GAAATCTTCCTCAACGCGGGTGTCACACCATATGTTTATGAGTTTGGTAGTATCGGAGCCAGT
GGTGATCTTGTTCCCCTGAGTTATATTACGGGTTCATTGATTGGTTTAGACCCGTCCTTTAAA
GTGGATTTTAACGGGAAAGAAATGGACGCCCCGACCGCTTTACGACAGCTTAATCTGAGCCCA
CTTACTTTGCTCCCTAAAGAAGGTCTTGCCATGATGAATGGCACCTCTGTGATGACTGGAATT
GCCGCGAATTGTGTGTATGACACGCAGATCCTAACGGCCATTGCCATGGGTGTTCACGCGTTG
GACATTCAAGCCCTGAATGGTACAAACCAGTCGTTTCATCCGTTTATCCATAATTCAAAACCC
CATCCGGGACAGCTTTGGGCTGCTGATCAGATGATCTCACTCCTGGCCAATAGTCAACTGGTT
CGGGACGAGCTCGACGGCAAACATGATTATCGCGATCATGAGCTCATCCAGGACCGGTATTCA
CTTCGTTGTCTCCCACAATACCTGGGGCCTATCGTTGATGGTATATCTCAAATTGCGAAGCAA
ATTGAAATTGAGATCAATAGCGTAACCGACAACCCGCTTATCGATGTTGATAATCAGGCCTCT
TATCACGGTGGCAATTTTCTGGGCCAGTATGTTGGTATGGGGATGGATCACCTGCGGTACTAT
ATTGGGCTTCTGGCTAAACATCTTGATGTGCAGATTGCCTTATTAGCTTCACCAGAATTTTCA
AATGGACTGCCGCCATCATTGCTCGGTAACAGAGAAAGGAAAGTAAATATGGGCCTTAAGGGC
CTTCAGATATGTGGTAACTCAATCATGCCCCTCCTGACCTTTTATGGGAACTCAATTGCTGAT
CGTTTTCCGACACATGCTGAACAGTTTAACCAAAACATTAACTCACAGGGCTATACATCCGCG
ACGTTAGCGCGTCGGTCCGTGGATATCTTCCAGAATTATGTTGCTATCGCTCTGATGTTCGGC
GTACAGGCCGTTGATTTGCGCACTTATAAAAAAACCGGTCACTACGATGCTCGGGCTTGCCTG
TCGCCTGCCACCGAGCGGCTTTATAGCGCCGTACGTCATGTTGTGGGTCAGAAACCGACGTCG
GACCGCCCCTATATTTGGAATGATAATGAACAAGGGCTGGATGAACACATCGCCCGGATATCT
GCCGATATTGCCGCCGGAGGTGTCATCGTCCAGGCGGTACAAGACATACTTCCTTGCCTGCAT TAA
101 Photorhabdus
ATGAAAGCTAAAGATGTTCAGCCAACCATTATTATTAATAAAAATGGCCTTATCTCTTTGGAA
luminescens
GATATCTATGACATTGCGATAAAACAAAAAAAAGTAGAAATATCAACGGAGATCACTGAACTT
PAL3
TTGACGCATGGTCGTGAAAAATTAGAGGAAAAATTAAATTCAGGAGAGGTTATATATGGAATC
AATACAGGATTTGGAGGGAATGCCAATTTAGTTGTGCCATTTGAGAAAATCGCAGAGCATCAG
CAAAATCTGTTAACTTTTCTTTCTGCTGGTACTGGGGACTATATGTCCAAACCTTGTATTAAA
GCGTCACAATTTACTATGTTACTTTCTGTTTGCAAAGGTTGGTCTGCAACCAGACCAATTGTC
GCTCAAGCAATTGTTGATCATATTAATCATGACATTGTTCCTCTGGTTCCTCGCTATGGCTCA
GTGGGTGCAAGCGGTGATTTAATTCCTTTATCTTATATTGCACGAGCATTATGTGGTATCGGC
AAAGTTTATTATATGGGCGCAGAAATTGACGCTGCTGAAGCAATTAAACGTGCAGGGTTGACA
CCATTATCGTTAAAAGCCAAAGAAGGTCTTGCTCTGATTAACGGCACCCGGGTAATGTCAGGA
ATCAGTGCAATCACCGTCATTAAACTGGAAAAACTATTTAAAGCCTCAATTTCTGCGATTGCC
CTTGCTGTTGAAGCATTACTTGCATCTCATGAACATTATGATGCCCGGATTCAACAAGTAAAA
AATCATCCTGGTCAAAACGCGGTGGCAAGTGCATTGCGTAATTTATTGGCAGGTTCAACGCAG
GTTAATCTATTATCTGGGGTTAAAGAACAAGCCAATAAAGCTTGTCGTCATCAAGAAATTACC
CAACTAAATGATACCTTACAGGAAGTTTATTCAATTCGCTGTGCACCACAAGTATTAGGTATA
GTGCCAGAATCTTTAGCTACCGCTCGGAAAATATTGGAACGGGAAGTTATCTCAGCTAATGAT
AATCCATTGATAGATCCAGAAAATGGCGATGTTCTACACGGTGGAAATTTTATGGGGCAATAT
GTCGCCCGAACAATGGATGCATTAAAACTGGATATTGCTTTAATTGCCAATCATCTTCACGCC
ATTGTGGCTCTTATGATGGATAACCGTTTCTCTCGTGGATTACCTAATTCACTGAGTCCGACA
CCCGGCATGTATCAAGGTTTTAAAGGCGTCCAACTTTCTCAAACCGCTTTAGTTGCTGCAATT
CGCCATGATTGTGCTGCATCAGGTATTCATACCCTCGCCACAGAACAATACAATCAAGATATT
GTCAGTTTAGGTCTGCATGCCGCTCAAGATGTTTTAGAGATGGAGCAGAAATTACGCAATATT
GTTTCAATGACAATTCTGGTAGTTTGTCAGGCCATTCATCTTCGCGGCAATATTAGTGAAATT
GCGCCTGAAACTGCTAAATTTTACCATGCAGTACGCGAAATCAGTTCTCCTTTGATCACTGAT
CGTGCGTTGGATGAAGATATAATCCGCATTGCGGATGCAATTATTAATGATCAACTTCCTCTG
CCAGAAATCATGCTGGAAGAATAA 102 Legionella
ATGGAGTTTAGTAGCCGGTATGTCGCACATGTCCCTGATGCTCAGGGTTTAGTCGATTATTCG
pneumophila
GCACAAGAAAATAGAATTTGGAATATTTTATTTGAGAGGCAACTCAAGTTATTGCCAGGAAGA
phhA
GCTTGTGATGAATTTCTGTCTGGATTACAGACTTTAGGACTTAACTCCTCGACTATTCCACAA
CTTCCAGAAGTAAGTGAGCGATTAAAGGCCAAAACGGGATGGCAAGTAGCGCCAGTTGCTGCT
TTAATTTCAGCCAGGGAATTTTTTGAATTATTAGCAGAAAAATATTTTCCTGCGGCGACTTTT
ATTCGAAGTGAAGAAGAATTGGATTATGTTCAAGAACCTGATATTTTTCATGAGCTTTTTGGT
CATTGTCCTATGTTAACCGATAGAGTCTATGCTGAATTTGTCCATGATTACGCATGTAAGGTA
TTAACTTTTCCTGAACAGGATTGGCCTTTATTGCAAAGAATGTTTTGGTTTACTGTAGAGTTT
GGATTGATTAAAACGCCTAAAGGGCTTAGAGCATACGGCGGGGGAATTTTATCTTCTATCAGT
GAAACGGTATATTGTGTGGAAAGTGATATTCCTGTGCGAATTTTATTTGATCCAGTGGTGGCT
TTTCGAATGCCTTATCGGATTGACCAGCTACAACCTGTTTATTTCGTTATTGACAGCTATCAA
AATTTATATGATTTCGTGCTTTCTGACATGGGTAAATTCATGGATCGTGCGCGAGAGTTAGGT
GAATTTCCACCGTATTTTGATGTGGATCCGGATAATCCAAATATTCATATAAGGGCTTGTTAA 103
Escherichia
GTGATCGAAATCTTACATGAATACTGGAAACCGCTGCTGTGGACCGACGGTTATCGCTTTACT
coli hisM
GGTGTGGCGATCACTCTGTGGCTGCTTATTTTGTCGGTAGTGATAGGCGGAGTCCTGGCGCTG
TTTCTGGCGATTGGTCGTGTCTCCAGTAATAAATACATCCAGTTTCCAATCTGGTTATTTACC
TATATTTTTCGCGGTACGCCGCTGTATGTTCAGTTGCTGGTGTTCTATTCCGGCATGTACACG
CTTGAGATTGTTAAGGGAACCGAATTCCTTAACGCTTTCTTCCGCAGTGGCCTGAACTGTACC
GTGCTGGCGCTGACGCTTAACACCTGCGCTTACACTACCGAGATTTTTGCTGGGGCAATCCGT
TCGGTTCCGCATGGGGAAATTGAAGCCGCCAGAGCCTATGGCTTCTCGACTTTTAAAATGTAT
CGCTGCATTATTTTGCCTTCTGCGCTGCGTATTGCGTTACCGGCATACAGCAACGAAGTGATC
CTGATGCTGCACTCTACTGCGTTGGCATTTACTGCCACGGTGCCGGATCTGCTGAAAATAGCC
CGCGATATTAACGCCGCCACGTATCAACCTTTTACCGCCTTCGGCATTGCCGCGGTGCTCTAT
TTAATCATCTCTTATGTCCTGATCAGCCTCTTTCGCAGAGCGGAAAAACGCTGGTTGCAGCAT
GTGAAACCTTCTTCAACGCACTGA 104 clbA (wild-
caaatatcacataatcttaacatatcaataaacacagtaaagtttcatgtgaaaaacatcaaa
type)
cataaaatacaagctcggaatacgaatcacgctatacacattgctaacaggaatgagattatc
taaatgaggattgatatattaattggacatactagtttttttcatcaaaccagtagagataac
ttccttcactatctcaatgaggaagaaataaaacgctatgatcagtttcattttgtgagtgat
aaagaactctatattttaagccgtatcctgctcaaaacagcactaaaaagatatcaacctgat
gtctcattacaatcatggcaatttagtacgtgcaaatatggcaaaccatttatagtttttcct
cagttggcaaaaaagattttttttaacctttcccatactatagatacagtagccgttgctatt
agttctcactgcgagcttggtgtcgatattgaacaaataagagatttagacaactcttatctg
aatatcagtcagcatttttttactccacaggaagctactaacatagtttcacttcctcgttat
gaaggtcaattacttttttggaaaatgtggacgctcaaagaagcttacatcaaatatcgaggt
aaaggcctatctttaggactggattgtattgaatttcatttaacaaataaaaaactaacttca
aaatatagaggttcacctgtttatttctctcaatggaaaatatgtaactcatttctcgcatta
gcctctccactcatcacccctaaaataactattgagctatttcctatgcagtcccaactttat
caccacgactatcagctaattcattcgtcaaatgggcagaattgaatcgccacggataatcta
gacacttctgagccgtcgataatattgattttcatattccgtcggtggtgtaagtatcccgca
taatcgtgccattcacatttag 105 clbA knock-
ggatggggggaaacatggataagttcaaagaaaaaaacccgttatctctgcgtgaaagacaag out
tattgcgcatgctggcacaaggtgatgagtactctcaaatatcacataatcttaacatatcaa
taaacacagtaaagtttcatgtgaaaaacatcaaacataaaatacaagctcggaatacgaatc
acgctatacacattgctaacaggaatgagattatctaaatgaggattgaTGTGTAGGCTGGAG
CTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGGAATAGG
AACTAAGGAGGATATTCATATGtcgtcaaatgggcagaattgaatcgccacggataatctaga
cacttctgagccgtcgataatattgattttcatattccgtcggtgg 106 Prp
TTACCCGTCTGGATTTTCAGTACGCGCTTTTAAACGACGCCACAGCGTGGTACGGCTGATCCC
promoter
CAAATAACGTGCGGCGGCGCGCTTATCGCCATTAAAGCGTGCGAGCACCTCCTGCAATGGAAG
(prpR
CGCTTCTGCTGACGAGGGCGTGATTTCTGCTGTGGTCCCCACCAGTTCAGGTAATAATTGCCG
sequence -
CATAAATTGTCTGTCCAGTGTTGGTGCGGGATCGACGCTTAAAAAAAGCGCCAGGCGTTCCAT
underlined;
CATATTCCGCAGTTCGCGAATATTACCGGGCCAATGATAGTTCAGTAGAAGCGGCTGACACTG
Ribosome
CGTCAGCCCATGACGCACCGATTCGGTAAAAGGGATCTCCATCGCGGCCAGCGATTGTTTTAA
binding
AAAGTTTTCCGCCAGAGGCAGAATATCAGGCTGTCGCTCGCGCAAGGGGGGAAGCGGCAGACG
site -
CAGAATGCTCAAACGGTAAAACAGATCGGTACGAAAACGTCCTTGCGTTATCTCCCGATCCAG
lower case;
ATCGCAATGCGTGGCGCTGATCACCCGGACATCTACCGGGATCGGCTGATGCCCGCCAACGCG
start codon
GGTGACGGCTTTTTCCTCCAGTACGCGTAGAAGGCGGGTTTGTAACGGCAGCGGCATTTCGCC of
gene of
AATTTCGTCAAGAAACAGCGTGCCGCCGTGGGCGACCTCAAACAGCCCCGCACGTCCACCTCG
interest
TCTTGAGCCGGTAAACGCTCCCTCCTCATAGCCAAACAGTTCAGCCTCCAGCAACGACTCGGT
(italicized
AATCGCGCCGCAATTAACGGCGACAAAGGGCGGAGAAGGCTTGTTCTGACGGTGGGGCTGACG
atg)
GTTAAACAACGCCTGATGAATCGCTTGCGCCGCCAGCTCTTTCCCGGTCCCTGTTTCCCCCTG
AATCAGCACTGCCGCGCGGGAACGGGCATAGAGTGTAATCGTATGGCGAACCTGCTCCATTTG
TGGTGAATCGCCGAGGATATCGCTCAGCGCATAACGGGTCTGTAATCCCTTGCTGGAGGTATG
CTGGCTATACTGACGCCGTGTCAGGCGGGTCATATCCAGCGCATCATGGAAAGCCTGACGTAC
GGTGGCCGCTGAATAAATAAAGATGGCGGTCATTCCTGCCTCTTCCGCCAGGTCGGTAATTAG
TCCTGCCCCAATTACAGCCTCAATGCCGTTAGCTTTGAGCTCGTTAATTTGCCCGCGAGCATC
CTCTTCAGTGATATAGCTTCGCTGTTCAAGACGGAGGTGAAACGTTTTCTGAAAGGCGACCAG
AGCCGGAATGGTCTCCTGATAGGTCACGATTCCCATTGAGGAAGTCAGCTTTCCCGCTTTTGC
CAGAGCCTGTAATACATCGAATCCGCTGGGTTTGATGAGGATGACAGGTACCGACAGTCGGCT
TTTTAAATAAGCGCCGTTGGAACCTGCCGCGATAATCGCGTCGCAGCGTTCGGTTGCCAGTTT
TTTGCGAATGTAGGCTACTGCCTTTTCAAAACCGAGCTGAATAGGCGTGATCGTCGCCAGATG
ATCAAACTCCAGGCTGATATCCCGAAATAGTTCGAACAGGCGCGTTACCGAGACCGTCCAGAT
CACCGGTTTATCGCTATTATCGCGCGAAGCGCTATGCACAGTAACCATCGTCGTAGATTCATG
TTTAAGGAACGAATTCTTGTTTTATAGATGTTTCGTTAATGTTGCAATGAAACACAGGCCTCC
GTTTCATGAAACGTTAGCTGACTCGTTTTTCTTGTGACTCGTCTGTCAGTATTAAAAAAGATT
TTTCATTTAACTGATTGTTTTTAAATTGAATTTTATTTAATGGTTTCTCGGTTTTTGGGTCTG
GCATATCCCTTGCTTTAATGAGTGCATCTTAATTAACAATTCAATAACAAGAGGGCTGAATag
taatttcaacaaaataacgagcattcgaatg 107 Tsx -
Atgaaaaaaactttactcgcagtcagcgcagcgctggcgctcacctcatcttttactgctaac
Salmonella
gcagcagaaaatgatcagccgcagtatttgtccgactggtggcaccagagcgtaaacgtggta
enterica
ggcagctaccatacccgtttctcgccgaaattgaacaacgacgtctatctggaatatgaagca
subsp.
tttgccaaaaaagactggtttgatttctacggctatatcgatattcccaaaacctttgattgg
enterica
ggtaacggcaacgataaaggtatctggtccgacggttctccgctgttcatggaaatcgaaccg
serovar
cgtttctcaattgataagctgaccggcgcagacctgagcttcggcccgtttaaagagtggtat
Typhimurium
ttcgccaacaactacatctacgatatgggcgataacaaagccagccgccagagcacgtggtat LT2
atgggtctggggaccgatatcgacaccggcctgccgatgggtctgtcgctgaacgtgtatgcg
(STM0413)
aaatatcagtggcaaaactacggcgcgtccaatgaaaacgaatgggacggctaccgtttcaaa
gtgaaatacttcgtccccatcaccgatctgtggggcggtaaactgagctatatcggctttacc
aactttgactggggatctgatttaggcgacgatccgaaccgtaccagcaactccatcgcttcc
agccatatcctggcgctgaactacgatcactggcactactcggtcgttgcgcgttacttccat
aacggcggacagtggcagaatggcgcaaaactgaactggggcgacggcgatttcagcgcgaaa
tctaccggctggggcggctacctggtcgtgggttacaacttctaa 136 Tsx -
MKKTLLAVSAALALTSSFTANAAENDQPQYLSDWWHQSVNVVGSYHTRFSPKLNNDVYLEYEA
Salmonella
FAKKDWFDFYGYIDIPKTFDWGNGNDKGIWSDGSPLFMEIEPRFSIDKLTGADLSFGPFKEWY
enterica
FANNYIYDMGDNKASRQSTWYMGLGTDIDTGLPMGLSLNVYAKYQWQNYGASNENEWDGYRFK
subsp.
VKYFVPITDLWGGKLSYIGFTNFDWGSDLGDDPNRTSNSIASSHILALNYDHWHYSVVARYFH
enterica NGGQWQNGAKLNWGDGDFSAKSTGWGGYLVVGYNF serovar Typhimurium
LT2 (STM0413) 108 Tsx -
atgaaaaaaacattactggcagccggtgcggtactggcgctctcttcgtcttttactgtc
Escherichia
aacgcagctgaaaacgacaaaccgcagtatctttccgactggtggcaccagagcgttaac coli
K-12 gttgtcggaagctatcacacccgtttcggaccgcagatccgcaacgatacctaccttgag
MG1655 tacgaagcattcgctaaaaaagactggttcgacttctatggttatgcggatgcgccggta
(b0411)
ttcttcggcggtaactccgatgctaaaggtatctggaaccacggttctccgctgtttatg
gaaatcgaaccacgtttctccatcgacaagctgaccaatactgaccttagcttcggtccg
ttcaaagagtggtacttcgcgaacaactacatttacgacatgggtcgtaataaagatggt
cgccagagcacctggtacatgggtctgggtaccgatatcgacactggcctgccgatgagc
ctgtccatgaacgtctatgcgaaataccagtggcagaactatggcgcagcgaacgaaaac
gagtgggacggttaccgtttcaaaattaaatactttgtgccgattaccgatctgtggggc
ggtcagctgagctacatcggcttcaccaacttcgactggggttccgatttaggggatgac
agcggtaacgcaatcaacggtattaagacccgtactaataactctatcgcttccagccat
attctggctctgaactacgatcactggcactactctgtcgtagctcgttactggcacgac
ggtggtcagtggaacgacgatgcagaactgaacttcggcaacggcaacttcaacgttcgc
tctaccggctggggtggttacctggtagtaggttacaacttctga 109 BH1446 -
atgaatattttgtggggtttattaggaatcgtcgttgtttttctaatcgcttttgcattttcc
Bacillus
acaaatcgtcgtgcaattaaaccacgaacgatattaggtggtctcgcgattcagctattattt
halodurans
gcgattattgtattaaaaattccagctggacaagcgttacttgagagcttaaccaatgtagtt
(BAB05165)
ttgaacattattagttatgcgaatgaagggatcgacttcgtatttggtggatttttcgaagaa
ggttcaggcgtaggcttcgtttttgcaattaacgttttgtctgtcgtcattttcttctcagca
ctaatctcgatcctttattatttagggatcatgcaatttgtcattaaaattatcggtggtgcg
ctgtcctggctactcggaacatcaaaggcagaatcaatgtcagcagcagctaacattttcgtt
gggcaaacggaagcgccactcgttgttaagccatacttaccaaaaatgacgcaatccgagctc
tttgcggttatgaccgggggacttgcttctgttgctggttctgttttaatcggttattctctt
ttaggagtaccgctacaatatttattagcggcaagctttatggctgctcctgcgggcttgatt
atggcgaaaatgatcatgcctgaaacggagaaaacaaccgatgcagaagatgactttaagctc
gcaaaggatgaagagtccacgaacttgattgacgcggccgccaatggggcgagcactgggtta
atgctcgttctaaatattgcggcgatgttactagcgttcgttgcattgattgcattaattaat
ggaattcttggatggatcggaggattgtttggggcgtcgcaattgtctttagagttaatcctc
ggatacgtgtttgctccgcttgcgtttgtcatcggaattccttgggctgaagcgcttcaagcg
ggaagctacatcggacagaaactcgtagtgaacgaatttgttgcctacttaagctttgcacca
gaaattgaaaacctttcagataaagcggtgatggtgattagttttgccctttgcggatttgct
aacttctcatccctcggaatccttttaggaggattgggtaagcttgctccgagccgtcgccct
gatattgcccgtctcggattacgcgcgatccttgcaggtacgctagcttctttactcagcgcc
tccattgcgggaatgttattctaa 137 BH1446 -
MNILWGLLGIVVVFLIAFAFSTNRRAIKPRTILGGLAIQLLFAIIVLKIPAGQALLESLTNVV
Bacillus
LNIISYANEGIDFVFGGFFEEGSGVGFVFAINVLSVVIFFSALISILYYLGIMQFVIKIIGGA
halodurans
LSWLLGTSKAESMSAAANIFVGQTEAPLVVKPYLPKMIQSELFAVMTGGLASVAGSVLIGYSL
(BAB05165)
LGVPLQYLLAASFMAAPAGLIMAKMIMPETEKTTDAEDDFKLAKDEESTNLIDAAANGASTGL
MLVLNIAAMLLAFVALIALINGILGWIGGLFGASQLSLELILGYVFAPLAFVIGIPWAEALQA
GSYIGQKLVVNEFVAYLSFAPEIENLSDKAVMVISFALCGFANFSSLGILLGGLGKLAPSRRP
DIARLGLRAILAGTLASLLSASIAGMLF 110 nupC -
atgaagtatttgattgggattatcggtttaatcgtgtttttaggcctcgcgtggatcgcgagc
Bacillus
agcggcaaaaaaagaattaagatccgcccaattgttgttatgctcattttgcaatttattctt
Subtilis
ggctacattctcctcaataccggaatagggaatttcctcgtgggaggatttgcaaaaggattc
subsp.
ggttacctgcttgaatacgcggcagagggaattaactttgtgtttggcggcttggtgaatgcg
subtilis
gaccaaacgacattctttatgaatgttctcttgccaatcgtgtttatttccgctctgatcggg 168
attctgcaaaagtggaaagtcctcccgtttatcattagatatatcggccttgccctcagcaag
(BSU39410;
gtaaacggtatgggaagattggaatcgtataacgcagtggcttctgcgattttagggcagtca
CAA57663 )
gaagtatttatctccttgaagaaagaactcggtcttttaaatcagcagcgcttgtacacgctt
tgcgcatctgcgatgtcaactgtatcaatgtcgattgtcggtgcgtatatgacaatgctgaaa
ccggaatatgttgtaacagcgcttgttttgaacttatttggcggtttcattatcgcttctatt
atcaatccgtacgaggttgcaaaagaagaggatatgcttcgtgttgaggaagaagaaaaacaa
tccttcttcgaagtgctcggagaatacattcttgacggtttcaaagtagcggttgtcgtcgct
gcgatgctgattggatttgtcgcgattattgcattgatcaatggcatttttaatgcagtattc
ggtatttcgttccaaggcattcttggatatgtgtttgctccattcgcttttcttgtcggtatc
ccatggaatgaagctgttaatgcgggaagcattatggcaacaaaaatggtatcgaatgaattt
gtcgccatgacgtcgcttacgcaaaacggtttccatttcagcggccgtacaacagcgatcgta
tcggtattccttgtgtcatttgcgaacttctcctcaatcggaatcattgccggtgccgtaaaa
ggactgaatgaaaagcaaggaaatgtcgtcgctcgtttcggcttgaaattattatacggtgct
acgcttgtcagctttttatcagcagcaattgtgggcttgatttactga 138
MKYLIGIIGLIVFLGLAWIASSGKKRIKIRPIVVMLILQFILGYILLNTGIGNFLVGGFAKGF
GYLLEYAAEGINFVFGGLVNADQTTFFMNVLLPIVFISALIGILQKWKVLPFIIRYIGLALSK
VNGMGRLESYNAVASAILGQSEVFISLKKELGLLNQQRLYTLCASAMSTVSMSIVGAYMTMLK
PEYVVTALVLNLFGGFIIASIINPYEVAKEEDMLRVEEEEKQSFFEVLGEYILDGFKVAVVVA
AMLIGFVAIIALINGIFNAVFGISFQGILGYVFAPFAFLVGIPWNEAVNAGSIMATKMVSNEF
VAMTSLIQNGFHFSGRTTAIVSVFLVSFANFSSIGIIAGAVKGLNEKQGNVVARFGLKLLYGA
TLVSFLSAAIVGLIY 111 yutK -
atgaatgttctgtgggggctgctgggcgcagttgcgatcattgctatcgcgtttttattttca
Bacillus
gaaaagaaaagcaatattaagataagaaccgtcatcgttggtttatgcacacaggtggcgttt
Subtilis
ggatacatcgtgttgaaatgggaagcgggacgcgctgtttttttatggttttcaagccgtgta
subsp.
cagcttctgattgactatgcgaatgaaggcatcagttttatttttggaccgcttctaaaggtc
subtilis
ggagacagtccggcatttgcattaagtgtactgcccgttatcattttcttctcagcactgatt
168:
gcagttttatatcatttgaaaatcatgcagctcgttttccgtgtcattggcggcggattgtcg
BSU32180
aagctccttggaacaagcaaaacggaatctctggcggctgctgccaatatttttgtaggacaa
tcagaatctccgttagtgatcaaacccctgattgccgggctgacgcgctctgagttgtttacg
attatgacgagcggtctatcggcagttgcgggatctaccttgtttgggtacgcgcttctcggt
attccgattgagtacttgctggcggccagctttatggctgctccagctggactagtctttggt
aaattgattatacccgaaacggaaaaaacgcaaaccgtaaaaagcgatttcaaaatggatgaa
ggcgaaggcgcagccaatgtcattgacgcagctgcaaagggagcgtcaacaggactgcaaatt
gcgttaaatgttggggcgatgctgcttgcgtttgttgcgttaatcgctgtagtaaacggtatt
ctcggcggggctttcggcttgttcggtttaaaaggcgtaacattagaatccattctcggctat
gtgttttctcctatcgcctttttgattggcgtgccttggcatgaagcattgcaggcgggaagc
tatatcggccagaaattggtgctgaatgagtttgtcgcttattctaacttcggttcgcacatc
ggcgagttttctaagaaaactgctaccattatcagtttcgcgttatgcggattcgccaatttt
tcatcaattgcgattatgcttggtacgcttggcggtttagcgcccagccgccgttcagatatc
gcacgtctcggcctgaaggctgttcttgcaggaacattagccaatctgctcagcgcagccatt
gccggcatgtttatataa 139
MNVLWGLLGAVAIIAIAFLFSEKKSNIKIRTVIVGLCTQVAFGYIVLKWEAGRAVFLWFSSRV
QLLIDYANEGISFIFGPLLKVGDSPAFALSVLPVIIFFSALIAVLYHLKIMQLVFRVIGGGLS
KLLGTSKTESLAAAANIFVGQSESPLVIKPLIAGLTRSELFTIMTSGLSAVAGSTLFGYALLG
IPIEYLLAASFMAAPAGLVFGKLIIPETEKTQTVKSDFKMDEGEGAANVIDAAAKGASTGLQI
ALNVGAMLLAFVALIAVVNGILGGAFGLFGLKGVTLESILGYVFSPIAFLIGVPWHEALQAGS
YIGQKLVLNEFVAYSNFGSHIGEFSKKTATIISFALCGFANFSSIAIMLGTLGGLAPSRRSDI
ARLGLKAVLAGTLANLLSAAIAGMFI 112 yxjA -
atgtactttttattaaaccttgtcggtctcattgtgattatggcagttgtgttcctatgctcc
Bacillus
ccgcagaaaaagaaaatccagtggcgtccgatcattacgttaattgttctggaattgctgatt
Subtilis
acttggtttatgctgggaacaaaggtcgggagctgggccatcggtaaaattggtgatttcttc
subsp.
acttggctgattgcttgcgccagtgacggtatcgcgtttgccttcccgtcagtcatggcgaat
spizizenii
gaaacagtagactttttctttagtgcacttcttccaattatctttatcgtcacattctttgat W23
attttaacatatttcggcattttgccttggctgattgataaaatcggatgggtgatttcaaag
(BSUW23_19355)
gcttcccgcttgccgaaattagaaagctttttctctattcaaatgatgttcttgggaaatact
gaagcacttgcggtcatccgccagcagcttacggtattaaataacaaccgcttgcttacattt
ggcttaatgagcatgagcagcatcagcggctccattattggatcttacctgtcaatggtgccg
gcgacatacgtgtttacagcgattccattgaactgcttaaacgcgctgattattgcaaacctg
ctgaaccctgttcatgtgccggaggatgaagatatcatctatacaccgcctaaagaagagaag
aaagactttttctctacgatttctaacagtatgcttgtcggcatgaacatggttatcgttatt
ttggcaatggtgatcggatatgtagcattaacgtctgcagtcaatggcattcttggtgttttc
gtacacggcctgaccatccagacaatttttgcttatctcttcagtccgttcgcattcctgctt
ggtctgccagtacatgatgcaatgtatgtcgctcagctaatgggaatgaaattggcaacgaac
gagtttgttgcgatgcttgacttgaaaaacaatcttacaacacttccgcctcacacagttgcg
gtggcgacgacattcctgacgtcatttgccaacttcagtactgtcggcatgatttacggaacg
tacaactcgatccttgacggcgaaaagtcaacggtcatcgggaaaaacgtgtggaaattgctc
gtcagcggcattgcggtatctttactaagtgctgcgattgtcggcctgtttgtgtggtag 140
yxjA -
MYFLLNLVGLIVIMAVVFLCSPQKKKIQWRPIITLIVLELLITWFMLGTKVGSWAIGKIGDFF
Bacillus
TWLIACASDGIAFAFPSVMANETVDFFFSALLPIIFIVTFFDILTYFGILPWLIDKIGWVISK
subtilis
ASRLPKLESFFSIQMMFLGNTEALAVIRQQLTVLNNNRLLTFGLMSMSSISGSIIGSYLSMVP
sus.
ATYVFTAIPLNCLNALIIANLLNPVHVPEDEDIIYTPPKEEKKDFFSTISNSMLVGMNMVIVI
spizizenii
LAMVIGYVALTSAVNGILGVFVHGLTIQTIFAYLFSPFAFLLGLPVHDAMYVAQLMGMKLATN W23
EFVAMLDLKNNLTTLPPHTVAVATTFLTSFANFSTVGMIYGTYNSILDGEKSTVIGKNVWKLL
(BSUW23_19355) VSGIAVSLLSAAIVGLFVW 113 ccCNT
atgttccgtcccgagaacgttcaggccctcgcgggtctggcgctcaccctgggcctgtgctgg
(CC2089) -
ctcgtttccgagaatcgcaagcggttcccctggggcctggccatcggcgcggtcgtcattcag
Caulobacter
gtcctgctggtcctggtcctgttcggcctgccgcaagcccagcagatgctgcgcggcgtcaac
crescentus
ggcgcggtggagggccttgccgcctcgacccaggccggcaccgccttcgtgttcggctttctg
CB15
gccggcggcgaccagccctatccggtcagcaatccgggcgcgggcttcatcttcgccttccgc
(AAK24060 )
gtgctgccggtgatcctggtggtctgcgccctgtcggcgctgctgtggcactggaagattctc
aagtggctggctcagggcttcggctttgtgttccagaagacgctgggcctgcgcggcccgccg
gccctggccaccgccgcgaccatcttcatgggtcaggtcgaggggccgatcttcatccgcgcc
tatctcgacaagctgagccgctcggaactcttcatgctgatcgcggtcggcatggcctgcgtg
tcgggctcgaccatggtcgcctacgccaccatcctggccgacgtcctgcccaacgccgccgcc
cacgtgctgaccgcctcgatcatctcggctccggccggcgtgctgctggcccggatcattgtg
ccgtccgatccgatggagaagagcgccgatcttgatctgtcgaccgaggacaagacctatggc
agctcgatcgacgccgtgatgaagggcaccaccgacggcctgcagatcgcgctgaacgtcggc
gccaccctgatcgtcttcgtggccctggccaccatggtcgacaaggtcctgggcgccttcccg
ccggtgggcggcgagccgctgagcatcgcgcgcggcctgggcgtggtcttcgcgccgctggcc
tggtcgatgggcatcccgtggaaagaagcgggcacggccggcggtctgctgggcgtgaagctg
atcctgaccgagttcaccgccttcatccagctgtccaaggtgggcgaagccctgctggacgaa
cgcacccggatgatcatgacctacgctctgtgcggtttcgccaatatcggctcggtcggcatg
aacgtcgccggcttctcggtgctggtgccccagcgccggcaggaagtgctgggcctggtctgg
aaggcgatgatggccggcttcctggccacctgcctgaccgcctcgctggtcggcctgatgccg
cgaagcctgtttgggctgtaa 141 ccCNT
MFRPENVQALAGLALTLGLCWLVSENRKRFPWGLAIGAVVIQVLLVLVLFGLPQAQQMLRGVN
(CC2089) -
GAVEGLAASTQAGTAFVFGFLAGGDQPYPVSNPGAGFIFAFRVLPVILVVCALSALLWHWKIL
Caulobacter
KWLAQGFGFVFQKTLGLRGPPALATAATIFMGQVEGPIFIRAYLDKLSRSELFMLIAVGMACV
crescentus
SGSTMVAYATILADVLPNAAAHVLTASIISAPAGVLLARIIVPSDPMEKSADLDLSTEDKTYG
CB15
SSIDAVMKGTTDGLQIALNVGATLIVFVALATMVDKVLGAFPPVGGEPLSIARGLGVVFAPLA
(AAK24060)
WSMGIPWKEAGTAGGLLGVKLILTEFTAFIQLSKVGEALLDERTRMIMTYALCGFANIGSVGM
NVAGFSVLVPQRRQEVLGLVWKAMMAGFLATCLTASLVGLMPRSLFGL 114 yeiJ -
atggatgtcatgagaagtgttctgggaatggtggtattgctgacgattgcgtttttactgtca
Escherichia
gtaaacaagaagaagatcagcctgcgtaccgttggcgcggcgttagtgttacaggtcgtgatt
coli K-12
ggcggcattatgctttggttaccgccagggcgttgggtcgctgaaaaagtcgcttttggcgtg
W3110
cataaagtgatggcgtacagcgacgcgggtagcgcatttatcttcggttctctggtcggaccg
(AAC75222;
aaaatggataccttatttgatggtgcaggatttatctttggtttcagggtgttaccggcaatt
JW2148)
atcttcgtcaccgcgctggtgagtattctctactacatcggtgtgatggggattttaattcga
attctcggcggtatcttccagaaagcattaaatatcagcaagatcgagtcattcgtcgcggtc
accaccattttcctcgggcaaaacgaaattccggcaatcgtcaaaccctttatcgatcgtctg
aatcgcaatgaattatttacagcgatttgtagtggcatggcctcgattgctggttcgacaatg
attggttacgccgcactgggcgtgcctgtggaatatctgctggcggcatcattaatggcgatc
cctggcgggatcttgtttgcccgcctgttaagcccggcaacggaatcttcgcaggtttccttt
aataacctctctttcaccgaaacaccgccaaaaagcattattgaagccgctgcgacaggggca
atgaccgggctgaaaatcgccgcaggtgtggcaacagtggtgatggcatttgttgcaataatt
gcgttgattaacggtattatcggcggcgttggtggctggtttggttttgaacatgcctcgctg
gagtccattttaggttacctgctggctccactggcgtgggtgatgggtgtggactggagtgat
gcgaatcttgccgggagtttgattggacagaaactggcaataaatgaatttgtcgcttatctc
aatttctcaccctatctgcaaacggctggcactctcgatgctaaaactgtggcgattatttcc
ttcgcgttgtgcggtttcgctaactttggttctatcggggtggtggtgggggcgttttctgcg
gttgcgccacaccgtgcgccggaaatcgcccagcttggtttacgggcgctggcggcggcgacg
ctttccaacttgatgagtgcgaccattgccgggttctttattggtttagcttga 142 yeiJ -
MDVMRSVLGMVVLLTIAFLLSVNKKKISLRTVGAALVLQVVIGGIMLWLPPGRWVAEKVAFGV
Escherichia
HKVMAYSDAGSAFIFGSLVGPKMDTLFDGAGFIFGFRVLPAIIFVTALVSILYYIGVMGILIR
coli K-12
ILGGIFQKALNISKIESFVAVTTIFLGQNEIPAIVKPFIDRLNRNELFTAICSGMASIAGSTM
W3110
IGYAALGVPVEYLLAASLMAIPGGILFARLLSPATESSQVSFNNLSFTETPPKSIIEAAATGA
(AAC75222;
MTGLKIAAGVATVVMAFVAIIALINGIIGGVGGWFGFEHASLESILGYLLAPLAWVMGVDWSD
JW2148)
ANLAGSLIGQKLAINEFVAYLNFSPYLQTAGILDAKTVAIISFALCGFANFGSIGVVVGAFSA
VAPHRAPEIAQLGLRALAAATLSNLMSATIAGFFIGLA 115 yeiM -
atggatataatgagaagtgttgtggggatggtggtgttactggcaatagcatttctgttgtca
Escherichia
gtgaataaaaagagcatcagtttgcgcacggttggagccgcactgctgctgcaaatcgctatt
coli K-12
ggtggcatcatgctctacttcccaccgggaaaatgggcagtagaacaggcggcattaggcgtt
W3110
cataaagtgatgtcttacagtgatgccggtagcgccttcatttttggttcgctggttgggccg
(AAC75225;
aaaatggatgtcctgtttgacggtgcgggttttatcttcgcctttcgcgtacttccggcgatt
JW2151 )
attttcgttactgcgctcatcagtctgctgtactacattggcgtgatggggctgctgattcgc
atccttggcagcattttccagaaagccctcaacatcagcaaaatcgaatcttttgttgcggtt
actactattttcctcgggcaaaatgagatcccggcgatcgttaaaccgtttatcgatcgcatg
aatcgcaacgagttgtttaccgcaatttgtagcgggatggcgtccattgctggttcgatgatg
attggttatgccggaatgggcgtaccaattgactacctgttagcggcatcgctgatggcgatc
cctggcgggattttgtttgcacgtattcttagcccggcaaccgagccttcgcaggtcacattt
gaaaatctgtcgttcagcgaaacgccgccaaaaagctttatcgaagcggcggcgagcggtgcg
atgaccgggctaaaaatcgccgctggtgtggcgacggtggtaatggcgtttgtcgcaattatt
gcgctgatcaacggcattatcggcggaattggcggctggtttggtttcgccaatgcctctctg
gaaagtatttttggctatgtgctggcaccgctggcgtggatcatgggtgtggactggagtgat
gccaatcttgcgggtagcctgattgggcagaaactggcgattaacgaattcgtcgcttacctg
agtttctccccatacctgcaaacgggcggcacgctggaagtgaaaaccattgcgattatctcc
tttgcgctttgtggttttgctaactttggttctatcggtgttgtcgttggcgcattttcggct
atttcgccaaaacgcgcgccggaaatcgcccagcttggtttacgggcgctggcagcagcaacg
ctttccaacctgatgagtgcgactattgccgggttctttattggtctggcgtaa 143 yeiM -
MDIMRSVVGMVVLLAIAFLLSVNKKSISLRTVGAALLLQIAIGGIMLYFPPGKWAVEQAALGV
Escherichia
HKVMSYSDAGSAFIFGSLVGPKMDVLFDGAGFIFAFRVLPAIIFVTALISLLYYIGVMGLLIR
coli K-12
ILGSIFQKALNISKIESFVAVTTIFLGQNEIPAIVKPFIDRMNRNELFTAICSGMASIAGSMM
W3110
IGYAGMGVPIDYLLAASLMAIPGGILFARILSPATEPSQVTFENLSFSETPPKSFIEAAASGA
(AAC75225;
MTGLKIAAGVATVVMAFVAIIALINGIIGGIGGWFGFANASLESIFGYVLAPLAWIMGVDWSD
JW2151)
ANLAGSLIGQKLAINEFVAYLSFSPYLQTGGTLEVKTIAIISFALCGFANFGSIGVVVGAFSA
ISPKRAPEIAQLGLRALAAATLSNLMSATIAGFFIGLA 116 HI0519 -
atgagtgtgttaagcagcattttgggaatggtcgtattaatcgctattgccgtgttactttct
Haemophilus
aataatcgtaaagcgattagtattcgaaccgtagtaggggcgttagcaatccaagtaggattt
influenzae
gccgcccttattttatatgtgccagcaggtaaacaagcgttgggtgccgctgcggatatggta Rd
KW20
tccaatgttattgcctatggtaatgacgggattaatttcgttttcggcggattggcagatcca
serotype
agtaaaccatccggtttcatttttgcagtgaaagtattaccgattatcgtgttcttctctggc
d(AAC22177
ttaatttctgtgctttactatctcggcattatgcaagtcgtgattaaagtattaggtggcgca
ttacaaaaagcattgggtacgtcaaaagcggaatcaatgtcagcggcggcgaatatcttcgtc
ggtcaaactgaagcaccattagttgttcgcccttacattaaaaatatgacccaatctgaatta
tttgccattatggtgggtggtacagcgtctatcgcgggttcagtaatggcaggttatgctgga
atgggcgtgccattgacatacttaatcgctgcgtcatttatggcggcaccagcaggtttatta
tttgcgaaattaatgttcccacaaaccgaacaattcacagataaacaaccagaagacaatgat
tcagaaaaaccaactaacgtacttgaagcaatggcgggcggtgcgagtgcaggtatgcaactt
gcgttaaacgtaggtgcaatgttaatcgcattcgttggtttaattgcattaattaatggtatt
ttaagtggcgtaggcggatggttcggctatggcgacttaaccttacaatctatctttggttta
atttttaaaccattagcatacttaatcggtgtaactgatggtgctgaagcaggtattgcagga
caaatgatcgggatgaaattagcggttaatgaatttgtgggttatcttgaatttgcaaaatat
ttacaaccagattctgcaattgtattaactgaaaaaaccaaagcgattattactttcgcactt
tgtggttttgctaacttcagctcaattgcaatcttaattggtggtttaggtggtatggcacca
agccgtcgtagtgatgttgctcgtttaggtatcaaagccgttatcgctggtactctcgctaac
ttaatgagtgcaactattgctggtttatttatcggcttaggtgctgcagcactttaa 144
HI0519 -
MSVLSSILGMVVLIAIAVLLSNNRKAISIRTVVGALAIQVGFAALILYVPAGKQALGAAADMV
Haemophilus
SNVIAYGNDGINFVFGGLADPSKPSGFIFAVKVLPIIVFFSGLISVLYYLGIMQVVIKVLGGA
influenzae
LQKALGTSKAESMSAAANIFVGQTEAPLVVRPYIKNMTQSELFAIMVGGIASIAGSVMAGYAG Rd
KW 20
MGVPLTYLIAASFMAAPAGLLFAKLMFPQTEQFTDKQPEDNDSEKPTNVLEAMAGGASAGMQL
serotype
ALNVGAMLIAFVGLIALINGILSGVGGWFGYGDLTLQSIFGLIFKPLAYLIGVTDGAEAGIAG
d(AAC22177
QMIGMKLAVNEFVGYLEFAKYLQPDSAIVLTEKTKAIITFALCGFANFSSIAILIGGLGGMAP
SRRSDVARLGIKAVIAGTLANLMSATIAGLFIGLGAAAL 117 nupC
atgatttttagctctctttttagtgttgtagggatggcggtgctttttcttattgcttgg
(HP1180) -
gtgttttctagcaataaaagggctattaattatcgcacgattgtcagtgcctttgtgatt
Helicobacter
caagtggctttaggggcgttggctttatatgtgcctttgggtagggaaatgctgcaaggc pylori
ttagccagcggcatacaaagcgtgatttcttacggctatgagggggtgcgttttttattt 26695
ggcaatctcgctccaaacgctaagggcgatcaagggataggggggtttgtctttgcgatc
(AAD08224)
aatgttttagcgatcattatcttttttgctagcttgatttcacttctatattatttaaaa
atcatgcctttatttatcaatctcatcggtggggcgttgcaaaaatgcttaggcacttct
agagcagaaagcatgagtgcagcggctaatatttttgtagcgcacaccgaagcgccctta
gtcattaaaccttatttgaaaagcatgagcgattcagagatttttgcggtcatgtgcgtg
ggcatggctagcgttgcggggcctgtgttagccgggtatgcgagcatgggcattcctttg
ccttatttgatcgccgcttcgtttatgtccgctcctggggggttgttgttcgctaaaatc
atttacccacaaaacgaaaccatttctagccatgcagatgtttctatagaaaagcatgtc
aatgccatagaagctatcgctaatggggcaagcacagggctaaatttagccttgcatgtg
ggagcgatgcttttagcctttgtggggatgctcgcgctcattaacgggcttttaggggtt
gtagggggttttttaggcatggagcatttgtctttagggttgattttaggcacgctctta
aaacccttagcctttatgttaggcattccttggagccaggccgggattgccggagaaatc
ataggcattaaaatcgcgctcaatgaatttgtgggctatatgcagttattgccttatttg
ggcgataaccctcctttaatcttgagcgagaaaactaaagcgatcatcacttttgcgttg
tgcgggtttgctaatttaagctcagtcgctatgctcattggagggcttggcagtttagtg
cctaaaaagaaggatctcattgtaaggcttgctttaaaagcggtgcttgtaggcacgctt
tctaatttcatgagcgcgactatcgccgggttattcatagggctaaacgctcattaa 145 nup C
MIFSSLFSVVGMAVLFLIAWVFSSNKRAINYRTIVSAFVIQVALGALALYVPLGREMLQG
(HP1180) -
LASGIQSVISYGYEGVRFLFGNLAPNAKGDQGIGGFVFAINVLAIIIFFASLISLLYYLK
Helicobacter
IMPLFINLIGGALQKCLGTSRAESMSAAANIFVAHTEAPLVIKPYLKSMSDSEIFAVMCV pylori
GMASVAGPVLAGYASMGIPLPYLIAASFMSAPGGLLFAKIIYPQNETISSHADVSIEKHV 26695
NAIEAIANGASTGLNLALHVGAMLLAFVGMLALINGLLGVVGGFLGMEHLSLGLILGTLL
(AAD08224)
KPLAFMLGIPWSQAGIAGEIIGIKIALNEFVGYMQLLPYLGDNPPLILSEKTKAIITFAL
CGFANLSSVAMLIGGLGSLVPKKKDLIVRLALKAVLVGTLSNFMSATIAGLFIGLNAH 118 nupC
ATGTTTTTATTAATCAACATTATTGGTCTAATTGTATTTCTTGGTATTGCGGTATTATTTTCA
(SA0600) -
AGAGATCGCAAAAATATCCAATGGCAATCAATTGGGATCTTAGTTGTTTTAAACCTGTTTTTA
Staphylococcus
GCATGGTTCTTTATTTATTTTGATTGGGGTCAAAAAGCAGTAAGAGGAGCAGCCAATGGTATC
aureus
GCTTGGGTAGTTCAGTCAGCGCATGCTGGTACAGGTTTTGCATTTGCAAGTTTGACAAATGTT
subsp.
AAAATGATGGATATGGCTGTTGCAGCCTTATTCCCAATATTATTAATAGTGCCATTATTTGAT
aureus N315
ATCTTAATGTACTTTAATATTTTACCGAAAATTATTGGAGGTATTGGTTGGTTACTAGCTAAA
(BAB41833)
GTAACAAGACAACCTAAATTCGAGTCATTCTTTGGGATAGAAATGATGTTCTTAGGAAATACT
GAAGCATTAGCCGTATCAAGTGAGCAACTAAAACGTATGAATGAAATGCGTGTATTAACAATC
GCAATGATGTCAATGAGCTCTGTATCCGGAGCTATTGTAGGTGCGTATGTACAAATGGTACCA
GGAGAACTGGTACTAACGGCAATTCCACTAAATATCGTTAACGCGATTATTGTGTCATGCTTG
TTGAATCCAGTAAGTGTTGAAGAGAAAGAAGATATTATTTACAGTCTTAAAAACAATGAAGTT
GAACGTCAACCATTCTTCTCATTCCTTGGAGATTCTGTATTAGCAGCAGGTAAATTAGTATTA
ATCATCATCGCATTTGTTATTAGTTTTGTAGCGTTAGCTGATCTATTTGATCGTTTTATCAAT
TTGATTACAGGATTGATAGCAGGATGGATAGGCATAAAAGGTAGTTTCGGTTTAAACCAAATT
TTAGGTGTGTTTATGTATCCATTTGCGCTATTACTCGGTTTACCTTATGATGAAGCGTGGTTG
GTAGCACAACAAATGGCTAAGAAAATTGTTACAAATGAATTTGTTGTTATGGGTGAAATTTCT
AAAGATATTGCATCTTATACACCACACCATCGTGCGGTTATTACAACATTCTTAATTTCATTT
GCAAACTTCTCAACGATTGGTATGATTATCGGTACATTGAAAGGCATTGTTGATAAAAAGACA
TCAGACTTTGTATCTAAATATGTACCTATGATGCTATTATCAGGTATCCTAGTTTCATTATTA
ACAGCAGCTTTCGTTGGTTTATTTGCATGGTAA 146 nupC
MFLLINIIGLIVFLGIAVLFSRDRKNIQWQSIGILVVLNLFLAWFFIYFDWGQKAVRGAANGI
(SA0600) -
AWVVQSAHAGTGFAFASLTNVKMMDMAVAALFPILLIVPLFDILMYFNILPKIIGGIGWLLAK
Staphylococcus
VTRQPKFESFFGIEMMFLGNTEALAVSSEQLKRMNEMRVLTIAMMSMSSVSGAIVGAYVQMVP
aureus
GELVLTAIPLNIVNAIIVSCLLNPVSVEEKEDIIYSLKNNEVERQPFFSFLGDSVLAAGKLVL
subsp.
IIIAFVISFVALADLFDRFINLITGLIAGWIGIKGSFGLNQILGVFMYPFALLLGLPYDEAWL
aureus N315
VAQQMAKKIVTNEFVVMGEISKDIASYTPHHRAVITTFLISFANFSTIGMIIGTLKGIVDKKT
(BAB41833) SDFVSKYVPMMLLSGILVSLLTAAFVGLFAW 119 nup C
atgtttttattaatcaacattattggtctaattgtatttcttggtattgcggtattattt
(SAV0645) -
tcaagagatcgcaaaaatatccaatggcaatcaattgggatcttagttgttttaaacctg
Staphylococcus
tttttagcatggttctttatttattttgattggggtcaaaaagcagtaagaggagcagcc aureus
aatggtatcgcttgggtagttcagtcagcgcatgctggtacaggttttgcatttgcaagt subsp.
ttgacaaatgttaaaatgatggatatggctgttgcagccttattcccaatattattaata aureus
Mu50 gtgccattatttgatatcttaatgtactttaatattttaccgaaaattattggaggtatt
(BAB56807)
ggttggttactagctaaagtaacaagacaacctaaattcgagtcattctttgggatagaa
atgatgttcttaggaaatactgaagcattagccgtatcaagtgagcaactaaaacgtatg
aatgaaatgcgtgtattaacaatcgcaatgatgtcaatgagctctgtatccggagctatt
gtaggtgcgtatgtacaaatggtaccaggagaactggtactaacggcaattccactaaat
atcgttaacgcgattattgtgtcatgcttgttgaatccagtaagtgttgaagagaaagaa
gatattatttacagtcttaaaaacaatgaagttgaacgtcaaccattcttctcattcctt
ggagattctgtattagcagcaggtaaattagtattaatcatcatcgcatttgttattagt
tttgtagcgttagctgatctatttgatcgttttatcaatttgattacaggattgatagca
ggatggataggcataaaaggtagtttcggtttaaaccaaattttaggtgtgtttatgtat
ccatttgcgctattactcggtttaccttatgatgaagcgtggttggtagcacaacaaatg
gctaagaaaattgttacaaatgaatttgttgttatgggtgaaatttctaaagatattgca
tcttatacaccacaccatcgtgcggttattacaacattcttaatttcatttgcaaacttc
tcaacgattggtatgattatcggtacattgaaaggcattgttgataaaaagacatcagac
tttgtatctaaatatgtacctatgatgctattatcaggtatcctagtttcattattaaca
gcagctttcgttggtttatttgcatggtaa 147 nupC
MFLLINIIGLIVFLGIAVLFSRDRKNIQWQSIGILVVLNLFLAWFFIYFDWGQKAVRGAA
(SAV0645) -
NGIAWVVQSAHAGIGFAFASLINVKMMDMAVAALFPILLIVPLFDILMYFNILPKIIGGI
Staphylococcus
GWLLAKVIRQPKFESFFGIEMMFLGNTEALAVSSEQLKRMNEMRVLTIAMMSMSSVSGAI aureus
VGAYVQMVPGELVLTAIPLNIVNAIIVSCLLNPVSVEEKEDIIYSLKNNEVERQPFFSFL subsp.
GDSVLAAGKLVLIIIAFVISFVALADLFDRFINLITGLIAGWIGIKGSFGLNQILGVFMY aureus
Mu50 PFALLLGLPYDEAWLVAQQMAKKIVINEFVVMGEISKDIASYTPHHRAVITTFLISFANF
(BAB56807) STIGMIIGILKGIVDKKTSDFVSKYVPMMLLSGILVSLLTAAFVGLFAW 120
atgcaatttatttatagtattattggtattttattggtattaggaattgtgtatgcaatt nupC
tctttcaatcgtaagagtgtttctctaagtttaattggaaaagctcttatcgttcaattc
(SNupC) -
attattgcgctaatcttagtacgtatcccactaggccaacaaattgttagtgttgtttca
Streptococcus
actggagttactagcgtaatcaactgtggtcaagctggtttaaattttgtgtttgggtca
pyogenes
ttagcagatagtggcgcaaaaactggttttattttcgctattcaaacgcttggtaatatt SF370
gttttcttatctgccctagttagtctactttattatgtaggaatccttggatttgtagta
serotype M1
aaatggataggtaagggcgttggtaaaattatgaaatcctcagaggttgagagttttgtt
(AAK34582)
gctgtagctaatatgtttcttggtcaaacagacagtccaatcttggttagcaaataccta
ggtcgtatgactgatagtgagataatggttgtgttggtatcaggtatgggaagtatgtca
gtttctattcttggtggctatattgcattaggcattccaatggaatatctcttgattgct
tcaacaatggttcctattggcagtattctcattgctaaaatcttattgcctcaaacagaa
cctgttcaaaaaattgatgacattaagatggataataaaggtaataacgccaatgtgatt
gatgcaatcgctgagggtgcaagcacaggtgcacaaatggctttctcaattggtgctagt
ttgattgcctttgttggtttagtttctttgattaatatgatgttaagtggattgggaatc
cgcttagaacaaatcttttcatatgtttttgctccatttggttttcttatgggatttgac
cacaaaaacattcttctagaaggaaaccttcttggaagtaagttgattttaaatgagttt
gtttcgttccaacaattgggtcacctaatcaaatctttagattatcgtacagcattggta
gcaactatttcactctgtggttttgctaatttatcaagtttaggtatttgtgtttcaggt
attgctgttctttgcccggagaaacgtagcaccctagctcgacttgttttccgtgcaatg
attggtggtattgctgtaagtatgcttagcgcctttatcgtcggtattgtaactctattc taa
148 nupC
MQFIYSIIGILLVLGIVYAISFNRKSVSLSLIGKALIVQFIIALILVRIPLGQQIVSVVS
(SpNupC) -
TGVTSVINCGQAGLNFVFGSLADSGAKTGFIFAIQTLGNIVFLSALVSLLYYVGILGFVV
Streptococcus
KWIGKGVGKIMKSSEVESFVAVANMFLGQTDSPILVSKYLGRMTDSEIMVVLVSGMGSMS
pyogenes
VSILGGYIALGIPMEYLLIASTMVPIGSILIAKILLPQTEPVQKIDDIKMDNKGNNANVI SF370
DAIAEGASTGAQMAFSIGASLIAFVGLVSLINMMLSGLGIRLEQIFSYVFAPFGFLMGFD
serotype M1
HKNILLEGNLLGSKLILNEFVSFQQLGHLIKSLDYRTALVATISLCGFANLSSLGICVSG
(AAK34582) IAVLCPEKRSTLARLVFRAMIGGIAVSMLSAFIVGIVTLF 121 nupC
atgagcctgtttatgagcctcatcggcatggcagttctgctaggaatcgcagttctactg
(VC2352) -
tcaagtaaccgtaaagctatcaatctaagaactgtgggtggcgcttttgctatccaattt Vibrio
tcactgggtgcatttattctgtatgtgccttggggccaagagctacttcgtggcttttcg
cholerae O1
gatgccgtatcgaatgttattaactacggtaacgatggtacttcattcctcttcggtgga biovar
El ctggtatcaggcaaaatgtttgaagtgtttggcggcggcggtttcattttcgcattccgc Tor
N16961 tactaccaacactgatcttcttctcagcactgatttctgtactgtactacttgggtgtt
(AAF95495)
atgcaatgggttatccgcattcttggcggtggtctgcaaaaagcactgggtacatcacgc
gcggaatctatgtctgcggctgcaaacattttcgtgggtcaaactgaagcaccattagtt
gttcgtccattcgttccaaaaatgactcaatctgagctgtttgcggtaatgtgtggtggc
ttggcttctatcgcaggtggtgtacttgcgggttacgcttcaatgggcgttaagatcgaa
tacttggtagcggcgtcattcatggcggcaccgggtggtctgctgttcgcaaaactgatg
atgcctgaaactgaaaaaccacaagacaatgaagacattactcttgatggtggtgacgac
aaaccggctaacgttatcgatgcggctgctggcggtgcttctgctggtctgcaacttgct
ctgaacgttggtgcaatgttgattgcctttatcggtttgattgctctgatcaacggtatg
ttgggtggcatcggtggttggttcggtatgcctgaactgaaactggaaatgctactgggc
tggttgtttgcgcctctggctttcctgatcggtgttccttggaacgaagcaactgttgcg
ggtgagttcatcggtctaaaaaccgttgctaacgaattcgttgcttactctcagtttgcg
ccttacctgactgaagcggcaccagtggttctgtctgagaaaaccaaagcgatcatctct
ttcgctctgtgtggttttgcgaacctttcttctatcgcaattctgcttggtggtttgggt
agcttggcacctaagcgtcgtggcgacatcgctcgtatgggggtcaaagcggttatcgca
ggtactctatctaacctgatggcagcgaccatcgctggcttcttcctctctttctaa 149 nupC
MSLFMSLIGMAVLLGIAVLLSSNRKAINLRTVGGAFAIQFSLGAFILYVPWGQELLRGFS
(VC2352) -
DAVSNVINYGNDGTSFLFGGLVSGKMFEVFGGGGFIFAFRVLPTLIFFSALISVLYYLGV Vibrio
MQWVIRILGGGLQKALGTSRAESMSAAANIFVGQTEAPLVVRPFVPKMTQSELFAVMCGG
cholerae O1
LASIAGGVLAGYASMGVKIEYLVAASFMAAPGGLLFAKLMMPETEKPQDNEDITLDGGDD biovar
El KPANVIDAAAGGASAGLQLALNVGAMLIAFIGLIALINGMLGGIGGWFGMPELKLEMLLG Tor
N16961 WLFAPLAFLIGVPWNEATVAGEFIGLKTVANEFVAYSQFAPYLTEAAPVVLSEKTKAIIS
(AAF95495)
FALCGFANLSSIAILLGGLGSLAPKRRGDIARMGVKAVIAGTLSNLMAATIAGFFLSF 122 nupC
ttgggcggcgttatgtcatcactcctcggtatgggcgcaattttgctggttgcgtggcta
(VC1953) -
ttttctaccaatagaaaaaatatcaacttgcgtacagtttctttagcgttactgctgcaa Vibrio
atcttcttcgccttactggtgctgtatgtacctgcgggtaaagaggcactcaatcgtgtg
Cholerae O1
acgggcgcggtgtcacaactgatcaactatgggcaagatggtatcggttttgtgtttggt biovar
El ggcctcgccaatggcagcgtaggttttgtgtttgcgattaatgtccttggcatcatcatt Tor
N16961 ttcttctctgcactgatttctggcctttaccatttaggcatcatgccgaaagtgattaac
(AAF95101)
ctcatcggtggtggtttacagaaattgcttggcacaggccgtgcagaatccctttctgct
accgcaaacattttcgtgggtatgattgaagcgccgctggtggtgaaaccttatcttcat
aaaatgaccgattcgcaattctttgcagtgatgacgggcggcttagcgtcggttgctggc
ggtactttggttggttatgcctctttaggtgtggaattgaactatctgatcgcggcggct
ttcatgtctgcccctgcgggtcttttgatggcaaaaatcatgttgccagaaaccgaacac
gtcgatgccgcgattgcgcaagatgagttggatctgccgaaatccactaacgtcgtcgaa
gcgattgcggatggcgcgatgtcgggtgtgaaaattgctgttgcggtaggggcgactttg
ctcgctttcgtgagtgtgattgctctgttaaacggcttgctcggttggtttggtggctgg
tttggcatcgagctaagctttgaactgatcatggggtatgttttcgctccggtagcttgg
ctgattggtattccatggcatgaggcgatcacggcaggctcgctgattggtaacaaagtg
gtggtgaacgagtttgtcgctttcattcaactgattgaagtgaaagagcaattgagtgcg
cattcacaagcgatcgtgactttcgcgctgtgcggttttgcgaatatttctaccatggcg
attttgattggtggtttgggtagccttgtacctgaacgtcgctcttttatctcccaatac
ggcttccgtgcgattggcgcaggcgtattagctaacctaatgagtgcatcgatcgctgga
gtgattttgtctttgtga 150 nupC
MGGVMSSLLGMGAILLVAWLFSTNRKNINLRTVSLALLLQIFFALLVLYVPAGKEALNRV
(VC1953) -
TGAVSQLINYGQDGIGFVFGGLANGSVGFVFAINVLGIIIFFSALISGLYHLGIMPKVIN Vibrio
LIGGGLQKLLGTGRAESLSATANIFVGMIEAPLVVKPYLHKMTDSQFFAVMTGGLASVAG
cholerae O1
GTLVGYASLGVELNYLIAAAFMSAPAGLLMAKIMLPETEHVDAAIAQDELDLPKSTNVVE biovar
El AIADGAMSGVKIAVAVGATLLAFVSVIALLNGLLGWFGGWFGIELSFELIMGYVFAPVAW Tor
N16961 LIGIPWHEAITAGSLIGNKVVVNEFVAFIQLIEVKEQLSAHSQAIVTFALCGFANISTMA
(AAF 95101) ILIGGLGSLVPERRSFISQYGFRAIGAGVLANLMSASIAGVILSL 123 nupC
atggcgattttgtttggaatcatcggtgttacggtactgatcttatgcgcgtatctgctc
(VCA0179) -
tctgaaagccgcagtgcgattaattggaaaaccatttcccgagccttgttgttgcaaatt Vibrio
ggttttgcggctcttgtgctttatttcccattggggcaaaccgcgctaagcagcttgagt
cholerae O1
aatggggtttctggtttgcttggttttgccgatgtcggcattcgctttctgtttggtgat biovar
El cttgccgatacgggctttatttttgctgttcgtgtattacctatcatcatcttcttcagt Tor
N16961 gcgctgatttctgccctttattaccttggtgtgatgcaaaaagtgatcgccctgatcggc
(AAF96092 )
ggtggcattcaacgcttcttaggcaccagtaaggcggaatcactggtcgcgacaggcaat
attttcctatcacaaggcgaatcgccacttttggtgcgccccttccttgccaatatgaca
cgctctgaactgtttgcggtcatggcgggcggtatggcatcggtagcaggctctgtgctg
ggtggttacgcaggtttaggggttgagctgaaatacctgattgcagcgagtttcatggcg
gcgccgggcagtttaatgatggcgaaaatcatcgttcctgagcgtggtgtgccaatcgat
caaagccaagtcgagttagataaagcgcaagacagcaacttgattgatgctctcgctagc
ggtgcgatgaatggtatgaaagtcgccgttgcagtgggcactatgttgattgcgttcgtc
agcgtgatcgctatggtcaacactggccttgaaaatctgggcgatctggttgggtttagc
ggcattaccttacaagccatgttcggttatctgtttgctcctctggcatgggtgattggc
attccaagtcacgaagtgctggcggcaggttcctacatcggtcagaaagtggtgatgaac
gaatttgtggctttcattgactttgttgagcataaagcgctgctttctgagcatagccaa
gtcatcatcacgtttgcattgtgtggctttgccaacattggctctatcgcgatccaatta
ggctccattggcgtgatagcccctgagcgccgctcggaagtggcgaacctaggcataaaa
gcggtcattgctggcactttagccaacctaatgagcgcttgcttagcggggattttcatc
tcgctataa 124 yegT -
atgaaaacaacagcaaagctgtcgttcatgatgtttgttgaatggtttatctggggcgcg
Eschericia
tggtttgtgccattgtggttgtggttaagtaaaagcggttttagtgccggagaaattggc coli
K-12 tggtcgtatgcctgtaccgccattgcggcgatcctgtcgccaattctggttggctccatc
W3110 actgaccgctttttctcggcgcaaaaagtgctggcggtattgatgttcgcaggcgcgctg
(P76417;
ctgatgtatttcgctgcgcaacagaccacttttgccgggttcttcccgttactgctggcc
JW2085)
tactcgctaacctatatgccgaccattgcgctgactaacagcatcgcttttgccaacgtg
ccggatgttgagcgtgatttcccgcgcattcgtgtgatgggcactatcggctggattgcc
tccggtctggcatgtggtttcttgccgcaaatactggggtatgccgatatctcaccgact
aacatcccgctgctgattaccgccggaagttctgctctgctcggtgtgtttgcgtttttc
ctgcccgacacgccaccaaaaagcaccggcaaaatggatattaaagtcatgctcggcctg
gatgcgctgatcctgctgcgcgataaaaacttcctcgtctttttcttctgttcattcctg
tttgcgatgccactagcgttctattacatctttgccaacggttatctgaccgaagttggc
atgaaaaacgccaccggctggatgacgctcggccagttctctgaaatcttctttatgctg
gcattgccgtttttcactaaacgctttggtatcaaaaaggtattattgcttggtctggtc
accgctgcgatccgctatggcttctttatttacggtagtgcggatgaatatttcacctac
gcgttactgttcctcggtattttgcttcacggcgtaagttacgatttttactacgttacc
gcttacatctatgtcgataaaaaagcccccgtgcatatgcgtaccgctgcgcaggggctg
atcacgctctgctgccagggcttcggcagtttgctcggctatcgtcttggcggtgtgatg
atggaaaagatgttcgcttatcaggaaccggtaaacggactgactttcaactggtccggg
atgtggactttcggcgcggtgatgattgccattatcgccgtgctgttcatgatttttttc
cgcgaatccgacaacgaaattacggctatcaaggtcgatgatcgcgatattgcgttgaca
caaggggaagttaaatga 151 yegT -
MKTTAKLSFMMFVEWFIWGAWFVPLWLWLSKSGFSAGEIGWSYACTAIAAILSPILVGSI
Escherichia
TDRFFSAQKVLAVLMFAGALLMYFAAQQTTFAGFFPLLLAYSLTYMPTIALTNSIAFANV coli
K-12 PDVERDFPRIRVMGTIGWIASGLACGFLPQILGYADISPTNIPLLITAGSSALLGVFAFF
W3110 LPDTPPKSTGKMDIKVMLGLDALILLRDKNFLVFFFCSFLFAMPLAFYYIFANGYLTEVG
(P76417;
MKNATGWMTLGQFSEIFFMLALPFFTKRFGIKKVLLLGLVTAAIRYGFFIYGSADEYFTY
JW2085)
ALLFLGILLHGVSYDFYYVTAYIYVDKKAPVHMRTAAQGLITLCCQGFGSLLGYRLGGVM
MEKMFAYQEPVNGLTFNWSGMWTFGAVMIAIIAVLFMIFFRESDNEITAIKVDDRDIALT QGEVK
125 nupG -
atgaatcttaagctgcagctgaaaatcctctcttttctgcagttctgtctgtggggaagt
Escherichia
tggctgacgaccctcggctcctatatgtttgttaccctgaagtttgacggtgcttctatt coli
K-12 ggcgcagtttatagctcactgggtatcgcagcggtctttatgcctgcgctgctggggatt
W3110 gtggccgacaaatggttaagtgcgaaatgggtatatgccatttgccacaccattggcgct
JW2932;
atcacgctgttcatggcggcacaggtcacgacaccggaagcgatgttccttgtgatattg
(P09452
attaactcgtttgcttatatgccaacgcttgggttaatcaacaccatctcttactatcgc
ctgcaaaatgccgggatggatatcgttactgacttcccgccaatccgtatctggggcacc
atcggctttatcatggcaatgtgggtggtgagcctgtctggcttcgaattaagccacatg
cagctgtatattggcgcagcactttccgccattctggttctgtttaccctgactctgccg
catattccggttgctaaacagcaagcgaatcagagctggacaaccctgctgggcctcgat
gcattcgcgctgtttaaaaacaagcgtatggcaatcttctttatcttctcaatgctgctg
ggcgcggaactgcagattaccaacatgttcggtaataccttcctgcacagcttcgacaaa
gatccgatgtttgccagcagctttattgtgcagcatgcgtcaatcatcatgtcgatttcg
cagatctctgaaaccctgttcattctgaccatcccgttcttcttaagccgctacggtatt
aagaacgtaatgatgatcagtattgtggcgtggatcctgcgttttgcgctgtttgcttac
ggcgacccgactccgttcggtactgtactgctggtactgtcgatgatcgtttacggttgc
gcattcgacttcttcaacatctctggttcggtgtttgtcgaaaaagaagttagcccggca
attcgcgccagtgcacaagggatgttcctgatgatgactaacggcttcggctgtatcctc
ggcggcatcgtgagcggtaaagttgttgagatgtacacccaaaacggcattaccgactgg
cagaccgtatggttgattttcgctggttactccgtggttctggccttcgcgttcatggcg
atgttcaaatataaacacgttcgtgtcccgacaggcacacagacggttagccactaa 152 nupG
- MNLKLQLKILSFLQFCLWGSWLTTLGSYMFVTLKFDGASIGAVYSSLGIAAVFMPALLGI
Escherichia
VADKWLSAKWVYAICHTIGAITLFMAAQVTTPEAMFLVILINSFAYMPTLGLINTISYYR coli
K-12 LQNAGMDIVTDFPPIRIWGTIGFIMAMWVVSLSGFELSHMQLYIGAALSAILVLFTLTLP
W3110 HIPVAKQQANQSWTTLLGLDAFALFKNKRMAIFFIFSMLLGAELQITNMFGNTFLHSFDK
(P09452;
DPMFASSFIVQHASIIMSISQISETLFILTIPFFLSRYGIKNVMMISIVAWILRFALFAY JW2932
GDPTPFGTVLLVLSMIVYGCAFDFFNISGSVFVEKEVSPAIRASAQGMFLMMTNGFGCIL
GGIVSGKVVEMYTQNGITDWQTVWLIFAGYSVVLAFAFMAMFKYKHVRVPTGTQTVSH 126 xapB
- atgagcatcgcgatgcgcttaaaggtaatgtcctttttgcaatattttatctgggggagc
Escherichia
tggctggttaccctcggctcttacatgattaatactcttcatttcaccggcgctaatgtt coli
K-12 ggcatggtttacagttccaaagggatcgccgcgattattatgcctggtataatggggatc
W3110 atcgcagacaaatggctgcgcgcagaacgtgcatacatgctgtgtcacctggtgtgtgcg
(P45562;
ggcgtacttttttatgcggcatccgtaactgatccggatatgatgttttgggtgatgtta
JW2397)
gtcaatgcgatggcgtttatgccgactattgcgttatcgaacagcgtctcttattcctgt
cttgcccaggcagggcttgacccggtgaccgctttcccgcccattcgcgtttttggtacg
gtggggttcattgtcgcgatgtgggcagtaagcctgctgcatctggaattgagtagtctg
cagctgtatatcgcgtccggtgcgtcattgctgctgtcggcttatgcgctgactttgccg
aagattccggttgcggagaaaaaagcgaccacatcgcttgccagcaagctgggtctggat
gccttcgtgctgtttaaaaatccacgcatggccatctttttcctctttgccatgatgctg
ggtgcggtactgcaaattaccaacgtttttggtaatccgttcctacatgatttcgcccgt
aacccggagtttgctgacagttttgtggtgaaatatccctccattttactgtcagtttca
cagatggcagaagtgggctttatactgactatcccattctttttaaagcgatttggcatt
aaaaccgtcatgctgatgagtatggtggcctggacgctgcgctttggcttcttcgcctat
ggcgatccgtcaacaaccggatttattttgctgctgctgtcgatgattgtttatggctgt
gcattcgatttcttcaatatttctggttcggtatttgtcgaacaggaagttgattccagc
attcgtgccagcgcgcaggggctctttatgaccatggtaaatggtgtcggcgcatgggtt
ggctcgattctgagtggcatggcagtagattacttttcggtggatggcgtaaaagactgg
caaactatctggctggtgtttgcaggatatgctctttttctcgcagtgatatttttcttt
gggtttaaatataatcatgaccctgaaaagataaagcatcgagcggtgactcattaa 153 xapB
- MSIAMRLKVMSFLQYFIWGSWLVTLGSYMINTLHFTGANVGMVYSSKGIAAIIMPGIMGI
Escherichia
IADKWLRAERAYMLCHLVCAGVLFYAASVTDPDMMFWVMLVNAMAFMPTIALSNSVSYSC coli
K-12 LAQAGLDPVTAFPPIRVFGTVGFIVAMWAVSLLHLELSSLQLYIASGASLLLSAYALTLP
W3110 KIPVAEKKATTSLASKLGLDAFVLFKNPRMAIFFLFAMMLGAVLQITNVFGNPFLHDFAR
(P45562;
NPEFADSFVVKYPSILLSVSQMAEVGFILTIPFFLKRFGIKTVMLMSMVAWTLRFGFFAY
JW2397)
GDPSTTGFILLLLSMIVYGCAFDFFNISGSVFVEQEVDSSIRASAQGLFMTMVNGVGAWV
GSILSGMAVDYFSVDGVKDWQTIWLVFAGYALFLAVIFFFGFKYNHDPEKIKHRAVTH 127
CC1628 -
ATGGGGACGAGTTTCCGTCTGTTCGTGATGATGGTGCTGCAGCTGGCGATCTGGGGCGCCTGG
Caulobacter
GCGCCCAAGATCTTCCCCTACATGGGCATGCTGGGCTTCGCGCCCTGGCAGCAGTCGCTGGTC
crescentus
GGCAGCGCCTGGGGCGTGGCGGCGCTGGTGGGCATCTTCTTCTCGAATCAGTTCGCCGACCGG
CB15
AACTTCTCGGCCGAGCGGTTCCTGGCGGTCAGCCACCTGATCGGCGGCGTGGCGCTGCTGGGC
(AAK23606)
ACGGCCTTCTCGACGGAGTTCTGGCCGTTCTTTGCCTGTTACCTCGTTTTCAGCCTGGTCTAT
GTGCCGACGCTGTCGGTCACCAACTCGATCGCCTTCGCCAATCTGCGCGATCCGGCGGCCGGC
TTCGGCGGGGTGCGGATGGGCGGAACCGTCGGCTGGGTGCTGGTCAGCTGGCCCTTCGTGTTC
CTGCTGGGCGCCCAAGCGACGGTGGAGCAGGTCCGCTGGATCTTCCTGGTGGCGGCGATCGTC
TCCTTCGTTTTCGCCGGTTACGCTCTGACCCTGCCGCACACGCCGCCGCGCAAGGCCGATGAC
GCTGTCGACAAGCTGGCCTGGCGACGGGCGTTCAAGCTACTGGGCGCGCCCTTCGTGTTTGTC
CTCTTTGTCGTGACCTTCATCGATTCCGTGATCCACAACGGCTACTTCGTGATGGCCGACGCC
TTCCTGACCAACCGGGTCGGGATCGCGGGCAATCTCAGCATGGTCGTGCTGAGCCTGGGCCAG
GTGGCCGAAATCATCACCATGCTGCTGTTGGGCCGCGTGCTGGCCAAGCTGGGCTGGAAGGTC
ACCATGATCGTCGGCGTGCTGGGCCACGCCGCGCGCTTTGCGGTCTTCGCCTACTTCGCCGAC
AGCGTGCCGGTCATCGTGGCGGTGCAGCTGCTGCACGGCGTCTGCTACGCCTTCTTCTTCGCC
ACGGTTTACATCTTCGTCGACGCCGTCTTCCCGAAAGATGTCCGCTCCAGCGCGCAGGGTCTG
TTCAACTTGCTGATCCTGGGCGTCGGCAATGTGGCCGCCAGCTTCATCTTCCCCGCGCTGATC
GGTCGCCTGACCACCGATGGGTCCGTCGACTACACGACGCTGTTCCTCGTGCCGACCGCCATG
GCTTTGGCGGCGGTCTGCCTGCTGGCGCTGTTCTTCCGGCCGCCCACGCGGGGACCTGTTTCG
GAGGCGGATTCCGCTTCATCCGCCGCCAGTTCGGCCCAAGCCTAG 154 CC1628 -
MGTSFRLFVMMVLQLAIWGAWAPKIFPYMGMLGFAPWQQSLVGSAWGVAALVGIFFSNQFADR
Caulobacter
NFSAERFLAVSHLIGGVALLGTAFSTEFWPFFACYLVFSLVYVPTLSVTNSIAFANLRDPAAG
crescentus
FGGVRMGGTVGWVLVSWPFVFLLGAQATVEQVRWIFLVAAIVSFVFAGYALTLPHTPPRKADD
CB15
AVDKLAWRRAFKLLGAPFVFVLFVVTFIDSVIHNGYFVMADAFLTNRVGIAGNLSMVVLSLGQ
(AAK23606)
VAEIITMLLLGRVLAKLGWKVTMIVGVLGHAARFAVFAYFADSVPVIVAVQLLHGVCYAFFFA
TVYIFVDAVFPKDVRSSAQGLFNLLILGVGNVAASFIFPALIGRLTTDGSVDYTTLFLVPTAM
ALAAVCLLALFFRPPTRGPVSEADSASSAASSAQA 128 codB -
gtgtcgcaagataacaactttagccaggggccagtcccgcagtcggcgcggaaaggggta
Escherichia
ttggcattgacgttcgtcatgctgggattaaccttcttttccgccagtatgtggaccggc coli
K-12 ggcactctcggaaccggtcttagctatcatgatttcttcctcgcagttctcatcggtaat
W3110 cttctcctcggtatttacacttcatttctcggttacattggcgcaaaaaccggcctgacc
(P25525;
actcatcttcttgctcgcttctcgtttggtgttaaaggctcatggctgccttcactgcta
JW0327)
ctgggcggaactcaggttggctggtttggcgtcggtgtggcgatgtttgccattccggtg
ggtaaggcaaccgggctggatattaatttgctgattgccgtttccggtttactgatgacc
gtcaccgtcttttttggcatttcggcgctgacggttctttcggtgattgcggttccggct
atcgcctgcctgggcggttattccgtgtggctggctgttaacggcatgggcggcctggac
gcattaaaagcggtcgttcccgcacaaccgttagatttcaatgtcgcgctggcgctggtt
gtggggtcatttatcagtgcgggtacgctcaccgctgactttgtccggtttggtcgcaat
gccaaactggcggtgctggtggcgatggtggcctttttcctcggcaactcgttgatgttt
attttcggtgcagcgggcgctgcggcactgggcatggcggatatctctgatgtgatgatt
gctcagggcctgctgctgcctgcgattgtggtgctggggctgaatatctggaccaccaac
gataacgcactctatgcgtcgggtttaggtttcgccaacattaccgggatgtcgagcaaa
accctttcggtaatcaacggtattatcggtacggtctgcgcattatggctgtataacaat
tttgtcggctggttgaccttcctttcggcagctattcctccagtgggtggcgtgatcatc
gccgactatctgatgaaccgtcgccgctatgagcactttgcgaccacgcgtatgatgagt
gtcaattgggtggcgattctggcggtcgccttggggattgctgcaggccactggttaccg
ggaattgttccggtcaacgcggtattaggtggcgcgctgagctatctgatccttaacccg
attttgaatcgtaaaacgacagcagcaatgacgcatgtggaggctaacagtgtcgaataa 155
codB - MSQDNNFSQGPVPQSARKGVLALTFVMLGLTFFSASMWTGGTLGTGLSYHDFFLAVLIGN
Escherichia
LLLGIYTSFLGYIGAKTGLTTHLLARFSFGVKGSWLPSLLLGGTQVGWFGVGVAMFAIPV coli
K-12 GKATGLDINLLIAVSGLLMTVTVFFGISALTVLSVIAVPAIACLGGYSVWLAVNGMGGLD
W3110 ALKAVVPAQPLDFNVALALVVGSFISAGTLTADFVRFGRNAKLAVLVAMVAFFLGNSLMF
(P25525;
IFGAAGAAALGMADISDVMIAQGLLLPAIVVLGLNIWTTNDNALYASGLGFANITGMSSK
JW0327)
TLSVINGIIGTVCALWLYNNFVGWLTFLSAAIPPVGGVIIADYLMNRRRYEHFATTRMMS
VNWVAILAVALGIAAGHWLPGIVPVNAVLGGALSYLILNPILNRKTTAAMTHVEANSVE 129
mctC -
ATGAATTCCACTATTCTCCTTGCACAAGACGCTGTTTCTGAGGGCGTCGGTAATCCGATTCTT
Corynebacterium
AACATCAGTGTCTTCGTCGTCTTCATTATTGTGACGATGACCGTGGTGCTTCGCGTGGGCAAG
AGCACCAGCGAATCCACCGACTTCTACACCGGTGGTGCTTCCTTCTCCGGAACCCAGAACGGT
CTGGCTATCGCAGGTGACTACCTGTCTGCAGCGTCCTTCCTCGGAATCGTTGGTGCAATTTCA
CTCAACGGTTACGACGGATTCCTTTACTCCATCGGCTTCTTCGTCGCATGGCTTGTTGCACTG
CTGCTCGTGGCAGAGCCACTTCGTAACGTGGGCCGCTTCACCATGGCTGACGTGCTGTCCTTC
CGACTGCGTCAGAAACCAGTCCGCGTCGCTGCGGCCTGCGGTACCCTCGCGGTTACCCTCTTT
TACTTGATCGCTCAGATGGCTGGTGCAGGTTCGCTTGTGTCCGTTCTGCTGGACATCCACGAG
TTCAAGTGGCAGGCAGTTGTTGTCGGTATCGTTGGCATTGTCATGATCGCCTACGTTCTTCTT
GGCGGTATGAAGGGCACCACATACGTTCAGATGATTAAGGCAGTTCTGCTGGTCGGTGGCGTT
GCCATTATGACCGTTCTGACCTTCGTCAAGGTGTCTGGTGGCCTGACCACCCTTTTAAATGAC
GCTGTTGAGAAGCACGCCGCTTCAGATTACGCTGCCACCAAGGGGTACGATCCAACCCAGATC
CTGGAGCCTGGTCTGCAGTACGGTGCAACTCTGACCACTCAGCTGGACTTCATTTCCTTGGCT
CTCGCTCTGTGTCTTGGAACCGCTGGTCTGCCACACGTTCTGATGCGCTTCTACACCGTTCCT
ACCGCCAAGGAAGCACGTAAGTCTGTGACCTGGGCTATCGTCCTCATTGGTGCGTTCTACCTG
ATGACCCTGGTCCTTGGTTACGGCGCTGCGGCACTGGTCGGTCCAGACCGCGTCATTGCCGCA
CCAGGTGCTGCTAATGCTGCTGCTCCTCTGCTGGCCTTCGAGCTTGGTGGTTCCATCTTCATG
GCGCTGATTTCCGCAGTTGCGTTCGCTACCGTTCTCGCCGTGGTCGCAGGTCTTGCAATTACC
GCATCCGCTGCTGTTGGTCACGACATCTACAACGCTGTTATCCGCAACGGTCAGTCCACCGAA
GCGGAGCAGGTCCGAGTATCCCGCATCACCGTTGTCGTCATTGGCCTGATTTCCATTGTCCTG
GGAATTCTTGCAATGACCCAGAACGTTGCGTTCCTCGTGGCCCTGGCCTTCGCAGTTGCAGCA
TCCGCTAACCTGCCAACCATCCTGTACTCCCTGTACTGGAAGAAGTTCAACACCACCGGCGCT
GTGGCCGCTATCTACACCGGTCTCATCTCCGCGCTGCTGCTGATCTTCCTGTCCCCAGCAGTC
TCCGGTAATGACAGCGCAATGGTTCCAGGTGCAGACTGGGCAATCTTCCCACTGAAGAACCCA
GGCCTCGTCTCCATCCCACTGGCATTCATCGCTGGTTGGATCGGCACTTTGGTTGGCAAGCCA
GACAACATGGATGATCTTGCTGCCGAAATGGAAGTTCGTTCCCTCACCGGTGTCGGTGTTGAA
AAGGCTGTTGATCACTAA 130 putP_6 -
atggatcttacgacattaataacttttatagtatatctactagggatgttggcgattggcctc
Virgibacillus
atcatgtattatcgaaccaataatttatcagattatgttcttggtggacgtgatcttggtcca sp.
ggcgtagctgcattgagtgctggtgcatcggatatgagtggttggctgttattaggtttgcct
ggagcgatttatgcatctggtatgtctgaagcttggatggggatcgggttagctgtaggtgct
tatttaaattggcaatttgtagctaagcgattacgcgtttataccgaggtatcaaataattcc
attacgatcccagattattttgaaaatcggtttaaagataactcacatattcttcgtgttata
tctgctatcgtaattttgttattcttcactttttatacatcttcaggaatggttgcaggagca
aaattatttgaggcttcattcggtctccaatacgaaactgctctgtggattggtgcggttgta
gttgtatcttatacgttacttggaggatttctagcggttgcatggacagactttattcaaggt
attcttatgttccttgcactaattgttgttccaatcgtcgcattagatcaaatgggtggctgg
aatcaagcggtacaagctgttggtgaaattaatccttcccacctcaatatggttgaaggtgtt
ggaataatggcaattatttcatcacttgcttggggcttaggttattttggacagccacatatt
attgttcgttttatggcattacgttcggcgaaagatgttccgaaagcgaaatttattggaaca
gcttggatgattttaggactttatggagcaatctttactggttttgtaggactagcatttatc
agtacacaagaagtaccgattctgtctgaattcgggattcaagtagttaatgagaatggttta
caaatgttagccgatcctgaaaagatatttattgctttctcccaaatactattccatccagta
gttgccggtatcttactagcggcaatcttgtctgcaattatgagtaccgttgattcacagtta
cttgtatcatcttcagcggttgcagaagatttctataaagctattttccgtaaaaaagctact
ggtaaagagcttgtttgggttggacgtattgctacagtgataattgcgattgttgctttaatt
attgcaatgaacccagatagctctgtattggatctagttagttatgcatgggctggatttggt
gcagcatttggaccaattatcatcttgtcattattctggaagagaatcacaagaaatggtgca
ctagcgggtatcattgtaggtgccattacggtaattgtatggggagactttctatctggaggt
atctttgacctctacgaaattgttccaggctttatcttaaatatgattgtcaccgttattgtg
agtcttatcgataaaccgaatccagatttagaagctgactttgatgaaaccgtagaaaaaatg
aaagaataa 131 cbsT1 -
ATGTCGACCACACCGACACAGCCATCATCACGAAAACAGGCTGTTTACCCGTACTTGATCGTG
Lactobacillus
CTGTCGGGCATCGTCTTCACGGCCATCCCGGTATCGCTGGTCTGCAGTTGCGCAGGTATCTTC
johnsonii
TTCACGCCTGTCAGCAGCTACTTCCATGTTCCCAAGGCCGCATTCACCGGATATTTCAGCATA
TTCAGCATCACCATGGTCGCCTTCCTGCCGGTGGCCGGATGGCTGATGCACCGCTACGATCTG
CGCATCGTACTGACCGCAAGCACCGTCCTGGCTGGACTGGGCTGCCTGGGTATGTCCCGATCA
TCCGCCATGTGGCAGTTCTATCTATGCGGAGTGGTTCTGGGAATCGGCATGCCGGCCGTCCTC
TATCTGTCAGTGCCAACACTCATCAACGCCTGGTTCCGCAAGCGGGTCGGGTTCTTCATCGGC
CTGTGCATGGCCTTCACCGGCATAGGCGGCGTGATCTTCAACCAGATAGGCACCATGATCATC
AGATCCGCCCCTGATGGATGGAGGCGGGGATATCTGGTTTTCGCTATTCTCATCCTGGTGATC
ACCCTGCCCTTCACCATTTTCGTCATTCGCAGCACACCCGAACAGATGGGTCTGCATCCCTAC
GGCGCCGACCAGGAGCCTGATGCAGCTGAGACGGCCACCAATAGTGCAGGCACCGGGAGCAAA
GACCAAAAGAGTCCTGAGCCTGCAGCGTCAACCGTAGGCATGACTGCCTCCCAGGCCTTGCGC
TCCCCTGCCTTCTGGGCGCTGGCGCTCTTCTGCGGTCTGATCACCATGAATCAGACCATTTAC
CAGTTCCTGCCCTCCTACGCGGCATCCCTGCCATCCATGGCAGCCTACACGGGACTGATCGCC
TCCTCCTGCATGGCCGGCCAGGCCATCGGCAAGATCATCCTGGGCATGGTCAACGACGGCAGC
ATCGTAGGCGGTCTCTGTCTGGGCATCGGCGGCGGCATTCTCGGCGTCTGCCTCATGGTCGCC
TTCCCCGGATTGCCCGTGCTCCTCCTGCTGGGAGCCTTTGCCTTCGGCCTTGTCTACGCCTGC
ACTACTGTGCAGACACCAATCCTGGTTACAGCGGTCTTCGGCTCGCGCGACTACACCAACATC
TATGCACGTATCCAGATGGTTGGGTCCCTAGCCTCGGCCTTCGCAGCTCTCTTCTGGGGCGCC
ATCGCTGACCAGCCCCACGGCTACATCATCATGTTCGGTCTGAGCATCCTGATCATGGTTGTG
GCCTTGTTCCTAGGCATTATCCCTCTGAAAGGTACGCGCAAGTTGACCGATCAGATCGCCTGA 132
cbsT2 -
atgtctactgatgccgctactaaagataaagtagtaagcaagggctataaatacttcatggtt
Lactobacillus
ttcctttgtatgttaacccaagctattccttatggaattgctcaaaacattcagcctttgttt
johnsonii
atccaccctttagttaatactttccactttaccttagcatcgtacacattaatttttacgttt
ggtgcggtttttgcttcagttgcttctccatttattggtaaggcattagaaaaagttaacttc
cgactaatgtatttaattggtattggtctttctgctattgcctacgtaatttttggaattagt
acaaaactacccggtttctatattgccgctatcatttgtatggttggttcaaccttttactcc
ggccaaggtgttccctgggttattaaccactggttcccagcaaagggacgtggggctgcctta
ggaattgccttctgcggtggttctattggtaatatctttttacaaccagcaacccaagctatt
ttaaaacactacatgacaggtaatactaagaccggtcatttaacctctatggcaccattcttt
atctttgccgtagctttattagtaatcggtgtaattatcgcctgcttcattagaacccctaag
aaagacgaaattgttgtttctgatgcagaactagctgaaagcaagaaagctgaagccgcagcc
aaagctaaagagtttaaaggctggactagtaaacaagtgttacaaatgaaatggttctggatt
ttcagccttggtttcttaatcattggtttaggcttagcttctttaaatgaagactatgccgcc
ttccttgatactaagctttctttaaccgatgttggtttagttgggtcaatgtacggtgttggt
tgtttaatcggaaatatttctggtggtttcttatttgataaatttggtacagcaaaatcaatg
acctatgctggttgtatgtatattttatctattctgatgatgatctttattagcttccagcca
tatggttcatctattagtaaggctgctggcattggctatgctatcttttgcggcttagctgta
tttagttacatgtcaggcccagccttcatggcaaaagacctctttggttcaagagatcaaggt
gtcatgcttggatacgttggtttagcttatgcaattggctatgccattggtgctccactattt
gggattattaagggagcggcaagctttacagttgcttggtactttatgattgcctttgttgca
attggttttatcattttagtatttgccgttatccaaattaagagataccaaaagaaatacatt
gcagagcaagcagcaaaagctaatgctaaataa 133 amtB -
atgaagatagcgacgataaaaactgggcttgcttcactggcgatgcttccgggactggta
Escherichia coli
atggctgcacctgcggtggccgataaagccgacaatgcgtttatgatgatttgtactgcg K-12
MG1655 ctggtgctgtttatgactattccggggattgccctgttttacggtgggttgattcgcggc
(B0451;
aaaaacgtgctgtcgatgctgacgcaggtgacggtgacatttgcactggtctgtattctc
945084)
tgggtggtttacggttactcgctggcgtttggtgagggcaacaacttcttcggcaacatt
aactggttgatgctgaaaaacatcgaactgacggcggtgatgggcagcatttatcagtat
atccacgtggcgtttcagggatcgtttgcctgcattaccgtcggcttgatagttggggcg
ctggcggaacgaatccgcttctcagctgtgttgattttcgtggtggtatggctgacgctc
tcttacattccgattgcgcatatggtgtggggcggtggtttgctggcttctcacggtgcg
ctggatttcgcgggtggcaccgtggtgcacattaacgccgcaatcgccggtctggtgggc
gcgtatctgataggaaaacgcgtgggcttcggtaaagaggcgtttaaaccgcacaacctg
ccgatggtcttcaccgggactgccattctctatatcggttggtttggctttaacgccggg
tcagcgggcacggcgaatgaaatcgcggcactggcatttgtgaatactgtggtcgcaacg
gcggcggcaattcttggctggatcttcggtgaatgggcgctgcgtggtaagccttcactg
ctgggggcgtgttctggcgcgattgccggtctggtcggcgtgacgccagcctgcggctac
attggggttggcggcgcgttgattatcggcgtggtagctggtctggcgggcttgtggggc
gttaccatgctcaaacgcttgctgcgggtggatgatccctgcgatgtcttcggtgtgcac
ggcgtttgtggcattgtcggctgtatcatgaccgggatttttgccgccagctcgctgggc
ggcgtgggcttcgctgaaggtgtgacgatgggccatcagttgctggtacagctggaaagc
atcgccattacgatcgtctggtccggtgttgtggcatttatcggctacaaattggcggat
ctgacggttggtctgcgtgtaccggaagagcaggagcgagaagggctggatgtcaacagc
cacggcgagaatgcctataacgcgtaa 156 amtB -
MKIATIKTGLASLAMLPGLVMAAPAVADKADNAFMMICTALVLFMTIPGIALFYGGLIRG
Escherichia coli
KNVLSMLTQVTVTFALVCILWVVYGYSLAFGEGNNFFGNINWLMLKNIELTAVMGSIYQY K-12
MG1655 IHVAFQGSFACITVGLIVGALAERIRFSAVLIVVVWLTLSYIPIAHMVWGGGLLASHGA
(B0451;
LDFAGGTVVHINAAIAGLVGAYLIGKRVGFGKEAFKPHNLPMVFTGTAILYIGWFGFNAG
945084)
SAGTANEIAALAFVNTVVATAAAILGWIFGEWALRGKPSLLGACSGAIAGLVGVTPACGY
IGVGGALIIGVVAGLAGLWGVTMLKRLLRVDDPCDVFGVHGVCGIVGCIMTGIFAASSLG
GVGFAEGVTMGHQLLVQLESIAITIVWSGVVAFIGYKLADLTVGLRVPEEQEREGLDVNS
HGENAYNA 134 GABA permease
atggggcaatcatcgcaaccacatgagttaggcggcgggctgaagtcacgccacgtcaccatg
GabP -
ttgtctattgccggtgttatcggcgcaagtctgtttgtcggttccagcgtcgccatcgccgaa
Escherichia coli
gcgggcccggcggtattactggcctatctgttcgccggattactggtggttatgattatgcgg
atgttggcggaaatggcagttgccacgcccgataccggttcgttttccacctatgccgataaa
gccattggccgctgggcgggttataccatcggctggctgtactggtggttttgggtactggtt
atcccgctggaagccaacatcgccgctatgatcctgcactcgtgggttccaggcattcccatc
tggttattttccctcgtcattaccctcgccttaactggcagtaatttattaagcgttaaaaac
tacggcgaatttgagttctggctggcgctgtgcaaagtcatcgctatcctggcctttattttc
cttggtgcagtcgcaattagcggtttttacccttatgccgaagtgagcgggatctcaagattg
tgggatagcggcggctttatgcccaacggtttcggtgcggtattaagcgcgatgttgatcacc
atgttctcgtttatgggcgcagaaattgtcaccattgccgccgcggaatccgacacgccggaa
aaacatattgtccgcgccactaactcggttatctggcgtatttctatcttctatttgtgctct
atttttgtcgtagtggcgttaataccgtggaatatgccggggctgaaagccgttggttcttat
cgctcggttctggaattgctcaatattccccatgcgaaattaatcatggactgcgtgatatta
ctttccgtaaccagctgtctgaactcggcgctgtataccgcgtcaaggatgctctactcctta
agccgtcgcggtgatgcgcccgcggtaatgggcaaaatcaaccgcagtaaaaccccgtatgtg
gcggtgttactctccaccggagcggcatttttaacggtggtggtgaactattacgcacctgcg
aaagtgtttaaattcctgatagacagctccggtgctatcgccctgctggtttatttagtcatc
gccgtttcacagttgcggatgcgtaaaattctgcgagcagaaggaagcgaaattcgcttgcgc
atgtggctttacccgtggctcacctggctggtaataggctttattacctttgtgttggtagtg
atgctattccgcccggcgcaacagttagaagtgatctctaccggcttattagcgatagggatt
atctgtaccgtgccgattatggcgcgctggaaaaagctggtattgtggcaaaaaacacccgtt
cataatacgcgctga 157 GABA permease
MGQSSQPHELGGGLKSRHVTMLSIAGVIGASLFVGSSVAIAEAGPAVLLAYLFAGLLVVMIMR
GabP -
MLAEMAVATPDTGSFSTYADKAIGRWAGYTIGWLYWWFWVLVIPLEANIAAMILHSWVPGIPI
Escherichia coli
WLFSLVITLALTGSNLLSVKNYGEFEFWLALCKVIAILAFIFLGAVAISGFYPYAEVSGISRL
WDSGGFMPNGFGAVLSAMLITMFSFMGAEIVTIAAAESDTPEKHIVRATNSVIWRISIFYLCS
IFVVVALIPWNMPGLKAVGSYRSVLELLNIPHAKLIMDCVILLSVTSCLNSALYTASRMLYSL
SRRGDAPAVMGKINRSKTPYVAVLLSTGAAFLTVVVNYYAPAKVFKFLIDSSGAIALLVYLVI
AVSQLRMRKILRAEGSEIRLRMWLYPWLTWLVIGFITFVLVVMLFRPAQQLEVISTGLLAIGI
ICTVPIMARWKKLVLWQKTPVHNTR 135 mtnH -
atgacgaactatcgcgttgagagtagcagcggacgggcggcgcgcaagatgaggctcgcatta
Escherichia coli
atgggacctgcgttcattgcggcgattggttatatcgatcccggtaactttgcgaccaatatt
caggcgggtgccagcttcggctatcagctactgtgggttgtcgtttgggccaacctgatggcg
atgctgattcagatcctctctgccaaactagggattgccaccggtaaaaatctggcggagcag
attcgcgatcactatccgcgtcccgtagtgtggttctattgggttcaggcagaaattattgcg
atggcaaccgacctggcggaatttattggtgcggcgatcggttttaaactcattcttggtgtc
tcgttgttgcagggcgcggtgctgacggggatcgcgactttcctgattttaatgctgcaacgt
cgcgggcaaaaaccgctggagaaagtgattggcgggttactgttgtttgttgccgcggcttac
attgtcgagttgattttctcccagcctaacctggcgcagctgggtaaaggaatggtgatcccg
agtttacctacttcggaggcggtcttcctggcagcaggcgtgttaggggcgacgattatgccg
catgtgatttatttgcactcctcgctcactcagcatttacatggcggttcgcgtcaacaacgt
tattccgccaccaaatgggatgtggctatcgccatgacgattgccggttttgtcaatctggcg
atgatggctacagctgcggcggcgttccacttttctggtcatactggtgttgccgatcttgat
gaggcttatctgacgctgcaaccgctgttaagccatgctgcggcaacggtctttgggttaagt
ctggttgctgccggactgtcctcaacggtggtggggacactggcggggcaggtggtgatgcag
ggattcattcgcttccatatcccgctgtgggtgcgtcgtacagtcaccatgttgccgtcattt
attgtcattctgatgggattagatccgacacggattctggttatgagtcaggtgctgttaagt
tttggtatcgccctggcgctggttccactgctgattttcaccagtgacagcaagttgatgggc
gatctggtgaacagcaaacgcgtaaaacagacaggctgggtgattgtagtgctggtcgtggcg
ctgaatatctggttgttggtggggacggcgctgggattgtag 158 mtnH -
MTNYRVESSSGRAARKMRLALMGPAFIAAIGYIDPGNFATNIQAGASFGYQLLWVVVWANLMA
Escherichia coli
MLIQILSAKLGIATGKNLAEQIRDHYPRPVVWFYWVQAEIIAMATDLAEFIGAAIGFKLILGV
SLLQGAVLTGIATFLILMLQRRGQKPLEKVIGGLLLFVAAAYIVELIFSQPNLAQLGKGMVIP
SLPTSEAVFLAAGVLGATIMPHVIYLHSSLTQHLHGGSRQQRYSATKWDVAIAMTIAGFVNLA
MMATAAAAFHFSGHTGVADLDEAYLTLQPLLSHAAATVFGLSLVAAGLSSTVVGTLAGQVVMQ
GFIRFHIPLWVRRTVTMLPSFIVILMGLDPTRILVMSQVLLSFGIALALVPLLIFTSDSKLMG
DLVNSKRVKQTGWVIVVLVVALNIWLLVGTALGL
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 172 <210> SEQ ID NO 1 <211> LENGTH: 290
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(290) <223> OTHER INFORMATION: FNR Responsive
Promoter <400> SEQUENCE: 1 gtcagcataa caccctgacc tctcattaat
tgttcatgcc gggcggcact atcgtcgtcc 60 ggccttttcc tctcttactc
tgctacgtac atctatttct ataaatccgt tcaatttgtc 120 tgttttttgc
acaaacatga aatatcagac aattccgtga cttaagaaaa tttatacaaa 180
tcagcaatat accccttaag gagtatataa aggtgaattt gatttacatc aataagcggg
240 gttgctgaat cgttaaggta ggcggtaata gaaaagaaat cgaggcaaaa 290
<210> SEQ ID NO 2 <211> LENGTH: 173 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(173)
<223> OTHER INFORMATION: FNR Responsive Promoter <400>
SEQUENCE: 2 atttcctctc atcccatccg gggtgagagt cttttccccc gacttatggc
tcatgcatgc 60 atcaaaaaag atgtgagctt gatcaaaaac aaaaaatatt
tcactcgaca ggagtattta 120 tattgcgccc gttacgtggg cttcgactgt
aaatcagaaa ggagaaaaca cct 173 <210> SEQ ID NO 3 <211>
LENGTH: 305 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(305) <223> OTHER INFORMATION: FNR
Responsive Promoter <400> SEQUENCE: 3 gtcagcataa caccctgacc
tctcattaat tgttcatgcc gggcggcact atcgtcgtcc 60 ggccttttcc
tctcttactc tgctacgtac atctatttct ataaatccgt tcaatttgtc 120
tgttttttgc acaaacatga aatatcagac aattccgtga cttaagaaaa tttatacaaa
180 tcagcaatat accccttaag gagtatataa aggtgaattt gatttacatc
aataagcggg 240 gttgctgaat cgttaaggat ccctctagaa ataattttgt
ttaactttaa gaaggagata 300 tacat 305 <210> SEQ ID NO 4
<211> LENGTH: 180 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(180) <223> OTHER
INFORMATION: FNR Responsive Promoter <400> SEQUENCE: 4
catttcctct catcccatcc ggggtgagag tcttttcccc cgacttatgg ctcatgcatg
60 catcaaaaaa gatgtgagct tgatcaaaaa caaaaaatat ttcactcgac
aggagtattt 120 atattgcgcc cggatccctc tagaaataat tttgtttaac
tttaagaagg agatatacat 180 <210> SEQ ID NO 5 <211>
LENGTH: 199 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(199) <223> OTHER INFORMATION: FNR
Responsive Promoter <400> SEQUENCE: 5 agttgttctt attggtggtg
ttgctttatg gttgcatcgt agtaaatggt tgtaacaaaa 60 gcaatttttc
cggctgtctg tatacaaaaa cgccgtaaag tttgagcgaa gtcaataaac 120
tctctaccca ttcagggcaa tatctctctt ggatccctct agaaataatt ttgtttaact
180 ttaagaagga gatatacat 199 <210> SEQ ID NO 6 <211>
LENGTH: 1647 <212> TYPE: DNA <213> ORGANISM:
Lactococcus lactis <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1647) <223> OTHER
INFORMATION: kivD (Lactococcus lactis IFPL730) <400>
SEQUENCE: 6 atgtatacag taggagatta cctattagac cgattacacg agttaggaat
tgaagaaatt 60 tttggagtcc ctggagacta taacttacaa tttttagatc
aaattatttc ccacaaggat 120 atgaaatggg tcggaaatgc taatgaatta
aatgcttcat atatggctga tggctatgct 180 cgtactaaaa aagctgccgc
atttcttaca acctttggag taggtgaatt gagtgcagtt 240 aatggattag
caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300
acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt
360 aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact
gacagcagaa 420 aatgcaaccg ttgaaattga ccgagtactt tctgcactat
taaaagaaag aaaacctgtc 480 tatatcaact taccagttga tgttgctgct
gcaaaagcag agaaaccctc actccctttg 540 aaaaaggaaa actcaacttc
aaatacaagt gaccaagaaa ttttgaacaa aattcaagaa 600 agcttgaaaa
atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660
ttagaaaaaa cagtcactca atttatttca aagacaaaac tacctattac gacattaaac
720 tttggtaaaa gttcagttga tgaagccctc ccttcatttt taggaatcta
taatggtaca 780 ctctcagagc ctaatcttaa agaattcgtg gaatcagccg
acttcatctt gatgcttgga 840 gttaaactca cagactcttc aacaggagcc
ttcactcatc atttaaatga aaataaaatg 900 atttcactga atatagatga
aggaaaaata tttaacgaaa gaatccaaaa ttttgatttt 960 gaatccctca
tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020
gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg
1080 caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca
agggacatca 1140 ttctttggcg cttcatcaat tttcttaaaa tcaaagagtc
attttattgg tcaaccctta 1200 tggggatcaa ttggatatac attcccagca
gcattaggaa gccaaattgc agataaagaa 1260 agcagacacc ttttatttat
tggtgatggt tcacttcaac ttacagtgca agaattagga 1320 ttagcaatca
gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380
gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac
1440 tcaaaattac cagaatcgtt tggagcaaca gaagatcgag tagtctcaaa
aatcgttaga 1500 actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag
cagatccaaa tagaatgtac 1560 tggattgagt taattttggc aaaagaaggt
gcaccaaaag tactgaaaaa aatgggcaaa 1620 ctatttgctg aacaaaataa atcataa
1647 <210> SEQ ID NO 7 <211> LENGTH: 5739 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic: Tet-bkd
construct sequence <400> SEQUENCE: 7 gtaaaacgac ggccagtgaa
ttcgttaaga cccactttca catttaagtt gtttttctaa 60 tccgcatatg
atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa 120
taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc
180 ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa
aatgccccac 240 agcgctgagt gcatataatg cattctctag tgaaaaacct
tgttggcata aaaaggctaa 300 ttgattttcg agagtttcat actgtttttc
tgtaggccgt gtacctaaat gtacttttgc 360 tccatcgcga tgacttagta
aagcacatct aaaactttta gcgttattac gtaaaaaatc 420 ttgccagctt
tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat 480
ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc
540 tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga
cctcattaag 600 cagctctaat gcgctgttaa tcactttact tttatctaat
ctagacatca ttaattccta 660 atttttgttg acactctatc attgatagag
ttattttacc actccctatc agtgatagag 720 aaaagtgaac tctagaaata
attttgttta actttaagaa ggagatatac atatgagtga 780 ttacgagccg
ttgcgtctgc atgtcccgga gcccaccggg cgtcctggct gcaagaccga 840
cttttcctat ctgcacctgt cccccgccgg cgaggtacgc aagccgccgg tggatgtcga
900 gcccgccgaa accagcgacc tggcctacag cctggtacgt gtgctcgacg
acgacggcca 960 cgccgtcggt ccctggaatc cgcagctcag caacgaacaa
ctgctgcgcg gcatgcgggc 1020 gatgctcaag acccgcctgt tcgacgcgcg
catgctcacc gcgcaacggc agaaaaagct 1080 ttccttctat atgcaatgcc
tcggcgagga agccatcgcc accgcccaca ccctggccct 1140 gcgcgacggc
gacatgtgct ttccgaccta tcgccagcaa ggcatcctga tcacccgcga 1200
atacccgctg gtggacatga tctgccagct tctctccaac gaggccgacc cgctcaaggg
1260 ccgccagctg ccgatcatgt actcgagcaa ggaggcaggt ttcttctcca
tctccggcaa 1320 cctcgccacc cagttcatcc aggcggtcgg ctggggcatg
gcctcggcga tcaagggcga 1380 cacgcgcatc gcctcggcct ggatcggcga
cggcgccacc gccgagtcgg acttccacac 1440 cgccctcacc ttcgcccatg
tctaccgcgc gccggtaatc ctcaacgtgg tcaacaacca 1500 gtgggcgatc
tccaccttcc aggccatcgc cggcggcgaa ggcaccacct tcgccaaccg 1560
tggcgtgggc tgcgggatcg cctcgctgcg ggtcgacggc aatgacttcc tggcggtcta
1620 cgccgcctcc gagtgggccg ccgagcgcgc ccggcgcaac ctcgggccga
gcctgatcga 1680 atgggtcacc taccgcgccg gcccgcactc gacttcggac
gacccgtcca agtaccgccc 1740 cgccgacgac tggaccaact tcccgctggg
cgacccgatc gcccgcctga agcggcacat 1800 gatcggcctc ggcatctggt
cggaggaaca gcacgaagcc acccacaagg ccctcgaagc 1860 cgaagtactg
gccgcgcaga aacaggcgga gagccatggc accctgatcg acggccgggt 1920
gccgagcgcc gccagcatgt tcgaggacgt ctatgcagaa ctgccggagc atctgcgccg
1980 gcaacgccag gagctcgggg tatgaatgcc atgaacccgc aacacgagaa
cgcccagacg 2040 gtcaccagca tgaccatgat ccaggcgctg cgctcggcga
tggacatcat gctcgagcgc 2100 gacgacgacg tggtggtatt cggccaggac
gtcggctact tcggcggcgt gttccgctgc 2160 accgaaggcc tgcagaagaa
atacggcacc tcgcgggtgt tcgatgcgcc gatctccgag 2220 agcggcatca
tcggcgccgc ggtcggcatg ggtgcctacg gcctgcgccc ggtggtggag 2280
atccagttcg ccgactacgt ctacccggcc tccgaccagt tgatctccga ggcggcgcgc
2340 ctgcgctatc gctcggccgg cgacttcatc gtgccgatga ccgtacgcat
gccctgtggc 2400 ggcggcatct acggcgggca aacgcacagc cagagcccgg
aggcgatgtt cacccaggtc 2460 tgcggcctgc gcacggtgat gccgtccaac
ccctacgacg ccaagggcct gctgatcgcc 2520 tgcatcgaga acgacgaccc
ggtgatcttc ctcgagccca agcgcctcta caacggcccg 2580 ttcgatggcc
accacgaccg cccggtgacg ccctggtcca agcatccggc cagccaggtg 2640
ccggacggct actacaaggt gccgctggac aaggcggcga tcgtccgccc cggcgcggcg
2700 ctgaccgtgc tgacctacgg caccatggtc tacgtggccc aggccgcggc
cgacgaaacc 2760 ggcctggacg ccgagatcat cgacctgcgc agcctctggc
cgctggacct ggaaaccatc 2820 gtcgcctcgg tgaagaagac cggccgctgc
gtcatcgccc acgaggcgac ccgcacctgt 2880 gggttcggcg ccgagctgat
gtcgctggtg caggagcact gcttccacca cctggaggcg 2940 ccgatcgagc
gcgtcaccgg ttgggacacc ccctacccgc atgcccagga gtgggcgtat 3000
ttccccggcc ccgcgcgcgt cggcgcggca ttcaagcgtg tgatggaggt ctgaatgggt
3060 acccatgtga tcaagatgcc ggacatcggg gaaggcatcg ccgaggtcga
actggtggag 3120 tggcatgtcc aggtcggcga ctcggtcaat gaagaccagg
tcctcgccga ggtgatgacc 3180 gacaaggcca cggtggagat tccctcgccg
gtggccggac gcatcctcgc cctcggcggc 3240 cagccgggcc aggtgatggc
ggtgggcggc gaactgatcc gcctggaggt ggaaggcgcc 3300 ggcaacctcg
ccgagagtcc ggccgcggcg acgccggccg cgcccgtcgc cgccaccccg 3360
gagaaaccga aggaagcccc ggtcgcggcg ccgaaagccg ccgccgaagc gccgcgcgcc
3420 ttgcgcgaca gcgaggcgcc acggcagcgg cgccagcccg gcgaacgccc
gctggcctcc 3480 cccgcggtgc gccagcgcgc ccgcgacctg ggcatcgagt
tgcagttcgt gcagggcagc 3540 ggtcccgccg gacgcgtcct ccacgaggac
ctcgatgcct acctgaccca ggatggcagc 3600 gtcgcgcgca gcggcggcgc
cgcgcagggg tatgccgagc gacacgacga acaggcggtg 3660 ccggtgatcg
gcctgcgtcg caagatcgcc cagaagatgc aggacgccaa gcgacgcatc 3720
ccgcatttca gctatgtcga ggaaatcgac gtcaccgatc tggaagccct gcgcgcccat
3780 ctcaaccaga aatggggtgg ccagcgcggc aagctgaccc tgctgccgtt
cctggtccgc 3840 gccatggtcg tggcgctgcg cgacttcccg cagttgaacg
cgcgctacga cgacgaggcc 3900 gaggtggtca cccgctacgg cgcggtgcac
gtcggcatcg ccacccagag cgacaacggc 3960 ctgatggtgc cggtgctgcg
ccacgccgaa tcgcgcgacc tctggggcaa cgccagcgaa 4020 gtggcgcgcc
tggccgaagc cgcacgcagc ggcaaggcgc aacgccagga gctgtccggc 4080
tcgaccatca ccctgagcag cctcggcgtg ctcggcggga tcgtcagcac accggtgatc
4140 aaccatccgg aggtggccat cgtcggcgtc aaccgcatcg tcgagcgacc
gatggtggtc 4200 ggcggcaaca tcgtcgtgcg caagatgatg aacctctcct
cctccttcga ccaccgggtg 4260 gtcgacggga tggacgcggc ggccttcatc
caggccgtgc gcggcctgct cgaacatccc 4320 gccaccctgt tcctggagta
agcgatgagc cagatcctga agacttccct gctgatcgtc 4380 ggcggcggtc
ccggcggcta cgtcgcggcg atccgtgccg ggcaactggg cattcccacc 4440
gtactggtgg agggcgccgc cctcggcggc acctgtctga acgtcggctg catcccgtcg
4500 aaggcgctga tccacgccgc cgaggaatac ctcaaggccc gccactatgc
cagccggtcg 4560 gcgctgggca tccaggtaca ggcgccgagc atcgacatcg
cccgcaccgt ggaatggaag 4620 gacgccatcg tcgaccgcct caccagcggc
gtcgccgcgc tgctgaagaa acacggggtc 4680 gatgtcgtcc agggctgggc
gaggatcctc gacggcaaaa gcgtggcggt cgaactcgcc 4740 ggcggcggca
gccagcgcat cgagtgcgag catctgctgc tggccgccgg ctcgcagagc 4800
gtcgagctac cgatcctgcc gctgggcggc aaggtgatct cctccaccga ggcgctggcg
4860 cccggcagcc tgcccaagcg cctggtggtg gtcggcggcg gctacatcgg
cctggagctg 4920 ggtaccgcct accgcaagct cggcgtcgag gtggcggtgg
tggaagcgca accacgcatc 4980 ctgccgggct acgacgaaga actgaccaag
ccggtggccc aggccttgcg caggctgggc 5040 gtcgagctgt acctcgggca
cagcctgctg ggcccgagcg agaacggcgt gcgggtccgc 5100 gacggcgccg
gcgaggagcg cgagatcgcc gccgaccagg tactggtggc ggtcggccgc 5160
aagccgcgca gcgaaggctg gaacctggaa agcctgggcc tggacatgaa cggccgggcg
5220 gtgaaggtcg acgaccagtg ccgcacctcg atgcgcaatg tctgggccat
aggcgatctc 5280 gccggcgagc cgatgctcgc gcaccgggcc atggcccagg
gcgagatggt cgccgagctg 5340 atcgccggca agcgtcgcca gttcgccccg
gtggcgatcc ccgcggtgtg cttcaccgat 5400 ccggaagtgg tggtcgccgg
gttgtccccg gagcaggcga aggatgccgg cctggactgc 5460 ctggtggcga
gcttcccgtt cgccgccaac ggtcgcgcca tgaccctgga ggccaacgaa 5520
ggcttcgtcc gcgtggtggc gcgtcgcgac aaccacctgg tcgtcggctg gcaggcggtg
5580 ggcaaggcgg tttcggaact gtccacggcc ttcgcccagt cgctggagat
gggcgcccgc 5640 ctggaagaca tcgccggcac catccacgcc catccgaccc
tcggcgaagc ggtccaggaa 5700 gccgccctgc gcgcgctggg acacgccctg
cacatctga 5739 <210> SEQ ID NO 8 <211> LENGTH: 6780
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic:
Tet-ldh-bkd construct <400> SEQUENCE: 8 gtaaaacgac ggccagtgaa
ttcgttaaga cccactttca catttaagtt gtttttctaa 60 tccgcatatg
atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa 120
taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc
180 ttctttagcg acttgatgct cttgatcttc caatacgcaa cctaaagtaa
aatgccccac 240 agcgctgagt gcatataatg cattctctag tgaaaaacct
tgttggcata aaaaggctaa 300 ttgattttcg agagtttcat actgtttttc
tgtaggccgt gtacctaaat gtacttttgc 360 tccatcgcga tgacttagta
aagcacatct aaaactttta gcgttattac gtaaaaaatc 420 ttgccagctt
tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat 480
ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc
540 tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga
cctcattaag 600 cagctctaat gcgctgttaa tcactttact tttatctaat
ctagacatca ttaattccta 660 atttttgttg acactctatc attgatagag
ttattttacc actccctatc agtgatagag 720 aaaagtgaac tctagaaata
attttgttta actttaagaa ggagatatac atatgttcga 780 catgatggac
gcggcccggc tcgagggtct gcacctcgcc caagacccgg ccacgggact 840
caaggccatt atcgccatcc acagcacgcg actcggcccg gcgctgggtg gttgtcgcta
900 cctgccttac cccaacgacg aagccgccat cggcgacgcc atccgcctgg
cccagggcat 960 gagctacaag gcggccctgg ccgggctgga gcagggcggc
ggcaaggcgg tgatcatccg 1020 cccgccgcac ctggacaatc gcggcgcgct
gttcgaggcc ttcgggcgct tcatcgaaag 1080 cctcggcgga cgctacatca
ctgcggtgga cagcggtacc tccagcgccg acatggactg 1140 catcgcccag
cagacccgcc acgtcaccag caccacccag gccggcgacc cctcgccgca 1200
taccgccctc ggcgtgttcg ccgggattcg cgccagcgcc caggcgcgcc tcggcagcga
1260 cgacctggaa ggcctgcggg tcgcggtgca ggggctcggc cacgtcggct
acgcattggc 1320 cgagcaactg gcggcggtcg gcgccgagct gctggtctgc
gacctcgatc ccggccgggt 1380 gcaactggcc gtcgagcagc tcggtgccca
tccgctggcg ccggaggcat tgctctccac 1440 cccttgcgac atcctcgcgc
cctgcggcct gggcggcgtg ctcaccagcc agagcgtcag 1500 ccagttgcgc
tgcgcggcgg tggccggggc ggcgaacaac cagttggagc ggccggaggt 1560
cgccgacgag ctggaggcgc gcggcatcct ctatgcgccg gactacgtga tcaactccgg
1620 cggcctgatc tacgtcgccc tcaagcaccg cggcgccgat ccgcacagca
tcaccgcgca 1680 cctggcgcgg attcccgcgc ggctcaccga gatctatgcc
catgcccagg ccgaccacca 1740 gtcgccggcg cggatcgccg accgtctggc
ggaacggatt ctctacggcc cgcagtgaga 1800 aggagatata catatgagtg
attacgagcc gttgcgtctg catgtcccgg agcccaccgg 1860 gcgtcctggc
tgcaagaccg acttttccta tctgcacctg tcccccgccg gcgaggtacg 1920
caagccgccg gtggatgtcg agcccgccga aaccagcgac ctggcctaca gcctggtacg
1980 tgtgctcgac gacgacggcc acgccgtcgg tccctggaat ccgcagctca
gcaacgaaca 2040 actgctgcgc ggcatgcggg cgatgctcaa gacccgcctg
ttcgacgcgc gcatgctcac 2100 cgcgcaacgg cagaaaaagc tttccttcta
tatgcaatgc ctcggcgagg aagccatcgc 2160 caccgcccac accctggccc
tgcgcgacgg cgacatgtgc tttccgacct atcgccagca 2220 aggcatcctg
atcacccgcg aatacccgct ggtggacatg atctgccagc ttctctccaa 2280
cgaggccgac ccgctcaagg gccgccagct gccgatcatg tactcgagca aggaggcagg
2340 tttcttctcc atctccggca acctcgccac ccagttcatc caggcggtcg
gctggggcat 2400 ggcctcggcg atcaagggcg acacgcgcat cgcctcggcc
tggatcggcg acggcgccac 2460 cgccgagtcg gacttccaca ccgccctcac
cttcgcccat gtctaccgcg cgccggtaat 2520 cctcaacgtg gtcaacaacc
agtgggcgat ctccaccttc caggccatcg ccggcggcga 2580 aggcaccacc
ttcgccaacc gtggcgtggg ctgcgggatc gcctcgctgc gggtcgacgg 2640
caatgacttc ctggcggtct acgccgcctc cgagtgggcc gccgagcgcg cccggcgcaa
2700 cctcgggccg agcctgatcg aatgggtcac ctaccgcgcc ggcccgcact
cgacttcgga 2760 cgacccgtcc aagtaccgcc ccgccgacga ctggaccaac
ttcccgctgg gcgacccgat 2820 cgcccgcctg aagcggcaca tgatcggcct
cggcatctgg tcggaggaac agcacgaagc 2880 cacccacaag gccctcgaag
ccgaagtact ggccgcgcag aaacaggcgg agagccatgg 2940 caccctgatc
gacggccggg tgccgagcgc cgccagcatg ttcgaggacg tctatgcaga 3000
actgccggag catctgcgcc ggcaacgcca ggagctcggg gtatgaatgc catgaacccg
3060 caacacgaga acgcccagac ggtcaccagc atgaccatga tccaggcgct
gcgctcggcg 3120 atggacatca tgctcgagcg cgacgacgac gtggtggtat
tcggccagga cgtcggctac 3180 ttcggcggcg tgttccgctg caccgaaggc
ctgcagaaga aatacggcac ctcgcgggtg 3240 ttcgatgcgc cgatctccga
gagcggcatc atcggcgccg cggtcggcat gggtgcctac 3300 ggcctgcgcc
cggtggtgga gatccagttc gccgactacg tctacccggc ctccgaccag 3360
ttgatctccg aggcggcgcg cctgcgctat cgctcggccg gcgacttcat cgtgccgatg
3420 accgtacgca tgccctgtgg cggcggcatc tacggcgggc aaacgcacag
ccagagcccg 3480 gaggcgatgt tcacccaggt ctgcggcctg cgcacggtga
tgccgtccaa cccctacgac 3540 gccaagggcc tgctgatcgc ctgcatcgag
aacgacgacc cggtgatctt cctcgagccc 3600 aagcgcctct acaacggccc
gttcgatggc caccacgacc gcccggtgac gccctggtcc 3660 aagcatccgg
ccagccaggt gccggacggc tactacaagg tgccgctgga caaggcggcg 3720
atcgtccgcc ccggcgcggc gctgaccgtg ctgacctacg gcaccatggt ctacgtggcc
3780 caggccgcgg ccgacgaaac cggcctggac gccgagatca tcgacctgcg
cagcctctgg 3840 ccgctggacc tggaaaccat cgtcgcctcg gtgaagaaga
ccggccgctg cgtcatcgcc 3900 cacgaggcga cccgcacctg tgggttcggc
gccgagctga tgtcgctggt gcaggagcac 3960 tgcttccacc acctggaggc
gccgatcgag cgcgtcaccg gttgggacac cccctacccg 4020 catgcccagg
agtgggcgta tttccccggc cccgcgcgcg tcggcgcggc attcaagcgt 4080
gtgatggagg tctgaatggg tacccatgtg atcaagatgc cggacatcgg ggaaggcatc
4140 gccgaggtcg aactggtgga gtggcatgtc caggtcggcg actcggtcaa
tgaagaccag 4200 gtcctcgccg aggtgatgac cgacaaggcc acggtggaga
ttccctcgcc ggtggccgga 4260 cgcatcctcg ccctcggcgg ccagccgggc
caggtgatgg cggtgggcgg cgaactgatc 4320 cgcctggagg tggaaggcgc
cggcaacctc gccgagagtc cggccgcggc gacgccggcc 4380 gcgcccgtcg
ccgccacccc ggagaaaccg aaggaagccc cggtcgcggc gccgaaagcc 4440
gccgccgaag cgccgcgcgc cttgcgcgac agcgaggcgc cacggcagcg gcgccagccc
4500 ggcgaacgcc cgctggcctc ccccgcggtg cgccagcgcg cccgcgacct
gggcatcgag 4560 ttgcagttcg tgcagggcag cggtcccgcc ggacgcgtcc
tccacgagga cctcgatgcc 4620 tacctgaccc aggatggcag cgtcgcgcgc
agcggcggcg ccgcgcaggg gtatgccgag 4680 cgacacgacg aacaggcggt
gccggtgatc ggcctgcgtc gcaagatcgc ccagaagatg 4740 caggacgcca
agcgacgcat cccgcatttc agctatgtcg aggaaatcga cgtcaccgat 4800
ctggaagccc tgcgcgccca tctcaaccag aaatggggtg gccagcgcgg caagctgacc
4860 ctgctgccgt tcctggtccg cgccatggtc gtggcgctgc gcgacttccc
gcagttgaac 4920 gcgcgctacg acgacgaggc cgaggtggtc acccgctacg
gcgcggtgca cgtcggcatc 4980 gccacccaga gcgacaacgg cctgatggtg
ccggtgctgc gccacgccga atcgcgcgac 5040 ctctggggca acgccagcga
agtggcgcgc ctggccgaag ccgcacgcag cggcaaggcg 5100 caacgccagg
agctgtccgg ctcgaccatc accctgagca gcctcggcgt gctcggcggg 5160
atcgtcagca caccggtgat caaccatccg gaggtggcca tcgtcggcgt caaccgcatc
5220 gtcgagcgac cgatggtggt cggcggcaac atcgtcgtgc gcaagatgat
gaacctctcc 5280 tcctccttcg accaccgggt ggtcgacggg atggacgcgg
cggccttcat ccaggccgtg 5340 cgcggcctgc tcgaacatcc cgccaccctg
ttcctggagt aagcgatgag ccagatcctg 5400 aagacttccc tgctgatcgt
cggcggcggt cccggcggct acgtcgcggc gatccgtgcc 5460 gggcaactgg
gcattcccac cgtactggtg gagggcgccg ccctcggcgg cacctgtctg 5520
aacgtcggct gcatcccgtc gaaggcgctg atccacgccg ccgaggaata cctcaaggcc
5580 cgccactatg ccagccggtc ggcgctgggc atccaggtac aggcgccgag
catcgacatc 5640 gcccgcaccg tggaatggaa ggacgccatc gtcgaccgcc
tcaccagcgg cgtcgccgcg 5700 ctgctgaaga aacacggggt cgatgtcgtc
cagggctggg cgaggatcct cgacggcaaa 5760 agcgtggcgg tcgaactcgc
cggcggcggc agccagcgca tcgagtgcga gcatctgctg 5820 ctggccgccg
gctcgcagag cgtcgagcta ccgatcctgc cgctgggcgg caaggtgatc 5880
tcctccaccg aggcgctggc gcccggcagc ctgcccaagc gcctggtggt ggtcggcggc
5940 ggctacatcg gcctggagct gggtaccgcc taccgcaagc tcggcgtcga
ggtggcggtg 6000 gtggaagcgc aaccacgcat cctgccgggc tacgacgaag
aactgaccaa gccggtggcc 6060 caggccttgc gcaggctggg cgtcgagctg
tacctcgggc acagcctgct gggcccgagc 6120 gagaacggcg tgcgggtccg
cgacggcgcc ggcgaggagc gcgagatcgc cgccgaccag 6180 gtactggtgg
cggtcggccg caagccgcgc agcgaaggct ggaacctgga aagcctgggc 6240
ctggacatga acggccgggc ggtgaaggtc gacgaccagt gccgcacctc gatgcgcaat
6300 gtctgggcca taggcgatct cgccggcgag ccgatgctcg cgcaccgggc
catggcccag 6360 ggcgagatgg tcgccgagct gatcgccggc aagcgtcgcc
agttcgcccc ggtggcgatc 6420 cccgcggtgt gcttcaccga tccggaagtg
gtggtcgccg ggttgtcccc ggagcaggcg 6480 aaggatgccg gcctggactg
cctggtggcg agcttcccgt tcgccgccaa cggtcgcgcc 6540 atgaccctgg
aggccaacga aggcttcgtc cgcgtggtgg cgcgtcgcga caaccacctg 6600
gtcgtcggct ggcaggcggt gggcaaggcg gtttcggaac tgtccacggc cttcgcccag
6660 tcgctggaga tgggcgcccg cctggaagac atcgccggca ccatccacgc
ccatccgacc 6720 ctcggcgaag cggtccagga agccgccctg cgcgcgctgg
gacacgccct gcacatctga 6780 <210> SEQ ID NO 9 <211>
LENGTH: 5597 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic: Tet-livKHMGF construct <400> SEQUENCE: 9
ccagtgaatt cgttaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat
60 caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata
attcgatagc 120 ttgtcgtaat aatggcggca tactatcagt agtaggtgtt
tccctttctt ctttagcgac 180 ttgatgctct tgatcttcca atacgcaacc
taaagtaaaa tgccccacag cgctgagtgc 240 atataatgca ttctctagtg
aaaaaccttg ttggcataaa aaggctaatt gattttcgag 300 agtttcatac
tgtttttctg taggccgtgt acctaaatgt acttttgctc catcgcgatg 360
acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc
420 cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg
ctaaggcgtc 480 gagcaaagcc cgcttatttt ttacatgcca atacaatgta
ggctgctcta cacctagctt 540 ctgggcgagt ttacgggttg ttaaaccttc
gattccgacc tcattaagca gctctaatgc 600 gctgttaatc actttacttt
tatctaatct agacatcatt aattcctaat ttttgttgac 660 actctatcat
tgatagagtt attttaccac tccctatcag tgatagagaa aagtgaactc 720
tagaaataat tttgtttaac tttaagaagg agatatacat atgaaacgga atgcgaaaac
780 tatcatcgca gggatgattg cactggcaat ttcacacacc gctatggctg
acgatattaa 840 agtcgccgtt gtcggcgcga tgtccggccc gattgcccag
tggggcgata tggaatttaa 900 cggcgcgcgt caggcaatta aagacattaa
tgccaaaggg ggaattaagg gcgataaact 960 ggttggcgtg gaatatgacg
acgcatgcga cccgaaacaa gccgttgcgg tcgccaacaa 1020 aatcgttaat
gacggcatta aatacgttat tggtcatctg tgttcttctt ctacccagcc 1080
tgcgtcagat atctatgaag acgaaggtat tctgatgatc tcgccgggag cgaccaaccc
1140 ggagctgacc caacgcggtt atcaacacat tatgcgtact gccgggctgg
actcttccca 1200 ggggccaacg gcggcaaaat acattcttga gacggtgaag
ccccagcgca tcgccatcat 1260 tcacgacaaa caacagtatg gcgaagggct
ggcgcgttcg gtgcaggacg ggctgaaagc 1320 ggctaacgcc aacgtcgtct
tcttcgacgg tattaccgcc ggggagaaag atttctccgc 1380 gctgatcgcc
cgcctgaaaa aagaaaacat cgacttcgtt tactacggcg gttactaccc 1440
ggaaatgggg cagatgctgc gccaggcccg ttccgttggc ctgaaaaccc agtttatggg
1500 gccggaaggt gtgggtaatg cgtcgttgtc gaacattgcc ggtgatgccg
ccgaaggcat 1560 gttggtcact atgccaaaac gctatgacca ggatccggca
aaccagggca tcgttgatgc 1620 gctgaaagca gacaagaaag atccgtccgg
gccttatgtc tggatcacct acgcggcggt 1680 gcaatctctg gcgactgccc
ttgagcgtac cggcagcgat gagccgctgg cgctggtgaa 1740 agatttaaaa
gctaacggtg caaacaccgt gattgggccg ctgaactggg atgaaaaagg 1800
cgatcttaag ggatttgatt ttggtgtctt ccagtggcac gccgacggtt catccacggc
1860 agccaagtga tcatcccacc gcccgtaaaa tgcgggcggg tttagaaagg
ttaccttatg 1920 tctgagcagt ttttgtattt cttgcagcag atgtttaacg
gcgtcacgct gggcagtacc 1980 tacgcgctga tagccatcgg ctacaccatg
gtttacggca ttatcggcat gatcaacttc 2040 gcccacggcg aggtttatat
gattggcagc tacgtctcat ttatgatcat cgccgcgctg 2100 atgatgatgg
gcattgatac cggctggctg ctggtagctg cgggattcgt cggcgcaatc 2160
gtcattgcca gcgcctacgg ctggagtatc gaacgggtgg cttaccgccc ggtgcgtaac
2220 tctaagcgcc tgattgcact catctctgca atcggtatgt ccatcttcct
gcaaaactac 2280 gtcagcctga ccgaaggttc gcgcgacgtg gcgctgccga
gcctgtttaa cggtcagtgg 2340 gtggtggggc atagcgaaaa cttctctgcc
tctattacca ccatgcaggc ggtgatctgg 2400 attgttacct tcctcgccat
gctggcgctg acgattttca ttcgctattc ccgcatgggt 2460 cgcgcgtgtc
gtgcctgcgc ggaagatctg aaaatggcga gtctgcttgg cattaacacc 2520
gaccgggtga ttgcgctgac ctttgtgatt ggcgcggcga tggcggcggt ggcgggtgtg
2580 ctgctcggtc agttctacgg cgtcattaac ccctacatcg gctttatggc
cgggatgaaa 2640 gcctttaccg cggcggtgct cggtgggatt ggcagcattc
cgggagcgat gattggcggc 2700 ctgattctgg ggattgcgga ggcgctctct
tctgcctatc tgagtacgga atataaagat 2760 gtggtgtcat tcgccctgct
gattctggtg ctgctggtga tgccgaccgg tattctgggt 2820 cgcccggagg
tagagaaagt atgaaaccga tgcatattgc aatggcgctg ctctctgccg 2880
cgatgttctt tgtgctggcg ggcgtcttta tgggcgtgca actggagctg gatggcacca
2940 aactggtggt cgacacggct tcggatgtcc gttggcagtg ggtgtttatc
ggcacggcgg 3000 tggtcttttt cttccagctt ttgcgaccgg ctttccagaa
agggttgaaa agcgtttccg 3060 gaccgaagtt tattctgccc gccattgatg
gctccacggt gaagcagaaa ctgttcctcg 3120 tggcgctgtt ggtgcttgcg
gtggcgtggc cgtttatggt ttcacgcggg acggtggata 3180 ttgccaccct
gaccatgatc tacattatcc tcggtctggg gctgaacgtg gttgttggtc 3240
tttctggtct gctggtgctg gggtacggcg gtttttacgc catcggcgct tacacttttg
3300 cgctgctcaa tcactattac ggcttgggct tctggacctg cctgccgatt
gctggattaa 3360 tggcagcggc ggcgggcttc ctgctcggtt ttccggtgct
gcgtttgcgc ggtgactatc 3420 tggcgatcgt taccctcggt ttcggcgaaa
ttgtgcgcat attgctgctc aataacaccg 3480 aaattaccgg cggcccgaac
ggaatcagtc agatcccgaa accgacactc ttcggactcg 3540 agttcagccg
taccgctcgt gaaggcggct gggacacgtt cagtaatttc tttggcctga 3600
aatacgatcc ctccgatcgt gtcatcttcc tctacctggt ggcgttgctg ctggtggtgc
3660 taagcctgtt tgtcattaac cgcctgctgc ggatgccgct ggggcgtgcg
tgggaagcgt 3720 tgcgtgaaga tgaaatcgcc tgccgttcgc tgggcttaag
cccgcgtcgt atcaagctga 3780 ctgcctttac cataagtgcc gcgtttgccg
gttttgccgg aacgctgttt gcggcgcgtc 3840 agggctttgt cagcccggaa
tccttcacct ttgccgaatc ggcgtttgtg ctggcgatag 3900 tggtgctcgg
cggtatgggc tcgcaatttg cggtgattct ggcggcaatt ttgctggtgg 3960
tgtcgcgcga gttgatgcgt gatttcaacg aatacagcat gttaatgctc ggtggtttga
4020 tggtgctgat gatgatctgg cgtccgcagg gcttgctgcc catgacgcgc
ccgcaactga 4080 agctgaaaaa cggcgcagcg aaaggagagc aggcatgagt
cagccattat tatctgttaa 4140 cggcctgatg atgcgcttcg gcggcctgct
ggcggtgaac aacgtcaatc ttgaactgta 4200 cccgcaggag atcgtctcgt
taatcggccc taacggtgcc ggaaaaacca cggtttttaa 4260 ctgtctgacc
ggattctaca aacccaccgg cggcaccatt ttactgcgcg atcagcacct 4320
ggaaggttta ccggggcagc aaattgcccg catgggcgtg gtgcgcacct tccagcatgt
4380 gcgtctgttc cgtgaaatga cggtaattga aaacctgctg gtggcgcagc
atcagcaact 4440 gaaaaccggg ctgttctctg gcctgttgaa aacgccatcc
ttccgtcgcg cccagagcga 4500 agcgctcgac cgcgccgcga cctggcttga
gcgcattggt ttgctggaac acgccaaccg 4560 tcaggcgagt aacctggcct
atggtgacca gcgccgtctt gagattgccc gctgcatggt 4620 gacgcagccg
gagattttaa tgctcgacga acctgcggca ggtcttaacc cgaaagagac 4680
gaaagagctg gatgagctga ttgccgaact gcgcaatcat cacaacacca ctatcttgtt
4740 gattgaacac gatatgaagc tggtgatggg aatttcggac cgaatttacg
tggtcaatca 4800 ggggacgccg ctggcaaacg gtacgccgga gcagatccgt
aataacccgg acgtgatccg 4860 tgcctattta ggtgaggcat aagatggaaa
aagtcatgtt gtcctttgac aaagtcagcg 4920 cccactacgg caaaatccag
gcgctgcatg aggtgagcct gcatatcaat cagggcgaga 4980 ttgtcacgct
gattggcgcg aacggggcgg ggaaaaccac cttgctcggc acgttatgcg 5040
gcgatccgcg tgccaccagc gggcgaattg tgtttgatga taaagacatt accgactggc
5100 agacagcgaa aatcatgcgc gaagcggtgg cgattgtccc ggaagggcgt
cgcgtcttct 5160 cgcggatgac ggtggaagag aacctggcga tgggcggttt
ttttgctgaa cgcgaccagt 5220 tccaggagcg cataaagtgg gtgtatgagc
tgtttccacg tctgcatgag cgccgtattc 5280 agcgggcggg caccatgtcc
ggcggtgaac agcagatgct ggcgattggt cgtgcgctga 5340 tgagcaaccc
gcgtttgcta ctgcttgatg agccatcgct cggtcttgcg ccgattatca 5400
tccagcaaat tttcgacacc atcgagcagc tgcgcgagca ggggatgact atctttctcg
5460 tcgagcagaa cgccaaccag gcgctaaagc tggcggatcg cggctacgtg
ctggaaaacg 5520 gccatgtagt gctttccgat actggtgatg cgctgctggc
gaatgaagcg gtgagaagtg 5580 cgtatttagg cgggtaa 5597 <210> SEQ
ID NO 10 <211> LENGTH: 1104 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1104)
<223> OTHER INFORMATION: livJ (Escherichia coli) <400>
SEQUENCE: 10 atgaacataa agggtaaagc gttactggca ggatgtatcg cgctggcatt
cagcaatatg 60 gctctggcag aagatattaa agtcgcggtc gtgggcgcaa
tgtccggtcc ggttgcgcag 120 tacggtgacc aggagtttac cggcgcagag
caggcggttg cggatatcaa cgctaaaggc 180 ggcattaaag gcaacaaact
gcaaatcgta aaatatgacg atgcctgtga cccgaaacag 240 gcggttgcgg
tggcgaacaa agtcgttaac gacggcatta aatatgtgat tggtcacctc 300
tgttcttcat caacgcagcc tgcgtctgac atctacgaag acgaaggcat tttaatgatc
360 accccagcgg caaccgcgcc ggagctgacc gcccgtggct atcagctgat
cctgcgcacc 420 accggcctgg actccgacca ggggccgacg gcggcgaaat
atattcttga gaaagtgaaa 480 ccgcagcgta ttgctatcgt tcacgacaaa
cagcaatacg gcgaaggtct ggcgcgagcg 540 gtgcaggacg gcctgaagaa
aggcaatgca aacgtggtgt tctttgatgg catcaccgcc 600 ggggaaaaag
atttctcaac gctggtggcg cgtctgaaaa aagagaatat cgacttcgtt 660
tactacggcg gttatcaccc ggaaatgggg caaatcctgc gtcaggcacg cgcggcaggg
720 ctgaaaactc agtttatggg gccggaaggt gtggctaacg tttcgctgtc
taacattgcg 780 ggcgaatcag cggaagggct gctggtgacc aagccgaaga
actacgatca ggttccggcg 840 aacaaaccca ttgttgacgc gatcaaagcg
aaaaaacagg acccaagtgg cgcattcgtt 900 tggaccacct acgccgcgct
gcaatctttg caggcgggcc tgaatcagtc tgacgatccg 960 gctgaaatcg
ccaaatacct gaaagcgaac tccgtggata ccgtaatggg accgctgacc 1020
tgggatgaga aaggcgatct gaaaggcttt gagttcggcg tatttgactg gcacgccaac
1080 ggcacggcga ccgatgcgaa gtaa 1104 <210> SEQ ID NO 11
<211> LENGTH: 639 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(639) <223> OTHER
INFORMATION: leucine exporter gene leuE (Escherichia coli Nissle
1917) <400> SEQUENCE: 11 gtgttcgctg aatacggggt tctgaattac
tggacctatc tggttggggc catttttatt 60 gtgttggtgc cagggccaaa
taccctgttt gtactcaaaa atagcgtcag tagcggtatg 120 aaaggcggtt
atcttgcggc ctgtggtgta tttattggcg atgcggtatt gatgtttctg 180
gcatgggctg gagtggcgac attaattaag accaccccga tattattcaa catcgtacgt
240 tatcttggtg cgttttattt gctctatctg gggagtaaaa ttctctacgc
gaccctgaaa 300 ggtaaaaata gcgagaccaa atccgatgag ccccaatacg
gtgccatttt taaacgcgcg 360 ttaattttga gcctgactaa tccgaaagcc
attttgttct atgtgtcgtt tttcgtacag 420 tttatcgatg ttaatgcccc
acatacggga atttcattct ttattctggc gacgacgctg 480 gaactggtga
gtttctgcta tttgagcttc ctgattattt ctggggcttt tgtcacgcag 540
tacatacgta ccaaaaagaa actggctaaa gtgggcaact cactgattgg tttgatgttc
600 gtgggtttcg ccgcccgact ggcgacgctg caatcctga 639 <210> SEQ
ID NO 12 <211> LENGTH: 2268 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(2268)
<223> OTHER INFORMATION: Arginine decarboxylase (Escherichia
coli) <400> SEQUENCE: 12 atgatgaaag tgttaatcgt ggaatctgaa
tttctgcacc aggatacgtg ggtcggtaac 60 gctgttgaac gtctggccga
tgctttaagc cagcaaaatg tgacagttat caaatccacc 120 tcttttgacg
atggctttgc cattctgtca agcaatgaag ccatcgattg tctgatgttc 180
tcgtaccaga tggaacaccc cgatgagcac caaaatgttc gtcagctgat cggcaaactt
240 cacgaacgtc aacagaacgt accggtcttt ctgttaggcg accgcgaaaa
ggccttggcg 300 gctatggatc gcgatctgct ggagttggtc gacgagtttg
cctggattct cgaggatacg 360 gcggatttta ttgccggtcg cgcagtcgcc
gccatgacgc gctaccgcca acagctgctc 420 ccgccgctgt tttctgccct
gatgaaatac tcggacattc acgaatacag ctgggcagct 480 cccgggcacc
agggcggcgt tggcttcacg aaaaccccag ctggtcgctt ttatcatgac 540
tactacggcg agaatttatt tcgtaccgac atgggcattg aacgtaccag cctgggctcg
600 ctgctggacc acacgggcgc ttttggggaa tcagagaaat atgcagcacg
cgtgttcggt 660 gcggaccgca gttggtccgt cgtggtgggc accagtggta
gcaaccgcac cattatgcag 720 gcgtgcatga ccgataatga tgtggtagtg
gtggatcgca attgtcataa gagcatcgaa 780 caaggcttga tgctgactgg
cgctaaacca gtctatatgg tgccgtcccg taatcgctat 840 ggtattatcg
gcccgattta tcctcaggag atgcagccgg aaaccctcca gaagaaaatc 900
tcagagtccc cgttaactaa agataaagct gggcaaaaac cgagttattg tgtagtaact
960 aattgtacgt atgatggtgt ttgctataac gctaaggagg cccaagatct
tctggaaaaa 1020 acaagtgatc gtcttcattt tgatgaagct tggtacggtt
atgcgcgttt caaccctatt 1080 tacgccgacc actatgcgat gcgtggtgaa
cctggggatc ataatggccc tactgtgttt 1140 gccacccatt ctacgcataa
actcctgaat gcgttgtcac aggcgagtta catccacgta 1200 cgcgaaggcc
gtggcgctat taattttagc cgctttaacc aggcctatat gatgcacgcg 1260
acgacaagtc cgctgtatgc gatttgcgcg tccaacgatg ttgcggtcag catgatggac
1320 ggcaacagcg gtctgtcgtt aacccaggaa gtgattgatg aagcggtcga
ctttcgccag 1380 gcgatggccc gtctgtacaa agaattcacc gccgatggct
cgtggttctt caaaccctgg 1440 aataaagaag tcgtgactga cccgcagacg
ggcaaaactt atgattttgc agatgccccg 1500 acgaagcttc ttactacggt
ccaggattgc tgggtgatgc acccggggga gtcttggcat 1560 ggcttcaaag
atatccctga taactggtct atgctcgacc caatcaaagt ttcaatttta 1620
gctccaggca tgggcgaaga tggcgaactg gaagagacgg gggtaccagc tgcgttggtt
1680 accgcctggt taggccgcca tggtattgtt ccaacacgta ccactgattt
tcagattatg 1740 tttctgttca gtatgggtgt gacgcgcggt aaatggggga
cgctggtcaa cactctctgc 1800 tcctttaaac gccattatga tgcgaacacg
cccctggcgc aagtcatgcc agagctggtg 1860 gaacaatacc ctgatactta
tgcgaacatg ggtatccacg atctgggaga tactatgttc 1920 gcctggctta
aagaaaataa cccgggggcc cgcctgaacg aagcatatag tggcctgccc 1980
atggcggaaa ttactccgcg tgaagcctat aatgccatcg ttgataataa cgtcgaatta
2040 gtatccatcg agaacctccc cggtcgtatt gcggcaaata gcgtaatccc
gtacccgccg 2100 ggtattccca tgctgctcag cggcgaaaac ttcggtgata
aaaattcccc gcaagtttct 2160 tatctgcgca gcctgcaatc gtgggaccat
cactttcccg ggtttgagca tgaaactgaa 2220 gggacagaga tcatcgatgg
catttatcat gtgatgtgcg tcaaggcg 2268 <210> SEQ ID NO 13
<211> LENGTH: 780 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(780) <223> OTHER
INFORMATION: ArgT (Escherichia coli) <400> SEQUENCE: 13
atgaaaaaaa gcatcctcgc gctgtcactg ttagtgggtc tcagcgccgc ggccagcagc
60 tatgctgctc ttcctgaaac ggtgcgcatc gggacggata ccacttatgc
accgtttagc 120 agcaaagatg ctaaaggaga cttcgtaggg tttgatatcg
atttaggcaa cgagatgtgc 180 aaacgtatgc aagtgaaatg tacctgggtg
gcttcagact ttgatgcatt aatcccgagt 240 ttgaaagcaa aaaaaattga
cgcaattatt tcgagcctga gcattacaga taagcgccaa 300 caagaaattg
ccttctcaga taaattatat gccgctgatt cgcgtcttat cgcggctaaa 360
ggctccccta tccaaccaac gttggacagc ctgaagggga aacatgtagg ggttctgcaa
420 gggtccacgc aggaagctta cgccaatgaa acctggcgtt cgaaaggggt
cgatgtggtg 480 gcgtacgcca atcaggactt ggtgtattcc gatctggccg
caggtcgtct ggacgcagct 540 ctgcaggacg aagtggcggc gagtgagggt
ttcctgaaac agccagcagg caaagatttt 600 gcgttcgccg gctcgagtgt
aaaggataaa aaatatttcg gggatggcac gggtgtcggt 660 ttacgcaaag
atgatgcaga actgaccgcg gcgtttaata aagcccttgg cgaactgcgc 720
caagacggca catatgataa aatggcgaaa aagtactttg acttcaatgt ttatggtgat
780 <210> SEQ ID NO 14 <211> LENGTH: 726 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(726) <223> OTHER INFORMATION: artP (Escherichia coli)
<400> SEQUENCE: 14 atgtctattc aattaaatgg catcaactgt
ttctacggtg cacatcaagc cttatttgac 60 atcacgcttg attgcccgca
aggggagaca ctggtgctgc tgggcccgag tggagccggc 120 aaatcgtcgt
tgctgcgggt gttgaacctg ttggagatgc cgcgctcagg caccctgaat 180
atcgcgggca accatttcga ttttacgaaa acaccgtccg ataaagctat tcgtgatctt
240 cgtcgcaacg tcggcatggt gtttcagcag tataatttat gtgctcatct
gacggttcag 300 caaaatctga tcgaagcacc gtgtcgtgtg ttgggcctga
gcaaagacca agccctggcc 360 agcgcagaaa aattattaga gcgcctgcgc
ttgaaaccat attcggatcg gtacccactt 420 cacttaagcg ggggccagca
acagcgcgtt gccatcgctc gtgcgctgat gatggagccg 480 caagttctcc
tttttgatga acctaccgca gcgcttgatc cggagatcac ggcgcagatc 540
gtcagcatca ttcgtgaact cgctgagacg aatattacac aagttattgt gacacatgag
600 gtagaagtgg ctcgcaagac cgcgtctcgc gtagtgtata tggaaaacgg
tcatatcgtg 660 gagcaagggg acgcctcatg ttttacagag ccgcagacag
aggcattcaa aaattatctg 720 agccac 726 <210> SEQ ID NO 15
<211> LENGTH: 732 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(732) <223> OTHER
INFORMATION: artI (Escherichia coli) <400> SEQUENCE: 15
atgaaaaaag tgcttattgc cgccctgatt gcgggcttct ctctgtctgc caccgcggcc
60 gaaaccatcc gttttgccac tgaagcgtca tatccccctt tcgaaagcat
tgacgccaac 120 aaccaaattg tcggtttcga cgttgacctc gcgcaggccc
tgtgcaaaga aattgatgcc 180 acctgcacct tctctaacca agcgtttgac
tcattgattc cttcgctgaa atttcgtcgc 240 gtggaagccg tcatgggcgg
catggatatc acccccgagc gcgaaaaaca ggtcttgttt 300 actacaccgt
actacgacaa ctcggctttg tttgtcggcc agcaaggcaa gtatacttct 360
gtcgaccagc tgaaaggtaa aaaagtccgt tcagtccaga acggcaccac tcaccagaaa
420 ttcatcatgg acaaacatcc tgagatcact accgtgccgt atgattctta
ccagaacgcg 480 aagttagatc tggaaaatgg tcggattgat ggcgtctttg
gcgacaccgc tgtggtacat 540 gaatggctga aagacaatcc taaattagtg
gttgtgggag ataaggttac ggataaggat 600 tattttggca ccggtctcgg
cattgcagtc cgccaaggta ataccgaatt gcaacagaaa 660 ttgaataccg
cgctggaaaa agtgaaaaaa gacggtacat acgaaaccat ttacaacaaa 720
tggtttcaaa aa 732 <210> SEQ ID NO 16 <211> LENGTH: 714
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(714) <223> OTHER INFORMATION: artQ
(Escherichia coli) <400> SEQUENCE: 16 atgaacgaat tcttccctct
cgcgtctgcg gcaggtatga ccgtgggttt ggcggtttgt 60 gcgctgattg
tcggtctcgc tctggcaatg ttctttgccg tatgggagtc agcgaaatgg 120
cgtccggtcg cctgggcagg ttccgccctg gtaaccattc tgcgtggtct gccagagatc
180 ctggtagttc tgtttatcta ctttggctct tctcagttac tgttaacact
gtctgacggg 240 tttacgatta acctgggttt tgtccagatt ccggtccaga
tggatattga aaatttcgac 300 gtctcccctt ttctctgtgg cgtcatcgcg
ctgagcttgc tctacgctgc atatgcatca 360 cagacccttc gtggtgcatt
aaaagcggtg ccagtaggac agtgggaaag cggccaggcc 420 cttggcctga
gcaagagcgc aatttttttc cgccttgtta tgccggccga tgtccgccat 480
gcgttaccag gtctgggtaa tcaatggctg gtgttgttga aagacaccgc ccttgtctcg
540 ctgattagcg tgaacgattt aatgctgcaa accaaatcga ttgcaacccg
cactcaggaa 600 ccgtttacct ggtacatcgt ggcggcagca atctatctgg
tgatcacact tctgagccag 660 tatattttaa aacgtattga cctgcgtgcc
acccgctttg agcgccgccc tagc 714 <210> SEQ ID NO 17 <211>
LENGTH: 666 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(666) <223> OTHER INFORMATION:
artM (Escherichia coli) <400> SEQUENCE: 17 atgtttgaat
atctgccgga actgatgaaa ggtttgcata ctagtctgac gctgaccgtc 60
gcgagtctga tcgttgcgct tatcctggca ctgatcttca ccattattct gactctcaag
120 accccggtcc tggtgtggct ggtccgcggt tacattacct tattcaccgg
gaccccgctc 180 ttggttcgca tttttcttat ttactatggt ccgggtcagt
ttccgacctt gcaagaatat 240 cctgcgttat ggcacctgct gtctgaaccg
tggctgtgcg ctctgattgc tctgagtgtt 300 aactcggcgg cctatacgac
acagctgttc tacggtgcta ttcgtgcgat cccagaaggt 360 caatggcagt
cttgtagcgc actgggcatg tcaaagaaag atactcttgc tattctgctg 420
ccgtacgctt ttaaacgctc tctgagctcg tacagcaatg aagttgtcct ggttttcaaa
480 agcactagct tagcgtatac gatcacgctg atggaagtca tgggttatag
ccagttatta 540 tatggtcgca cgtacgacgt catggtgttt ggtgcagcgg
gcattatcta tcttgtagtt 600 aatggattac tgacgttaat gatgcgcttg
atcgaacgca aagccgtggc attcgagcgg 660 cgtaat 666 <210> SEQ ID
NO 18 <211> LENGTH: 729 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(729) <223>
OTHER INFORMATION: artJ (Escherichia coli) <400> SEQUENCE: 18
atgaaaaaat tggtgcttgc agcactgctg gccagtttca ctttcggcgc ttcggcggcc
60 gaaaagatta atttcggtgt cagcgcaact tacccaccgt tcgaaagcat
cggtgcgaac 120 aatgagattg taggatttga tatcgatctg gccaaagcgt
tatgcaaaca aatgcaagcg 180 gagtgcactt ttaccaatca tgcgtttgat
agcctgatcc cgtcgctgaa gttccgtaaa 240 tacgacgccg tgatttcggg
gatggacatc acccctgagc gctcgaaaca ggtgagcttc 300 accactccat
attatgaaaa ctcagcggtg gtgattgcga aaaaagacac ctataaaaca 360
tttgccgacc tgaaagggaa atgtattggt atggagaacg gcaccaccca tcagaagtat
420 attcaagacc agcacccgga ggttaagacc gtaagctacg actcctacca
gaatgctttc 480 attgatttaa aaaatggtcg tattgatggt gtattcggag
atacagccgt ggtgaatgag 540 tggctgaaaa ccaatccgca gttgggtgtt
gcgaccgaaa aagtgacaga tccacaatac 600 tttgggactg gcctgggcat
cgcggtgcgc ccggataaca aagccctgtt ggagaaactg 660 aacaacgcgt
tagctgcgat taaagcggat gggacctatc agaagatttc agaccaatgg 720
ttcccgcaa 729 <210> SEQ ID NO 19 <211> LENGTH: 633
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(633) <223> OTHER INFORMATION: ArgO
(Escherichia coli) <400> SEQUENCE: 19 atgttctcgt actatttcca
aggcttagca ctgggtgcgg ccatgatctt accgctgggc 60 ccacaaaacg
cttttgttat gaaccaggga atccgccggc agtaccatat catgattgcg 120
ctgctgtgtg ccatctcgga tctggtcctg atttgcgccg gtatttttgg cgggtcggcg
180 ttacttatgc aaagcccttg gctgctggcg ctggtaacgt ggggcggcgt
agcatttctg 240 ctttggtatg gattcggcgc cttcaaaact gcgatgagtt
cgaatatcga gcttgcgagt 300 gctgaggtaa tgaaacaggg ccgttggaaa
attattgcga ccatgttagc cgtgacttgg 360 ttgaacccgc acgtgtacct
ggatactttt gtggtgttgg gttcactcgg tgggcaatta 420 gatgtggaac
cgaaacgctg gtttgccttg ggcacaatct cggccagttt tttgtggttc 480
ttcgggctgg cgctgctggc cgcgtggctg gcaccacgtt tacgcaccgc caaggcccag
540 cgcatcatca acttagtcgt gggctgtgtg atgtggttca ttgctctgca
actggcgcgc 600 gatggcattg cgcacgccca ggccctgttc tca 633 <210>
SEQ ID NO 20 <211> LENGTH: 1203 <212> TYPE: DNA
<213> ORGANISM: Flavobacterium limnosediminis JC2902
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1203) <223> OTHER INFORMATION: monofunctional
lysine-ketoglutarate reductase (Flavobacterium limnosediminis
JC2902) <400> SEQUENCE: 20 atgatgcgct ttggcatcat taaagaacgt
aagaacccgc cagatcgtcg tgtagtgttt 60 acaccgtccg aactgatcaa
actgaaagaa cagtttccgc tggccgaaat taaggtggaa 120 tcctcagata
ttcgcatttt ttctgatgat gagtatcgta aacttggatt tgaagtaacc 180
gatgacctga gtgattgtga tgtcttgatt ggcgtgaaag aagtaccgat cgatgccctg
240 ctgcccggga aaaagtattt ttttttctct cacacaatta aaaaacagcc
ttacaataaa 300 aaactgctga tcgcctgctt ggaaaaaaac atccgtctga
ttgatcatga gacgatcgtg 360 aatgaagata atcatcgttt gattgggttc
ggccgttacg caggtatcgt gggggcctat 420 aacggtttcc gtgcttttgg
tattaagtac gagctcttta acctgcccaa agcggaaacc 480 ttagcggaca
aaacggcact tgtggaacgc ctgcgtcggc cgatgctgcc gccaatcaaa 540
attgtgttga ccggtcacgg caaagtaggt atgggtgcaa aagagattct ggatgccatg
600 aaaatcaaac aagtttccgt ggaggactac ttaacaaaaa cctatgacaa
gccggtgtat 660 acgcagatcg acgttctgga ctataacaag cggaaagatg
gcaaaccggc ggaacgtgaa 720 cacttttatg ccaatccgca ggagtatgtc
tcggacttcg aacgctttac caaggtgtcg 780 gatctgttca tcgcaggcca
tttctatggc aacggtgcac cggtaattct gactcgcacc 840 atgcttaacg
cttctgataa taaaattaaa gtagttgcgg atattagctg tgatgtcggt 900
ggccctatcg aatgtacgct gcgcagcagc accatcgcag agccgtttta tggttattat
960 ccttccgaag gtaaagaagt cgacgtcaac catccgggcg cggtggttgt
gatggcggtg 1020 gacaatctgc cctgcgagct gcctaaagat gccagcgagg
gtttcggaga aatgtttctc 1080 aaacatgtga ttccagcctt ctacaacaac
gataaggacg gcattcttga gcgggccaaa 1140 atcaccgaaa acggcaaatt
aacaaaacgc ttctcctact tacaggacta tgtcgatggt 1200 gaa 1203
<210> SEQ ID NO 21 <211> LENGTH: 1356 <212> TYPE:
DNA <213> ORGANISM: Flavobacteriumsp. EM1321 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1356) <223> OTHER INFORMATION: saccharopine
dehydrogenase (Flavobacteriumsp. EM1321) <400> SEQUENCE: 21
atgcgtaata ttttgattat cggcgccggt cggtccgctt cctcgctgat tcagtactta
60 ttgaataagt cccaagaaga acagctgcat ttaaccattg ccgatttatc
actcgaactg 120 gctcagaaga aaaccaataa ccatccgaac gctaccgcgc
tggcgctgga tatttataat 180 aaggatgaac gtcgtgcggc catcgagaaa
gcggccattg tgatcagcat gttgccagcg 240 catctgcata tcgaaatcgc
ccgggattgc ctgtatttta aaaagaacct tgttacggcg 300 agctatatta
gtgacgcgat gcaggagctt gatgcggaag ttaaagagaa caaactgatc 360
tttatgaatg aggtcggttt agacccgggt attgatcata tgagcgccat gaaagtcatc
420 gatgaaattc gggaacaagg cggcaaaatg cttctcttcg aaagtttttg
cggcggcctg 480 gtggcaccag aatcagataa caatttatgg aactataaat
ttacctgggc cccacgtaac 540 gtagttctgg ctggccaggg tggtgtggca
aaattcattc aagaaggcac ctataaatat 600 atcccgtatg acagcttatt
tcgccggacc gagtttctgg aagtagaagg atacgggcgt 660 ttcgaagctt
attcgaatcg cgattctctc aaatatcgga gtatttatgg gctcgatgac 720
gttctcaccc tgtttcgtgg tacaatccgt cgcgttggct tctccaaagc ttggaacatg
780 tttgtgcaac tgggcatgac ggacgacagc tatgttatgg aagattctga
gaatatgtcc 840 tatcgtcaat ttattaactc attcctgcct tatcacccaa
ccgatagcgt tgaaattaag 900 acccgttttt tgttaaaaat cgatcaggat
gatatcatgt gggacaaact gctggaactg 960 gatcttttca acgataaaaa
aatggttggg ttgaaaaatg cgacgccggc acagatcctg 1020 gagaaaatcc
tgaacgattc gtggaccctg caaccggaag ataaagatat gatcgtgatg 1080
tatcataaat ttggttacca gatcaacggc gaaaaagtgc agatggattc acagatggtg
1140 tgtatcggcc aggaccaaac gtataccgcg atggcaaaaa ccgtcggcct
gcctgtggca 1200 atggcaactc tgctgattct gaacggtaaa atcaaaacaa
cgggagttca gttgccaatc 1260 aataaagaag tttacctgcc ggtcctggag
gaactggaga aatatggcgt tgtgttcaaa 1320 gaacagatgc tcccatatct
tggatacaaa tatagt 1356 <210> SEQ ID NO 22 <211> LENGTH:
1350 <212> TYPE: DNA <213> ORGANISM: Bacillus
methanolicus PB1 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1350) <223> OTHER
INFORMATION: Lysine aminotransferase (Bacillus methanolicus PB1)
<400> SEQUENCE: 22 atgaagaaaa atcattcctt gcagtcgctt
aaaaaccaag atgagcgttt catttggcac 60 tcgatgaagc cgtataaccc
cgacaagacg atcgttgtca ccaaggccga aggatcatgg 120 attacaacga
gtgatggaaa gaagtatctt gacgcaatgg ccggtctttg gtgcgttaac 180
gtggggtatg gacgcaaaga gcttgccgat gccgcgtacg aacagatgat ggaaatggca
240 tactatccac tgactcagtc acatgtaccc gccattcagt tagcggagaa
gttgaacgat 300 ctgctggaag acgaatacgt aatctttttt agcaattcgg
ggagtgaggc gaacgaggct 360 gcttttaaaa ttgctcgtca gtatcatcaa
caaaaaggag accacaatcg ctataagatt 420 gttgcacgct accgtgcata
tcatgggaac tcaattggag ccttggcagc gacagggcag 480 gcccagcgta
aatataagta tgagcctctg gcctttggat tcgtccatgt tgcccctcct 540
gactcctacc gtgatgaaac taacgtatcc gatccttcgc agttgtccgc agtcaaagaa
600 attgaccgtg taatgacgtg ggagctttcg gaaactatcg ccgcaatgat
catggaaccg 660 attattactg gtggaggcat cttagtgccc ccagaggggt
atatgaaagc ggctaaggag 720 gtttgtgaaa agcacggggc tcttttgatt
gtggacgagg tgatttgcgg gtttggtcgt 780 acgggtaagc cgttcggatt
catgaactat ggagtcaagc cggacattat caccatggct 840 aaaggcatca
ccagtgcgta tcttccgttg tcagcaactg cagtcaaaaa ggaaatctat 900
gatgccttta aaggtgagga cgaatatgag ttcttccgtc atgtcaacac tttcggaggg
960 tcacccgccg catgtgcgct ggctatcaag aacattcaga ttttggagga
ggaaaagctg 1020 tttgaccgct cgggcgacat gggcgaaaaa gttttaacag
aacttcagaa cttgttacgc 1080 gatcacccct acgttggcga cgttcgtgga
aagggtctgt taatcggaat tgaattggtt 1140 aaagacaagc agacgaaaga
gcccttaaat acaagcaaag ttgacgaagt aatcgctctt 1200 tgtaaacagg
aaggacttct gattggaaaa aatggcatga ccgtggcagg ctataacaac 1260
gtccttacac tgtcccctcc gcttaatatc ccagagaccg acttagactt tttgatcaaa
1320 gtactgacgg cgtccttgga gaagattaag 1350 <210> SEQ ID NO 23
<211> LENGTH: 1098 <212> TYPE: DNA <213>
ORGANISM: Agrobacterium tumefaciens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1098) <223> OTHER INFORMATION: lysine dehydrogenase
(Agrobacterium tumefaciens) <400> SEQUENCE: 23 atgaaaaata
tcgtagtgat cggggcaggg aatattggca gcgccattgc gtggatgttg 60
gcagctagcg gggattatcg cattactgta gcagaccgca gcgcggatca gttagctaat
120 gtaccggctc atgaacgtgt cgacattgtt gacattaccg accgccccgc
gctggaagca 180 ctgttaaaag ggaaatttgc ggtacttagc gccgctccca
ccgagtttca tttgactgcc 240 ggaattgcgg aagcggccgt cgcggtaggc
acgcactact tagacttaac agaagatgtg 300 gagtctaccc gcaaggtaaa
agcgctggct gagacggccg agacagcttt aatcccccaa 360 tgtgggctgg
caccaggttt tatttcgatt gttgctgccg atttggctgt gaagtttgat 420
aaattagatt ctgttcgtat gcgcgtcggg gcgctgcctc agtatcccag taacgcattg
480 aattacaatt tgacttggag cacagatggc cttatcaacg agtacattga
gccttgcgag 540 gggtttgtag aaggtcgctt gaccgcggtc ccggctttag
aggaacgcga ggaatttagt 600 cttgatggga tcacctacga ggcattcaac
acctcgggcg gacttgggac cttgtgcgcc 660 acccttgagg gtaaggtgcg
cacaatgaac taccgtacca tccgctatcc gggtcatgta 720 gcaatcatga
aggcacttct taatgacttg aacctgcgta atcgccgtga cgttttgaaa 780
gatctttttg aaaatgcact gcctggaacg atgcaagatg tcgtaattgt ttttgtaaca
840 gtgtgtggca ctcgcaacgg acgctttctg caagagactt atgccaataa
agtgtacgcg 900 gggcctgtgt caggccgcat gatgtccgcg atccagatca
caacagctgc tggaatttgc 960 acagtcctgg atttgttggc cgaaggcgcg
cttccgcaga agggcttcgt tcgtcaagag 1020 gaggtcgcac tgcctaagtt
tttggaaaat cgtttcggac gttattatgg ttctcacgaa 1080 ccgcttgctc
gtgttggt 1098 <210> SEQ ID NO 24 <211> LENGTH: 1179
<212> TYPE: DNA <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: lysine racemase (uncultured
bacterium) <400> SEQUENCE: 24 atggcacata caggccgtat
gtttaagatc gaagccgcgg agatcgtagt ggctcgcctg 60 ccgctgaaat
ttcgttttga gacatctttc ggtgtccaga cacataaagt ggtgccttta 120
ctgatcttac atggcgaagg tgttcaaggg gtcgcggagg ggacaatgga agctcgcccc
180 atgtaccgcg aagaaacgat tgccggagcc cttgatttgt tgcgtggaac
ttttttacct 240 gcgattctgg gccaaacctt tgccaatcca gaagcggtaa
gtgatgccct gggctcttac 300 cgcggcaatc gcatggcacg cgctatggtg
gagatggcag cttgggactt gtgggcccgc 360 acccttggtg tgcctttggg
cacactgttg ggtggtcaca aggaacaagt cgaggtgggt 420 gtatcgttgg
gaatccaggc agatgagcaa gctacagtag acttagtgcg tcgtcatgtt 480
gaacaaggat atcgtcgcat taagttgaag attaagcctg ggtgggacgt tcaacctgta
540 cgtgcgaccc gtgaggcatt catgttaaac acgcttaatg tcggcgcctc
tggttacgcg 600 ggcgcagaac tggttacata cgtgaaccgc cacccccata
tgaacattac ggcgttgacc 660 gtatcagcac agtcaaacga tgcagggaag
ttaatctccg atttgcatcc ccaattaaag 720 ggcatcgttg acttaccatt
gcagccgatg tccgacatct ctgaattcag ccccggggta 780 gatgtagtgt
tcctggctac agctcacgaa gtttcacacg acctggcccc gcaatttttg 840
gaggcgggtt gtgtggtctt tgatctgtcc ggcgcttttc gcgttaacga tgctacattt
900 tacgagaagt attacggttt cacccaccaa tacccagagc tgctggaaca
ggcggcctac 960 gggcttgctg agtggtgtgg caacaaactt aaggaagcta
atcttattgc agttcctgga 1020 tgttacccta ccgccgcaca gctggcgctg
aagccgttaa ttgatgctga cctgctggac 1080 ctgaaccaat ggccggtgat
caatgcgacc agtggcgtat ctggggcggg tcgtaaagcc 1140 gcaatttcaa
actccttctg cgaggttagc ttacaaccg 1179 <210> SEQ ID NO 25
<211> LENGTH: 1407 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1407)
<223> OTHER INFORMATION: Lysine transporter yvsh (Bacillus
subtilis) <400> SEQUENCE: 25 atggagcaga cgaagaaatg gggattttgg
ttactgacgg ccttcgtcgt gggcaacatg 60 gtgggtagtg gaatcttttc
tcttccatcc tccctggcga gcatcgcgtc gcctttcgga 120 gctacgtccg
cttggcttct gacaggtgcg ggggtgttaa tgatcgcctt agtattcgga 180
catttgtcca ttcgtaaacc cgaattgact gccgggcctc aatcatacgc ccgtgcattg
240 ttcagcgatc caaaaaaggg gaatgcggcc gggtttacta tggtttgggg
ttactgggtc 300 gcgagctgga tcagtaacgt agcaatcatt acatctctgg
cggggtatct gaccagcttc 360 ttccccatcc tggtagacaa acgcgaaatg
ttttctattg ggggtcaaga ggtcaccctg 420 gggcagctgc tgacttttgc
cgtttgcacc attctgttgt ggggcaccca tgcgattttg 480 gtcgcatcga
tcaatggcgc aagcaagctg aattttgtga ccacattatc caaggtcttg 540
ggattcgtgt ttttcattgt ggcagggtta ttcgtcttcc agacgacgct ttttggtcat
600 ttctatttcc cggtccaagg cgagaatgga acgagcatcg gtattggggg
acaggtgcat 660 aacgctgcga tttctacact ttgggctttc gtcggaatcg
aaagcgccgt tatcttgtct 720 ggccgcgcgc gcagccagcg cgatgttaaa
cgtgctacca ttaccggact tctgattgca 780 ctgtcgatct atattatcgt
cacgttaatc acgatgggtg ttttacccca cgacaaatta 840 gtaggaagtg
aaaagccatt tgtcgatgtt ttatatgcaa tcgtcgggaa cgctggttca 900
gtaatcatgg cactgctggc catcttgtgc ctttttggaa ccatgttggg gtggatttta
960 ctgggctcgg aggtgcccta ccaagcagcc aaagctggtg atttccccgc
cttctttgcc 1020 aaaactaata agaaaggttc tccagtgatt gcgcttatca
ttaccaatgt catgtcacag 1080 gttttcattt ttagcgtgat cagtcgtaca
atttccgatg cttttacttt tttgactaca 1140 gcggccacgt tggcctatct
gattccctac ttagtttcag cgatttatag tttgaaagtg 1200 gttattaaag
gcgaaaccta tgaccagttg aaaggcagtc gtgtacgtga tggtcttatc 1260
gctatcttgg catgtgcata ctcagtcttc gtaatcgtga cgggtaccgc cgatttgacg
1320 acctttattt taggtattgg gctttttttt gtgggcctta tcgtgtaccc
atttgtctcg 1380 aagaagtttc aaaaggagaa gcaggaa 1407 <210> SEQ
ID NO 26 <211> LENGTH: 1404 <212> TYPE: DNA <213>
ORGANISM: Klebsiella sp. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1404) <223> OTHER
INFORMATION: Lysine Transporter LysP (Klebsiella) <400>
SEQUENCE: 26 atgaccatga ttgctattgg cgggtcgatc ggcacagggc ttttcgttgc
atccggagca 60 acgattagtc aagcaggtcc aggcggggct ctgctgtctt
atattcttat cggcttaatg 120 gtgtattttc tgatgacctc tcttggagag
ctggccgctt ttatgccagt ctccggatcg 180 ttcgctacat atgggcaaaa
ctacgtagag gagggtttcg ggtttgcgct gggttggaat 240 tactggtata
attgggctgt gacgatcgca gttgacttgg tggcttcgca gcttgtgatg 300
agctattggt tccctgacac tccgggctgg atttggtctg ctttgttttt gggcatcatg
360 ttcttgctta actggatctc cgttcgcggg ttcggtgaag ctgagtactg
gttcagtctg 420 attaaagttg cgaccgttat tatcttcatc atcgttggcg
tgatgatgat tgtcggcatt 480 ttcaaagggg cgcaaccggc tggatggtcc
aactggggta tcgctgacgc cccatttgcg 540 gggggcttct cggcgatgat
tggcgttgcc atgattgtcg gtttttcctt tcagggtaca 600 gagttaattg
gaattgctgc tggtgaatcc gagaatcctg agaaaaatat tccacgtgcg 660
gtacgtcagg tattctggcg cattttactg ttttatgttt ttgcaatctt gattatctcg
720 ttgatcatcc cttatactga cccatcctta ttgcgtaacg atgtgaagga
tatttccgtg 780 tctcccttca cgttggtatt tcagtatgct gggctgctta
gtgccgctgc gatcatgaac 840 gcagtcattc ttacggctgt actgagcgct
ggaaactcgg gaatgtacgc ttcaacacgc 900 atgttatata ccttggcatg
tgacgggaaa gcaccgcgta tctttagcaa gctttcccgt 960 ggcggtgtgc
cacgcaatgc tctgtatgca acaactgtaa ttgctgcctt atgctttctt 1020
accagcatgt tcggcaacca aacggtttat ctgtggttgc tgaacacttc gggaatgaca
1080 gggttcatcg cctggctggg tattgctatt tctcactatc gtttccgtcg
cggctacgtg 1140 ctgcagggga atgatatcaa taatcttccg tatcgttcag
gattttttcc tcttggaccc 1200 atttttgcat ttgtattgtg tttgattatt
actcttggcc aaaattatga ggcgttctta 1260 aaagatacta tcgattgggg
tggggtagcc gcaacctaca tcgggattcc cttgttcctt 1320 gttatttggt
ttggatataa gttggctaag ggtacccgct ttgtccgtta ttccgaaatg 1380
accttcccag atcgttttaa acgc 1404 <210> SEQ ID NO 27
<211> LENGTH: 630 <212> TYPE: DNA <213> ORGANISM:
Pseudomonas sp. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(630) <223> OTHER
INFORMATION: Lysine Exporter (Pseudomonas) <400> SEQUENCE: 27
atgagtatgg aagtctggct ggggtttttt gcagcgtgtt gggtgattag tttgtcaccg
60 ggagccggag ccatcgcctc tatgtcatcg ggtttacaat atggcttctg
gcgtggctac 120 tggaatgcac ttggattgca gcttggttta attatgcaaa
ttgcaattat cgctgcgggc 180 gtcggagccg tcttggcggc ctcggctacg
gccttccagg taattaaatg gttcggagtt 240 gggtatcttg tgtatttagc
atacaaacaa tggcgtgcac tgcccatgga tatgtcggat 300 gaaagcgggg
tgcgtccaat cggcaaacca ttatcgctgg tatttcgtgg atttttggtg 360
aatatctcca acccaaaagc tttagtattc atgttggccg ttttacccca gttcctgaat
420 ccccacgccc ccttgttacc ccaatacgtg gctatcactg tgacaatggt
tacagttgac 480 ttgttagtga tggccggata cacaggttta gcatctcatg
tattacgtat gcttcgtacc 540 ccaaaacagc aaaaacgcct gaaccgcacc
ttcgccggtt tattcatcgg agcggccaca 600 ttccttgcca ctttgcgccg
cgcaccagta 630 <210> SEQ ID NO 28 <211> LENGTH: 1014
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1014) <223> OTHER INFORMATION: Asparaginase
(Escherichia coli) <400> SEQUENCE: 28 atgcagaaga aatcgatcta
cgtcgcgtac acgggcggca ccattgggat gcagcgttcg 60 gagcagggtt
acatccccgt ttccggtcac ttgcagcgcc agctggcctt gatgcccgag 120
ttccatcgcc ccgagatgcc agattttacc attcatgagt acactccact tatggattca
180 tcggacatga cgccggaaga ctggcaacac attgcagaag atatcaaggc
tcactatgat 240 gattatgacg gctttgttat tttacacggt actgacacaa
tggcatacac agcttctgca 300 ctttccttta tgcttgagaa ccttggtaag
cccgtgatcg tgaccgggtc gcagatcccc 360 cttgccgaat tgcgcagtga
cgggcagatc aatcttctta atgcgttata tgtggccgct 420 aactatccga
tcaatgaagt gactttattc ttcaataacc gcttgtaccg tggaaaccgc 480
actacgaaag cccatgctga tggctttgac gcctttgcat ccccaaatct gcctcccctt
540 ttggaagccg ggattcacat ccgtcgttta aatacacccc ccgccccaca
tggagagggg 600 gagcttatcg tacatccaat tacccctcaa cctatcggag
ttgtaacgat ttaccctggt 660 attagtgccg acgtagtccg caatttcctt
cgccagcccg tgaaagcatt gatcttacgt 720 tcctacggtg tagggaacgc
gccacagaat aaggcatttc tgcaagaatt acaagaggca 780 tcggatcgtg
gtatcgtggt agtcaacctg acacagtgca tgtcaggtaa agttaatatg 840
ggtggatacg caaccgggaa tgcattagct catgcagggg taattggagg cgctgatatg
900 acggtcgaag ctaccctgac gaagcttcat tatctgttat cccaggagtt
ggacaccgag 960 accattcgca aagctatgtc tcagaacctt cgcggtgagc
ttactcccga tgac 1014 <210> SEQ ID NO 29 <211> LENGTH:
1461 <212> TYPE: DNA <213> ORGANISM: Mycobacterium
bovis <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1461) <223> OTHER INFORMATION:
Asparagine transporter ansp2 (Mycobacterium bovis) <400>
SEQUENCE: 29 atgccgcctc tggacatcac cgacgaacgc ttgactcgcg aagatacagg
atatcacaaa 60 ggccttcact cccgtcagct tcagatgatc gctcttggag
gtgctattgg gaccggactt 120 tttctggggg caggcggacg tctggcttct
gccggaccgg gattattctt ggtttatggt 180 atctgtggca tttttgtctt
tcttattctt cgtgccttgg gagaacttgt gcttcaccgc 240 cctagttcag
gatcatttgt atcctacgcg ggggaatttt atggtgaaaa ggtcgcgttc 300
gtcgcggggt ggatgtattt tttgaattgg gcaatgactg ggattgtgga cactacagcc
360 atcgcccact attgccacta ttggcgcgct tttcaaccaa ttccacagtg
gacgttggcc 420 cttattgcgt tgttagttgt attatccatg aatctgatct
ccgtccgctt attcggggaa 480 cttgagtttt gggcctcgct tattaaagta
attgcgcttg ttacgttcct gattgtaggg 540 actgtattcc tggcggggcg
ttacaagatt gacgggcaag aaactggtgt atcattatgg 600 tcatctcatg
gcggaatcgt tcctacgggg ttactgccca ttgtccttgt gacctctgga 660
gttgtgttcg catacgcagc catcgagctg gtaggaatcg cagccgggga gacggccgaa
720 ccagccaaaa tcatgccccg cgcaatcaat tcggtcgtcc ttcgtattgc
gtgtttttat 780 gtgggatcta cggtgcttct ggcgttgctt ttaccataca
cggcttataa ggagcacgta 840 agtcccttcg taacattttt cagcaaaatt
ggaattgatg ccgcggggag tgtaatgaac 900 ttggtagtgc ttacggcagc
gttatctagt ttgaacgctg gtttgtattc cacaggacgc 960 atcctgcgct
caatggcgat caacggcagc ggaccacgct ttacggcacc catgagtaaa 1020
accggtgttc cttatggcgg tatcttgctt acagcaggta tcggtttatt gggaatcatt
1080 cttaatgcga tcaaaccctc gcaggcgttc gaaatcgttt tacacatcgc
tgctaccggc 1140 gtaatcgcag cctgggctac gatcgtggct tgtcagttgc
gcttacatcg catggccaac 1200 gctggccaac ttcagcgccc taagttccgt
atgcccttgt caccttttag cgggtacctg 1260 actttggcgt ttctggcggg
cgtgctgatt ctgatgtatt tcgatgagca gcacgggccc 1320 tggatgatcg
ccgcgacagt aattggggtt cctgccctta ttgggggttg gtacttggtt 1380
cgtaaccgtg tgactgccgt cgctcatcac gctattgacc acactaagag tgtagctgtg
1440 gttcattcgg cagatcccat t 1461 <210> SEQ ID NO 30
<211> LENGTH: 1344 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1344)
<223> OTHER INFORMATION: Serine ammonia lyase (Bacillus
subtilis) <400> SEQUENCE: 30 atgtccatta atcaggaggc gcttcacgta
ctgttgaagg atccttttat tcatcgtctt 60 attgatgctg agccagtgtt
ttgggcaaat ccaggtatga aggaggggct gctttttcac 120 gctgacgagt
gggaaagtga gattgccgaa gcagagaaac gcttgcgtcg ttttgcgcct 180
tatatcgcgg aggttttccc agagaccaaa gatgcaaagg gcatgatcga gtctccactg
240 tttgagatgc aacatatgaa aaagaaactg gaggcagcat accaacaacc
tttccccgga 300 cgttggctgc ttaagtgtga ccatgaactt cccatttccg
ggtcgattaa ggcccgcgga 360 ggtatctatg aagtcttgaa acatgccgaa
aagctggctc ttcaggaggg gatgcttcaa 420 gagtcggacg attatcgtat
gttgcaagaa gatcgtttcg cggccttctt cagccgctat 480 tctatcgcag
tgggctccac gggcaattta ggtttaagta tcgggattat tggcgctgct 540
cttggtttcc gcgtaacagt tcacatgagt gctgacgcta agcaatggaa gaaagatctg
600 ttacgtcaga aaggggtaac cgtaatggaa tatgagagcg attattcaga
agcagttaaa 660 gaaggtcgtc gccaagcaga acaagaccca ttctgttact
tcattgatga tgaacatagc 720 cgtcaattgt tcctgggcta cgcagttgcc
gcgtcccgcc ttaagacaca actggattgc 780 atggaaatcc aacctggtcc
cgaaacaccc ctgttcgtgt atcttccctg tggcgtaggt 840 ggggggccag
gaggtgtcgc tttcgggttg aaactgctgt atggagatca cgtccatgta 900
tttttcggag aaccgacgca atccccgtgc atgcttttag gcttatattc tggcttacac
960 gagcagattt cagttcaaga cattggattg gacaaccgta ccgcggcgga
tggcttggcg 1020 gtagggcgtc cctcaggatt cgtaggaaaa ttaatcgaac
cactgctgtc gggctgctat 1080 actgtagaag acgatacact gtatgcttta
ctgcacatgc tggcagcttc ggaatccaag 1140 tatcttgaac ctagcgcctt
ggcggggatg ttcggcccga tccagctgtt cagcacagaa 1200 gaaggacgtc
gctattctca gaaacataaa atggagcatg cggtgcacgt tatctggggg 1260
acggggggta gcatggtgcc aaaggaggag atggccgcat acaaccgcat cggggcggat
1320 ctgttaaaaa atgaaatgaa gaag 1344 <210> SEQ ID NO 31
<211> LENGTH: 1374 <212> TYPE: DNA <213>
ORGANISM: Pseudomonas fluorescens F113 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1374) <223> OTHER INFORMATION: SdaA (Pseudomonas
fluorescens F113) <400> SEQUENCE: 31 atgtcccttt cagtgtttga
tctttttaag atcggaattg gtccctcgtc ctctcatacc 60 gtaggaccta
tgcgtgcggc cgctcgtttt gccgaaggtc tgcgccgcga cgacctgctg 120
aactgtacta ctagcgtgaa agtcgagctg tacggatctc tgggcgcgac tggtaaaggg
180 cacggttcgg acaaagcagt gttactggga ttggagggag aacaccctga
cactgtcgac 240 accgagacgg ttgacgctcg tttacaggcg atccgcagtt
caggccgcct gaatttattg 300 ggggagcata gcattgagtt taatgaaaag
ctgcacttgg caatgattcg caagccgtta 360 gctttccatc cgaatggcat
gattttccgt gcgtttgatg ctgcgggctt acaggtacgt 420 tcccgtgagt
attactccgt cggcggaggg ttcgttgtag acgaggacgc agcgggtgcc 480
gaccgtatcg tcgaggatgc aacacctttg acattcccct tcaagagcgc gaaggatctt
540 ttaggtcatt gttctactta tggtttaagc atcagccaag tcatgcttac
aaacgagtct 600 gcgtggcgtc cggaagcgga gacccgcgca gggcttctta
aaatttggca ggtgatgcaa 660 gactgcgttg ccgcggggtg tcgcaatgag
ggcatccttc caggaggtct taaagtaaag 720 cgccgcgcgg ctgcgttgca
tcgtcaattg tgtaagaacc ccgaggctgc cctgcgcgat 780 ccgttaagtg
tattagattg ggtgaatttg tatgcgttag cggtaaatga agagaacgcc 840
tacggtggac gcgtggtcac ggcgcccact aatggagccg caggaatcat tcctgccgta
900 ttgcattact acatgcgctt tattccgggg gcatctgagg acggagtagt
ccgcttcctt 960 cttacagcgg cggcaatcgg gatcttgtat aaagagaacg
cctctattag tggggctgag 1020 gttggctgtc agggcgaagt aggagtggca
tgctccatgg cagcgggggc gttgtgcgaa 1080 gtcttgggag gctcggtcca
acaagtagaa aacgcagcag aaatcggaat ggagcataac 1140 cttggcttga
catgtgatcc tatcggcggg ttagtacagg tcccgtgtat cgagcgtaac 1200
gcaatgggat ctgttaaagc cattaacgca gtacgcatgg ctatgcgcgg ggacggtcac
1260 catttcgtct cccttgacaa agtaattcgt accatgcgtc aaactggggc
cgacatgaaa 1320 agcaagtaca aggaaaccgc gcgtggtgga cttgctgtca
acatcatcga gtgt 1374 <210> SEQ ID NO 32 <211> LENGTH:
1365 <212> TYPE: DNA <213> ORGANISM: Klebsiella
pneumoniae <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1365) <223> OTHER INFORMATION:
sdaB(Klebsiella pneumoniae) <400> SEQUENCE: 32 atgattagtg
tgtttgacat ctttaaaatc ggtatcggtc cgtcttcttc ccatacggtt 60
ggtcccatga aagcagggaa gcagtttacc gacgacttaa ttgctcgtgg actgctggca
120 gaggtcagta aggtcgtggt tgatgtttat ggctcccttt cattgacggg
caaaggtcac 180 catactgaca ttgctatcat tatgggtctg gcgggaaact
tgccagacac cgttgacatc 240 gacgccatcc ccggcttcat ccaagatgtt
aacactcacg gacgtctgat gttagcgaat 300 gggcagcatg aagttgattt
cccggtagac cagtgtatga attttcacgc tgacaacctg 360 tccttgcacg
agaatggaat gcgtattacg gctcttgcgg gagacaaagt gttgtactct 420
cagacttact actcaatcgg cggcggattc attgttgatg aggaacattt tggccaaaca
480 acggaggctc ctgtagccgt cccatatcca tacaaaaacg ccgctgattt
gcagcgtcat 540 tgccgtgaaa ctggtttgag tttatctgga cttatgatgc
aaaacgaact tgcattgcat 600 agcaaagaag ctctggaaca gcactttgct
gcagtttggg aggttatgtc tgccggcatt 660 gagcgcggca ttacaactga
aggtgtgttg cctggcaaat tacgtgtacc ccgccgcgcc 720 gcggcactgc
gtcgtatgtt agtctcgcaa gacacgacga actcggaccc tatggctgtt 780
gtagattgga tcaatatgtt cgcgttggcc gtcaacgagg agaacgcggc gggcggtcgc
840 gttgttacag cccccacaaa tggcgcgtgc ggaattgttc cggccgtgct
ggcatattat 900 gacaaattta tccgcaaagt caactccaac agtctggcgc
gttatatgct ggtggcaagt 960 gcaatcggct cactttataa gatgaatgcg
agcatctccg gcgcagaagt tggctgccaa 1020 ggtgaagtgg gggtcgcctg
ctctatggca gcggctggct tggcagagct gttgggcggg 1080 tcgccagggc
aagtgtgcat tgcggctgaa attgcgatgg agcataactt gggccttacg 1140
tgcgatcccg tagctggcca agtgcaggta ccgtgtatcg aacgcaatgc aattgcagcc
1200 gtaaaagcag taaatgcggc tcgcatggcc ttacgtcgta cttccgagcc
ccgtgtgtgc 1260 ttggataagg tgatcgaaac catgtatgag acaggtaagg
acatgaatgc aaagtatcgt 1320 gaaacgtctc gtggaggcct ggccatgaag
atcgtcgcgt gtgac 1365 <210> SEQ ID NO 33 <211> LENGTH:
1362 <212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1362) <223> OTHER INFORMATION: tdcG L-serine
dehydratase (Escherichia coli O157:H7 str. SS17) <400>
SEQUENCE: 33 atgattagtg cattcgatat tttcaagatt ggaatcggcc cctcgtcatc
gcacacggtg 60 ggcccaatga acgcaggtaa gtccttcatt gatcgccttg
agtcgagtgg cttattgaca 120 gcgacaagcc acattgtcgt ggacctgtac
gggagtctgt cgttgacggg caaaggccat 180 gcgaccgatg ttgctattat
catgggattg gccgggaatt caccgcagga cgtagtaatc 240 gatgaaatcc
cggccttcat tgagctggta actcgttcgg gccgtctgcc agtcgcaagc 300
ggagctcata tcgttgactt cccagttgcc aagaacatta tttttcaccc tgaaatgtta
360 cctcgccatg agaacggaat gcgtatcaca gcatggaaag ctcaggaaga
attattgagt 420 aagacgtatt actcggttgg tggcgggttc atcgtcgagg
aagagcactt cggtttatct 480 catgacgtag aaacaccagt accatacgac
ttccattcag caggtgagtt gttgaaaatg 540 tgcgattaca atggccttag
tatttcggga cttatgatgc ataacgaatt agcgcttcgt 600 tcgaaggccg
aaattgacgc cggcttcgca cgtatctggc aagttatgca tgatggcatc 660
gaacgtggta tgaacaccga aggtgtgtta ccaggaccct tgaatgttcc gcgtcgtgca
720 gtcgcactgc gtcgtcaact tgttagtagt gacaacattt ccaatgatcc
aatgaacgtg 780 attgactgga tcaacatgta cgcgctggcg gtctcggagg
aaaacgccgc tgggggtcgc 840 gtggtaacag cacctacgaa tggggcttgc
gggatcatcc ctgcggtatt ggcctattac 900 gataagtttc gccgtccagt
caatgagcgc tcaatcgctc gttacttcct ggcggcgggg 960 gctatcggcg
ctttatacaa gatgaacgcc tctatttcag gggcggaggt cggttgtcaa 1020
ggagagattg gggtcgcgtg ctctatggca gctgcaggtt tgacagaatt attaggcggc
1080 agcccagccc aagtttgcaa cgcggctgaa atcgcaatgg aacataatct
tggtctgacc 1140 tgtgaccctg tcgcaggtca ggtacagatt ccttgcattg
agcgtaatgc aatcaacgca 1200 gtaaaagctg ttaatgcggc gcgtatggct
atgcgtcgca catcagcccc gcgtgtgagc 1260 ctggataagg taatcgagac
catgtacgaa accggtaaag acatgaatga caaataccgc 1320 gaaacctctc
gcgggggtct tgcaattaaa gtcgtgtgtg gc 1362 <210> SEQ ID NO 34
<211> LENGTH: 1251 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1251)
<223> OTHER INFORMATION: glyA(Escherichia coliEPEC C342-62)
<400> SEQUENCE: 34 atgttgaaac gtgagatgaa tattgccgac
tatgatgcag aattatggca agctatggaa 60 caagagaaag tccgccagga
agaacatatt gaattaatcg cctctgaaaa ttacactagt 120 ccccgcgtta
tgcaagccca aggcagccaa ttaactaaca aatatgccga gggatatcct 180
gggaaacgct actatggagg ttgcgagtat gtagatattg tcgaacagtt agcaatcgac
240 cgcgcgaaag agcttttcgg cgcagactat gcaaacgtgc agccccattc
gggtagccaa 300 gcgaattttg cggtctatac cgcactgctg gaaccgggag
acacggtact gggtatgaat 360 ttagctcatg gtggtcactt aacgcacggg
tcccccgtta atttctctgg aaaactgtac 420 aacatcgtcc cctatggaat
cgatgctacc ggccacattg attacgcgga tcttgagaag 480 caagctaagg
aacataaacc aaagatgatc attggcggtt tttcagctta tagtggtgtc 540
gtcgactggg ctaagatgcg tgaaattgca gactctattg gcgcgtacct ttttgtcgac
600 atggcccacg tggctggctt ggtggcggca ggggtctacc cgaaccccgt
tccccatgcg 660 catgtcgtga ccaccacgac acataagaca ctggctgggc
ctcgtggtgg cttaatcttg 720 gccaaggggg ggtctgagga attatacaaa
aaacttaact cagccgtttt tccaggcgga 780 cagggtggtc cgttgatgca
cgtgattgct ggaaaggcgg tcgctcttaa ggaagccatg 840 gaacctgaat
tcaaaacgta ccaacagcag gttgcaaaaa acgccaaagc gatggttgag 900
gttttcctgg aacgtggtta caaagtcgtt agtgggggta ccgataatca tcttttctta
960 gttgacctgg tagataaaaa tttgaccgga aaggaggcgg acgctgcctt
aggccgtgcg 1020 aatattaccg tcaataaaaa ctcggtgcca aatgatccca
agtcgccttt cgtgacttca 1080 ggaatccgcg taggaactcc cgcaattaca
cgccgcgggt tcaaggaagc tgaggcgaag 1140 gagttagcag gatggatgtg
tgatgtttta gactcgatta acgatgaggc ggtgatcgaa 1200 cgtatcaaag
gtaaagtatt agatatttgc gcccgttatc cagtttatgc c 1251 <210> SEQ
ID NO 35 <211> LENGTH: 1287 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1287)
<223> OTHER INFORMATION: SdaCserine STP transporter
(Escherichia coliBL21(DE3) <400> SEQUENCE: 35 atggagacca
cgcagacttc tacaattgcg agcaaagata gccgttctgc ttggcgcaaa 60
actgatacta tgtggatgtt gggcctgtat ggaacagcta ttggggccgg ggtactgttt
120 ttgccaatca atgctggagt ggggggtatg atcccgctga tcattatggc
gattcttgct 180 ttcccaatga cattttttgc acatcgcggt cttacacgct
ttgtcctttc aggaaagaat 240 cctggggagg acattacgga ggttgtagaa
gaacattttg gcattggggc tgggaaactt 300 atcacattgc tgtatttttt
tgcaatctat cccattttgc ttgtctatag cgtagcaatc 360 acgaacaccg
tagaatcatt catgtcgcac cagttaggca tgacacctcc gccacgtgcg 420
attctgtcat tgatcttgat cgtgggaatg atgacaattg ttcgtttcgg agagcaaatg
480 atcgtgaaag ccatgtcaat tttggtattt ccgttcgtgg gagtcttaat
gttgctggca 540 ttgtatttaa ttccccagtg gaatggtgcc gctctggaga
ccttgtcgtt ggatacggcg 600 tcagcgaccg gtaatggtct ttggatgacg
ctttggttgg ccattccggt catggttttt 660 tcatttaacc actcaccgat
cattagctcg ttcgctgtgg cgaaacgcga agaatacggt 720 gatatggctg
aacaaaagtg ctcgaagatt ttggcattcg cccacatcat gatggtactt 780
acggtcatgt tcttcgtgtt ttcttgcgtc cttagtttaa ccccagcgga cctggcggct
840 gcaaaggaac aaaatatcag catcttaagc tatttggcga atcatttcaa
cgcgcctgtt 900 atcgcatgga tggcacccat tatcgctatc attgcaatta
ccaaatcttt cttagggcac 960 tacttgggtg cgcgcgaagg atttaacggg
atggttatca agtcgcttcg tgggaaagga 1020 aagagtatcg agatcaataa
acttaatcgc atcaccgcct tgttcatgtt agtaacaacg 1080 tggatcgtcg
ctacacttaa tccctccatt ctggggatga ttgaaacgct tgggggtcca 1140
atcatcgcaa tgatcttgtt tctgatgccg atgtacgcta tccagaaggt acccgcaatg
1200 cgtaaatact ctgggcatat ctccaacgtg tttgttgttg ttatgggatt
aatcgctatt 1260 tctgctatct tctatagtct gttctcc 1287 <210> SEQ
ID NO 36 <211> LENGTH: 438 <212> TYPE: DNA <213>
ORGANISM: Lactobacillus saniviri <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(438) <223>
OTHER INFORMATION: threonine Serine Exporter (Lactobacillus
saniviri JCM 17471 = DSM 24301) <400> SEQUENCE: 36 atggcgtatt
ctgtccagtt cctgatccaa ctgtccttct cgtaccttgc cactgtggct 60
tttgctatct gcatcaacgt tccacgtcgt gcgttaaatt ttgccggatg ggccggtgcc
120 atcgggtgga tctgctactg gctgctgaac acacatggca cgggccgcat
gttcgctaac 180 ctgattggcg ctgtcgcagt tggggtatgt ggtatcattt
tcgctcgcat caagaagatg 240 cccgtgatta ttttcaatat tccggggctg
gtgccattag tgcctggagc aaccgcctac 300 caggcagttc gcgctcttgc
gttgggaaat atggaccttg ctatccagct tggagttcgt 360 gttattatgg
tcgcaggggc aatcgcggtg ggattcatgg ttagtcagct tctgtcagag 420
ttgacttacc gcttgcac 438 <210> SEQ ID NO 37 <211>
LENGTH: 930 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(930) <223> OTHER INFORMATION:
glutaminase YbaS (Escherichia coli ST131) <400> SEQUENCE: 37
atgctggatg ctaataagct gcagcaggct gtcgatcagg cttatactca atttcattct
60 ttgaatggtg ggcagaatgc cgattacatt cctttcttgg ctaatgtccc
agggcaatta 120 gcagccgtag ctattgtaac atccgatggc aacgtgtatt
ctgccgggga ctcggactac 180 cgcttcgcac ttgagtctat cagtaaagtc
tgcactttgg cactggcgct ggaggacgtt 240 gggcctcagg ccgtgcagga
caaggttggg gctgatccta cagggctgcc attcaactca 300 gtaattgctt
tggaattaca cggtggaaaa ccactgtcac cgctggtgaa cgcgggggca 360
atcgctacca cgtctttgat taatgcagaa aatacggaac agcgttggca acgtattttg
420 catattcagc agcagcttgc tggtgagcaa gtcgcacttt ctgatgaagt
gaaccaaagt 480 gaacaaacta ctaattttca caaccgtgca attgcttggt
tactgtacag tgctggctac 540 ttgtactgtg acgcaatgga agcctgtgat
gtttatacac gtcagtgcag tactttgatc 600 aacacaatcg aattggcaac
attgggagct acgttagccg ctgggggcgt gaatccgttg 660 acacataaac
gcgttctgca agcggacaat gtgccctata ttttggctga aatgatgatg 720
gaagggcttt atggccgctc tggggactgg gcctaccgtg taggcttgcc aggaaagtcg
780 ggggtcggag gagggattct ggccgtggtg cccggcgtaa tgggaattgc
cgcgttttcg 840 cctcccttag acgaagaagg taacagcgtg cgcggacaaa
agatggttgc gagcgttgca 900 aagcagcttg ggtataacgt atttaaaggg 930
<210> SEQ ID NO 38 <211> LENGTH: 924 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(924)
<223> OTHER INFORMATION: Glutaminase (Escherichia coli
O145:H28 str. RM12581) <400> SEQUENCE: 38 atggccgtcg
caatggataa cgccatttta gagaatatcc tgcgccaagt gcgcccatta 60
atcggacaag gcaaggttgc ggattacatt ccggccttag ctacagtgga tgggagtcgc
120 ctgggaatcg ctatttgcac tgttgacggc caattgtttc aggcaggcga
cgcacaagag 180 cgcttctcca tccagagcat ttctaaagtg ttgtcattgg
ttgttgctat gcgtcactac 240 tctgaggagg aaatttggca gcgcgtgggg
aaggacccgt ccggcagtcc atttaattcg 300 ttggtacagt tggagatgga
acaaggaatc cctcgtaatc ccttcatcaa tgcaggtgct 360 cttgtagtct
gcgacatgtt acaaggtcgt ttatctgccc ctcgccaacg catgttggaa 420
gttgtgcgtg gtttgtctgg agttagcgat atcagctacg acacggtcgt ggctcgcagt
480 gaatttgaac actcagcacg caatgcagcg attgcgtggt taatgaagtc
gtttgggaat 540 tttcatcacg atgtgacgac agtccttcaa aattatttcc
actactgcgc attgaagatg 600 tcgtgcgtag agcttgcccg tacgttcgtc
tttcttgcga accagggcaa ggccatccat 660 atcgacgagc ccgtcgtaac
cccgatgcag gcgcgtcaaa tcaatgcgct gatggcgaca 720 tcgggaatgt
atcagaatgc gggggagttc gcctggcgtg tcggattacc agctaaatcc 780
ggtgtaggcg gtggaatcgt tgccattgtg ccccatgaaa tggctatcgc tgtgtggtcc
840 ccagaattag atgacgcagg aaattcgtta gcaggtattg cggttttaga
acaacttacg 900 aaacaattag gacgctcggt gtat 924 <210> SEQ ID NO
39 <211> LENGTH: 927 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(927) <223>
OTHER INFORMATION: ylaM (Bacillus subtilis subsp. subtilis str.
168) <400> SEQUENCE: 39 atggtgtgtc agcataatga tgaattagag
gctcttgtca agaaggcaaa aaaggttacg 60 gataaggggg aggtggctag
ttacattcca gctctggcta aggcggacaa acacgactta 120 agtgtcgcaa
tctactatag caataatgtg tgcctgtccg caggggacgt tgaaaagacg 180
ttcactctgc aatccatcag caaagttctg tcgttagctc tggtacttat ggagtatggg
240 aaggataagg tattcagtta tgttgggcag gaacctacag gtgatccctt
taacagcatc 300 attaaactgg agacagtcaa cccctctaag ccattaaatc
cgatgatcaa tgcgggcgcg 360 ttagtagtga ccagtcttat ccgcggacgt
acggtgaagg agcgtcttga ctatcttctt 420 agctttatcc gtcgtctgac
taataatcaa gaaattacat actgccgcga ggtagcggaa 480 agcgaatatt
ctacttcaat gattaaccgt gcgatgtgct attatatgaa acagtatgga 540
attttcgaag atgacgttga agcggttatg gacctttata caaagcaatg cgctattgaa
600 atgaactcac ttgatttggc taagatcggt tcggttttcg ccttgaacgg
acgccatcct 660 gaaaccgggg agcaagtgat ttcgaaggat gtagcccgta
tctgtaagac gtttatggtg 720 acgtgtggaa tgtataatgc ctctggtgaa
tttgcgatca aagttggtat ccctgcgaaa 780 tcgggagtgt caggtgggat
tatgggtatc tccccttacg atttcggaat cgggatcttt 840 ggacccgcgc
tggacgagaa ggggaatagt attgctggtg tgaagctttt agaaatcatg 900
agcgagatgt accgtcttag tatcttt 927 <210> SEQ ID NO 40
<211> LENGTH: 981 <212> TYPE: DNA <213> ORGANISM:
Bacillus subtilis <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(981) <223> OTHER
INFORMATION: ybgJ(Bacillus subtilis) <400> SEQUENCE: 40
atgaaagagt tgattaaaga gcatcaaaag gatatcaatc ctgcattaca actgcatgac
60 tgggtagaat actaccgtcc atttgcggca aatggccaaa gtgcaaacta
tatccccgct 120 ttagggaagg tgaacgacag ccagttaggg atctgcgtac
tggaaccgga tggcaccatg 180 attcacgctg gggattggaa tgtgtccttt
accatgcagt cgatttcaaa agtaattagc 240 ttcattgctg cctgcatgtc
gcgtggaatc ccgtatgtct tggatcgtgt agacgtggaa 300 cccacaggag
atgcttttaa tagtatcatc cgtttagaga tcaacaaacc aggaaagcct 360
ttcaatccta tgattaatgc cggagctttg actatcgcta gcattcttcc aggagagtcc
420 gcttacgaaa aacttgagtt tttgtatagc gtgatggaga ctttaatcgg
taaacgcccc 480 cgtattcacg aagaagtatt ccgttctgaa tgggagaccg
ctcatcgcaa tcgcgcctta 540 gcctactatc ttaaagaaac aaacttctta
gaggccgagg tcgaagagac actggaagta 600 tatttgaaac aatgcgcgat
ggaatcgacc acggaagaca tcgccctgat cgggttgatc 660 ctggcccacg
atgggtatca tcctatccgt catgagcagg tcattcccaa ggatgttgcc 720
aagttggcta aagcgttaat gttgacctgt ggcatgtata acgcttctgg aaagtatgcg
780 gctttcgttg gagtacccgc aaaatctgga gtttcgggtg gtattatggc
cttggtgcct 840 ccaagtgcgc gtcgcgaaca gccgttccag agcgggtgcg
gtatcgggat ttatggacct 900 gcaattgatg agtacgggaa tagcctgacg
ggcggcatgc ttttaaaaca catggcccaa 960 gagtgggaac tgagtatttt c 981
<210> SEQ ID NO 41 <211> LENGTH: 3436 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(3436) <223> OTHER INFORMATION: Glutamine permease
glnHPQ operon (Escherichia coli) <400> SEQUENCE: 41
ccatggcaga acgtgcagtg cagctgggcg gtgtagctct ggggaccact caagttatca
60 acagcaaaac cccgctgaaa agttacccgc tggacatcca caacgttcag
gatcacctga 120 aagaactggc tgaccgttac gcaatcgtcg ctaatgacgt
acgcaaagcg attggcgaag 180 cgaaagatga cgacaccgca gatatcctga
ccgccgcgtc tcgcgacctg gataaattcc 240 tgtggtttat cgagtctaac
atcgaataaa tccatcgctg atggtgcaga actttagtac 300 ccgataaaag
cggcttcctg acaggaggcc gttttgtttt gcagcccacc tcaacgcact 360
tatttagtgc atccatctgc tatctccagc tgattaagta aattttttgt atccacatca
420 tcacacaatc gttacataaa gattgttttt tcatcaggtt ttacgctaaa
taatcactgt 480 gttgagtgca caattttagc gcaccagatt ggtgccccag
aatggtgcat cttcagggta 540 ttgccctata aatcgtgcat cacgtttttg
ccgcatctcg aaaaatcaag gagttgcaaa 600 actggcacga ttttttcata
tatgtgaatg tcacgcaggg gatcgtcccg tggatagaaa 660 aaaggaaatg
ctatgaagtc tgtattaaaa gtttcactgg ctgcactgac cctggctttt 720
gcggtttctt ctcatgccgc ggataaaaaa ttagttgtcg cgacggatac cgccttcgtt
780 ccgtttgaat ttaaacaggg cgataaatat gtgggctttg acgttgatct
gtgggctgcc 840 atcgctaaag agctgaagct ggattacgaa ctgaagccga
tggatttcag tgggatcatt 900 ccggcactgc aaaccaaaaa cgtcgatctg
gcgctggcgg gcattaccat caccgacgag 960 cgtaaaaaag cgatcgattt
ctctgacggc tactacaaaa gcggcctgtt agtgatggtg 1020 aaagctaaca
ataacgatgt gaaaagcgtg aaagatctcg acgggaaagt ggttgctgtg 1080
aagagcggta ctggctccgt tgattacgcg aaagcaaaca tcaaaactaa agatctgcgt
1140 cagttcccga acatcgataa cgcctatatg gaactgggca ccaaccgcgc
agacgccgtt 1200 ctgcacgata cgccaaacat tctgtacttc atcaaaaccg
ccggtaacgg tcagttcaaa 1260 gcggtaggtg actctctgga agcgcagcaa
tacggtattg cgttcccgaa aggtagcgac 1320 gagctgcgtg acaaagtcaa
cggcgcgttg aaaaccctgc gcgagaacgg aacttacaac 1380 gaaatctaca
aaaaatggtt cggtactgaa ccgaaataat aacgctacac ctgtaaaacg 1440
cactggcagt tccctctccc ctatggggag aggattaggg tgaggggcgc aaacccgctc
1500 cggggccatt aattaccctg aatttgatta tttacaccac ggtaacagga
acaacatatg 1560 cagtttgact ggagtgccat ctggcctgcc attccgcttc
tgattgaagg tgccaaaatg 1620 accctgtgga tttcggtcct cggtctggca
ggcggtctgg taatcggatt gctggcaggt 1680 tttgcacgca ccttcggagg
ttggatagcc aaccacgtcg cgctggtctt tattgaagtg 1740 atccgcggca
cacctatcgt cgtccaggtg atgtttattt atttcgccct gccgatggcg 1800
tttaacgact tacgcatcga cccatttact gcggcggtgg tcaccatcat gatcaactcc
1860 ggcgcgtata ttgcggaaat cacgcgtggt gcggtgctgt ctatccacaa
aggttttcgt 1920 gaagcaggac tggcgctcgg tctttcacgt tgggaaacca
ttcgctacgt cattttaccg 1980 ctggcactgc gtcgtatgct gccgccgctg
ggtaaccagt ggatcatcag cattaaagac 2040 acctcgctgt ttattgtgat
cggcgtggcg gaactgaccc gtcaggggca agaaattatt 2100 gccggtaact
tccgcgccct tgagatctgg agcgccgtgg cggtgttcta tctgattatt 2160
accctggtgc tgagctttat tctgcgtcgt ctggaaagaa ggatgaaaat cctgtgattg
2220 aatttaaaaa cgtctccaag cactttggcc caacccaggt gctgcacaat
atcgatttga 2280 acattgccca gggcgaagtc gtggtgatta tcgggccgtc
cggttccggt aaatcgaccc 2340 tgctgcgctg catcaacaaa ctggaagaaa
tcacctccgg cgatctgatt gtcgatggcc 2400 tgaaggttaa cgatccgaaa
gttgacgagc gcctgattcg ccaggaagca ggtatggtgt 2460 tccagcagtt
ttacctcttc ccgcatctga cagcgctgga aaacgtcatg tttggcccgc 2520
tacgcgtgcg tggcgcgaac aaagaagagg cggaaaaact ggcacgtgag ctgctggcga
2580 aagtcggtct ggcagaacgt gcacatcact acccttccga actttctggt
ggtcaacagc 2640 agcgtgtggc gattgcccgc gcgctggcgg tgaagccgaa
aatgatgctg tttgatgaac 2700 cgacttccgc tcttgacccg gaactgcgcc
atgaagtgct gaaggttatg caggatctgg 2760 ctgaagaagg gatgacgatg
gtgatcgtga cccacgaaat cggttttgcc gagaaagtag 2820 cttcgcggct
gatctttatc gacaaaggcc ggattgcgga agatggcaat ccgcaggtgt 2880
tgatcaagaa cccgccgagc cagcgcttgc aggaattttt gcagcacgtc tcttaataag
2940 acacattgcc tgatcgtacg cttatcaggc ctacaggata tctggcaact
tattaaaatt 3000 gcatgaactt gtaggacgga taaggcgttc acgcgcatcc
ggcaaaaaag cccgcacgtt 3060 gtcagcaacc tgcttaatat cccttcctcc
ctttcacccg aaagggaggc acaccagatt 3120 cctctcattt aaaatcgccc
ctcctccagc atctatactt atctttttgc tctattttct 3180 cactggagga
gtcatgcggt ggatcctgtt catcctcttc tgcctgctgg gcgcacctgc 3240
ccacgcggta tccatacccg gcgttacaac cacaacgaca acggactcaa cgactgaacc
3300 ggccccggaa ccggatatcg aacaaaaaaa agcggcctat gcgcactggc
ggatgtgctg 3360 gataatgaca cctcgcgtaa agagttgatc gaccagttgc
gcaccgttgc cgctacgccc 3420 ctgctgaacc ggtacc 3436 <210> SEQ
ID NO 42 <211> LENGTH: 744 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(744) <223>
OTHER INFORMATION: Glutamine permease H glnH (Escherichia coliEPEC
C342-62) <400> SEQUENCE: 42 atgaaaagtg tacttaaagt gtcattggca
gcactgacac ttgcatttgc agtctccagt 60 catgctgcgg acaaaaagtt
agtcgtagcg actgacactg cgtttgttcc tttcgaattc 120 aagcaggggg
acaagtacgt cggctttgac gtagaccttt gggccgccat tgcaaaagag 180
cttaagttgg attacgagtt aaagcctatg gacttcagtg gtatcattcc cgccctgcaa
240 acgaaaaacg tggatcttgc gcttgcaggc attactatta ccgacgaacg
caagaaggcg 300 attgacttca gcgacggcta ttataagtcg ggtcttttag
ttatggtaaa agccaacaat 360 aatgatgtga aaagcgtgaa agatttggac
gggaaagtag tggcagttaa atcaggtaca 420 gggagtgtgg attacgcgaa
agctaatatc aaaaccaaag acttacgtca attcccgaat 480 atcgacaatg
cgtatatgga actggggacg aaccgtgcgg atgcggtgct gcacgataca 540
cccaacatcc tttatttcat taaaacagct ggtaatggtc aatttaaagc tgtaggcgac
600 agcctggaag cccagcaata cgggatcgcg ttccctaagg gctctgatga
gcttcgtgac 660 aaggtaaacg gggcgcttaa aacgctgcgt gaaaacggaa
cgtacaatga aatctataag 720 aagtggttcg gaaccgagcc caaa 744
<210> SEQ ID NO 43 <211> LENGTH: 657 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(657)
<223> OTHER INFORMATION: Glutamine permease P glnP
(Escherichia coliB354) <400> SEQUENCE: 43 atgcaattcg
attggagtgc gatttggcct gccattcccc ttctgattga gggtgcaaaa 60
atgactctgt ggatttcagt gctggggtta gccggaggtc ttgttattgg gttattagca
120 gggtttgcac gcactttcgg gggatggatt gcaaatcatg ttgcgctggt
cttcatcgaa 180 gtcattcgtg gcacccccat cgtggtccaa gtgatgttta
tttacttcgc gttgccaatg 240 gcatttaacg atcttcgtat tgatccattt
actgcggcag tggtgactat catgattaat 300 agtggggcgt acattgcgga
gattactcgc ggcgctgttc tttccattca caaaggtttt 360 cgtgaggccg
gtttagctct tgggctttcc cgctgggaaa caattcgtta tgttatcttg 420
ccgcttgcct tgcgccgtat gttgccgccg ctgggtaacc aatggatcat ttctatcaaa
480 gatacttcgc ttttcattgt tattggagtg gctgaattaa cacgccaagg
tcaagaaatc 540 atcgcgggga atttccgtgc attagagatc tggagtgctg
tcgccgtttt ctacttgatc 600 attacgctgg tgctgtcctt tattttgcgc
cgcttggagc gtcgcatgaa gattctt 657 <210> SEQ ID NO 44
<211> LENGTH: 720 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(720) <223> OTHER
INFORMATION: Glutamine Permease Q glnQ (Escherichia coliEPEC
C342-62) <400> SEQUENCE: 44 atgattgaat ttaagaatgt gtcgaagcat
ttcggcccca cccaagtact tcacaacatt 60 gaccttaaca tcgcccaggg
cgaggttgta gtaatcatcg gtccatctgg tagtggcaag 120 tccaccttgc
tgcgttgtat caataaactt gaggaaatca ccagcggaga cttaattgtg 180
gacggtctta aagtcaacga tccaaaagtg gacgaacgct tgattcgtca ggaagcgggt
240 atggttttcc agcagttcta cttgtttccg caccttacgg ctcttgagaa
cgtcatgttc 300 ggaccgttac gcgtgcgcgg ggccaataag gaggaggcgg
agaagttggc acgcgagctg 360 ttagcaaaag ttggcttggc tgaacgtgca
catcattacc cttctgagct gtcaggtggg 420 caacagcaac gtgtcgccat
cgcacgcgcg cttgctgtaa aaccaaagat gatgctgttc 480 gatgagccaa
cgtcggcgct tgacccggag ttgcgccatg aggtccttaa ggttatgcaa 540
gacttagctg aagagggaat gacgatggta atcgtgacgc acgagattgg attcgcagag
600 aaggtagcat ctcgtttgat cttcatcgac aaaggtcgca ttgcagaaga
cggcgaccca 660 caagttctga ttaagaaccc cccttcacag cgcctgcaag
aatttctgca acatgtctcc 720 <210> SEQ ID NO 45 <211>
LENGTH: 1437 <212> TYPE: DNA <213> ORGANISM: Ustilago
maydis 521 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1437) <223> OTHER INFORMATION:
tryptophanamino transferase (transaminase) (Ustilago maydis 521)
<400> SEQUENCE: 45 atgagttccg ccacaagtcc ggcactggat
tatgcattgc tgttgtcttc ttctgctcgt 60 aaccgtatgc cttctgcaat
ccgttccctg ttcccggcag aattaattcc aggcatggtc 120 tctcttttgt
caggtaaacc gaattcggag acctttccct ttcagcgcat cagtttggaa 180
cttaaaccct ccatccatct ggagggacag accgagacag tgagcatcga aggtagcgat
240 ttagacatcg ctcttcagta ttcagcaacg agtgggttgc caaagttggt
agactggatc 300 attaaatttc aatctcgcgt tcacgctcgt aagcaggtcg
atgagggcaa taagccgggt 360 gaagtatggc gctgtagctt tggcaacgga
tctcaagacc tgctgaccaa gacatttgag 420 gctttagttg acgccggtga
ttcagtagtc ctggaaagtc cggcttacag tggaattttg 480 ccgtcgttgg
ttgcgcataa agccaacctt ttcgaggcag aaactgacgc cgagggcgtt 540
gagcccacgg ctttagacac attgctgact aactggaaga ctgacagtgc aacacgtgac
600 tctcgttttc ccaagttttt atatactacc ccgactggtg caaatccgtc
cgggacatca 660 gcctctgata atcgcaagcg tgcgatcctt gatattatcc
gcaagcacaa tttacttctg 720 ctggaggatg atccttacta ttttttgtca
ttccaagggt tggaaccggg ggctgacgcg 780 gtcaaacgca ctcgtgggaa
gagctatttt cagttggaag ctcaggacga ctatggcgtc 840 ggccgtgttg
ttcgctttga ttcatttagt aagatcttgt ctgccggatt acgcctgggt 900
ttcgttacag gacccaaaga gattctggac gccatcgacc tggacacttc ctcccgcaat
960 ttgcagacaa gtggcacttc ccaggcaatc gcctatgctt tgttgtctaa
gtggggaatt 1020 gacggttttt tacatcatgc ggacaatgtc gcacgttttt
accaaaatcg cttagaacgc 1080 tttgaagcca gtgcccaggc aatcttaacc
ggaagcccta gcatcgcctc gtgggttcgt 1140 ccttcggcag ggatgttcct
gtggatcaag ttaaagttgc ctccgtcgcc cgactcggcg 1200 gagggtgata
gttttgacct gatctctaat aaagctaagg cagctggggt attggcttta 1260
cccggtgtgg ccttcaaacc accgagcagt tcaagtacgg gtggcaaacg taagacatcg
1320 gcatatgtcc gcacgtcatt ctcccaggtg cctctggacc aagtggatac
cgcattcaca 1380 cgcctgcgtc aggtggtaga ggaggcctgg cgtgaggctg
gacttcaaat ccccgcg 1437 <210> SEQ ID NO 46 <211>
LENGTH: 1242 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1242) <223> OTHER
INFORMATION: Mtrtryptophan ArAAP transporter (Escherichia
coliBL21(DE3)) <400> SEQUENCE: 46 atggctaccc ttactactac
tcaaacttcc ccatcgcttc ttggaggagt cgttatcatc 60 ggtggaacta
tcatcggagc agggatgttt tcactgccgg ttgtgatgtc gggagcatgg 120
ttcttttggt caatggcggc tcttatcttc acgtggttct gtatgttgca tagtggcctg
180 atgatcctgg aagcaaatct gaactaccgt attgggtcct cttttgatac
aattacaaag 240 gaccttctgg ggaaaggatg gaatgtagtt aatggaatta
gtatcgcgtt cgtcctttac 300 atcttgacct acgcgtatat ctctgcctca
gggagcatct tgcatcacac ttttgccgag 360 atgtcattga acgtgcccgc
acgcgctgct ggctttggtt ttgcactgct tgtggcattc 420 gtagtctggt
taagtacgaa ggctgtgagc cgtatgaccg ctatcgtcct tggggctaaa 480
gtaattacct tctttttaac attcggctcg ctgttaggac acgtgcagcc tgccactttg
540 ttcaatgtgg ctgaatcaaa cgcctcgtat gccccctatt tacttatgac
tttgccgttt 600 tgtctggctt ccttcggtta tcacggaaac gtgccatcac
tgatgaaata ttatggtaag 660 gatcctaaaa caattgtgaa gtgcttggta
tacgggacct taatggcact tgccctttac 720 acgatctggc ttcttgcaac
gatgggcaat attcctcgcc ctgaatttat cgggatcgca 780 gaaaaagggg
ggaatattga cgtgctggtc caggctttat cgggtgtctt gaatagccgc 840
tctttggatc ttttgttagt tgtcttttcc aattttgccg tggcatcgag tttcttaggt
900 gtgacgctgg gtctttttga ttacctggcc gatctgttcg gattcgacga
cagcgcggtg 960 ggccgtctta aaactgcttt attaacattt gcgccccctg
tagtgggagg tcttctgttt 1020 cctaacggat tcttatacgc catcggctac
gccggattgg cggccacgat ttgggcagct 1080 atcgtcccgg ctttattggc
acgtgcctca cgcaaacgct tcgggagtcc taaattccgt 1140 gtttggggcg
ggaagcctat gattgccctt attttagtgt ttggagtcgg taatgcactt 1200
gtgcacatct tgtcatcgtt caatctgctt cccgtttatc aa 1242 <210> SEQ
ID NO 47 <211> LENGTH: 1245 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1245)
<223> OTHER INFORMATION: tryptophan permeaseTnaB (Escherichia
colistr. K-12 substr. MC4100) <400> SEQUENCE: 47 atgaccgacc
aagctgaaaa gaagcattcg gcattctggg gagtaatggt cattgccggt 60
accgtgatcg gcggtgggat gtttgcttta cctgtggact tagcaggcgc gtggtttttt
120 tggggggcgt tcattctgat tattgcttgg ttttccatgc tgcatagtgg
cttgctgctt 180 cttgaagcga atcttaacta tccggtgggg tcaagtttca
ataccattac aaaggacctg 240 attggtaaca catggaatat catttcgggg
atcacggtag catttgtatt gtatattctt 300 acatatgctt atatcagtgc
gaatggcgca atcatttccg agacgatctc catgaacctg 360 gggtatcacg
cgaatccccg tattgtcggc atctgcacag cgatttttgt tgcgagcgta 420
ttatggctga gttcgttggc agcttcgcgt attacttccc ttttccttgg tttgaaaatc
480 atcagcttcg taattgtgtt tgggagtttt tttttccagg tcgactactc
cattcttcgc 540 gatgcaacaa gtagcacagc aggcaccagt tacttcccat
atatctttat ggccttaccg 600 gtttgtttag cgtcttttgg ttttcatggt
aatatcccct cattaattat ttgctacggc 660 aagcgcaagg acaaattaat
taagtctgtt gttttcggct ccttgttggc gcttgtaatc 720 tatttatttt
ggctttattg tacgatgggg aacatccctc gcgaatcctt taaggctatt 780
atttcttcag gaggcaacgt agacagtttg gtaaaaagtt ttttgggtac gaagcagcat
840 ggtatcatcg agttttgttt acttgttttc agtaatcttg ccgttgcttc
ctcattcttt 900 ggcgtgactc tggggctttt tgattatctg gcagatttat
tcaagatcga caactcgcat 960 ggcgggcgct tcaaaacggt tctgcttaca
tttcttcctc cagctttact ttacctgatc 1020 tttccgaatg gttttatcta
tggtattggg ggggcaggcc tgtgcgccac tatctgggca 1080 gttatcattc
ctgctgtatt ggctatcaag gcacgcaaaa agtttcccaa ccagatgttc 1140
accgtgtggg gcggcaattt gattccggca atcgtgatct tatttggtat cacggttatt
1200 ctttgctggt tcggcaatgt gtttaacgtc ctgcctaagt ttgga 1245
<210> SEQ ID NO 48 <211> LENGTH: 1368 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1368) <223> OTHER INFORMATION: aroP (Escherichia
coliO104:H4 str. C227-11) <400> SEQUENCE: 48 atggaagggc
agcagcatgg cgaacagctt aagcgtggcc tgaagaatcg tcatatccag 60
cttatcgcat taggcggagc tattgggacc ggcttgttct taggctctgc ttcagtcatt
120 cagtctgcgg ggccaggcat tattttaggc tacgcgattg cgggcttcat
cgccttttta 180 attatgcgcc agcttggcga gatggtggtg gaggaacccg
tggcaggcag tttctctcac 240 tttgcataca agtattgggg aagttttgca
ggctttgcga gcggttggaa ctactgggtt 300 ctgtacgttc tggtggccat
ggcggaactg acagcagtcg gtaaatatat tcaattctgg 360 taccctgaaa
ttcccacttg ggtctctgcc gctgtcttct ttgtcgtcat taatgcaatc 420
aacttgacca acgtcaaagt attcggcgag atggagtttt ggttcgctat tatcaaagtc
480 attgctgttg tggccatgat cattttcgga ggctggctgc ttttcagcgg
caacggaggt 540 ccccaggcaa ctgtatcgaa tctttgggac cagggtggtt
tcttgccaca tgggttcacg 600 gggttagtta tgatgatggc cattattatg
ttctcgtttg gagggcttga attggtgggc 660 atcactgctg ctgaagctga
taacccggag caaagcattc ctaaggccac aaatcaagtg 720 atctatcgca
tccttatctt ttacattgga tcgttggcag tattgctgag tttgatgccc 780
tggacccgtg tcaccgctga tacaagccct tttgttttga tttttcatga attaggggat
840 acttttgtgg caaatgcgtt aaacatcgtc gtattaactg ctgccttgtc
agtatataac 900 tcctgcgtat actgtaatag ccgtatgctg ttcggcttgg
ctcagcaggg gaacgctccg 960 aaagcactgg ccagtgtcga caagcgtgga
gtacctgtga atacgatttt agtttctgct 1020 ctggtcactg cactttgtgt
attgatcaac tacctggcgc ctgagtcggc gtttggcctg 1080 ctgatggcgc
tggtggttag cgcattggtc atcaattggg cgatgatctc cttggcacac 1140
atgaaattcc gccgtgctaa acaagaacag ggtgtggtta cacacttccc agcattatta
1200 taccctctgg gcaactggat ttgcttactt tttatggcag cggttctggt
catcatgctg 1260 atgacgcctg gtatggctat ttctgtttat ctgattccgg
tttggttaat cgtattaggg 1320 attggctatt tattcaagga aaaaactgca
aaggctgtca aagcgcat 1368 <210> SEQ ID NO 49 <211>
LENGTH: 879 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(879) <223> OTHER INFORMATION:
Aromatic amino acid exporterYddG (Escherichia coliTW10598)
<400> SEQUENCE: 49 atgacccgcc agaaggcgac tctgatcggt
ttgattgcta tcgtattatg gtccacaatg 60 gttggtttaa ttcgtggggt
ttctgagggg cttggcccgg tgggcggagc agcagctatc 120 tactccctga
gcggtctgtt attgatcttt acagttgggt ttccgcgtat ccgtcaaatc 180
cccaagggat acttattggc ggggagttta ctttttgtga gctatgaaat ttgccttgcc
240 ttgtctctgg gctacgcagc gacacgccat caagcaattg aggtagggat
ggttaattac 300 ctttggccgt cattgacgat tcttttcgca atcttattta
acggtcagaa gactaattgg 360 ttgattgtac cgggtttatt attagcgttg
gtgggagtat gctgggtgtt gggaggtgac 420 aatggtctgc attatgacga
gattattaat aatatcacaa catcgccctt atcctacttt 480 ctggctttca
ttggtgcctt tatctgggcc gcctattgca ccgtgacgaa taagtacgct 540
cgtggcttca acggaattac agtatttgtc ttgcttactg gtgcatcttt gtgggtatat
600 tatttcttga cccctcaacc agagatgatc ttctccaccc cggttatgat
caaattaatt 660 tcagcagctt tcactttggg attcgcatac gcagcttgga
atgtcggcat tcttcatggg 720 aatgtgacga ttatggcagt cggttcctac
ttcacgcccg tacttagttc cgctttagca 780 gcggtactgc tgtcggcgcc
tttgagtttt agtttctggc agggtgccct gatggtgtgt 840 gggggctccc
ttttgtgctg gcttgctacc cgccgtggt 879 <210> SEQ ID NO 50
<211> LENGTH: 1152 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1152)
<223> OTHER INFORMATION: S-adenosylmethioninesynthase
Escherichia coli (strain K12) <400> SEQUENCE: 50 atggcaaagc
accttttcac gtcggaatct gtatctgaag ggcatcccga caaaattgca 60
gatcaaatct ccgacgcggt acttgatgct attctggaac aagatcccaa agcccgcgtc
120 gcttgcgaaa cttatgtcaa gacaggcatg gtgttagtcg gcggcgagat
cactacctct 180 gcgtgggtgg atatcgagga aatcacgcgc aatacggtgc
gtgagattgg ctatgtacac 240 tcggacatgg ggttcgacgc caacagttgt
gcggttttaa gtgccattgg gaaacagtca 300 cctgatatta atcagggggt
ggatcgtgcg gaccctcttg aacaaggtgc tggtgaccaa 360 ggtctgatgt
tcggttatgc tacgaacgaa accgatgtgt tgatgcccgc cccgatcaca 420
tacgcccacc gtctggtcca acgccaggcg gaggtccgta aaaacggcac gcttccttgg
480 cttcgtccag atgctaagtc gcaggtcact ttccaatacg acgacgggaa
gattgtcgga 540 atcgacgccg tggtcttgtc aactcagcat tcagaggaga
tcgatcaaaa gagccttcag 600 gaagccgtca tggaagagat catcaagccg
attctgcctg cagaatggtt aacttccgcg 660 accaagttct ttattaaccc
caccgggcgt tttgtcattg gcggtcctat gggcgactgt 720 gggttgaccg
gccgtaaaat tattgtcgac acttatggcg gaatggctcg tcatggcggt 780
ggggcattca gtggcaagga cccgtcaaag gtagatcgtt cagccgccta tgccgcccgt
840 tacgtagcca agaacattgt tgctgcagga cttgctgacc gctgtgaaat
ccaagtgagc 900 tacgcgatcg gcgtagcaga acccacctcc attatggtgg
aaacttttgg caccgaaaaa 960 gtccccagtg agcaactgac cttattggtt
cgtgagtttt ttgatttgcg cccttacgga 1020 cttatccaaa tgttagacct
tttgcaccca atctacaaag aaactgcagc atacggtcac 1080 tttggacgcg
agcattttcc ctgggagaag acagacaaag cacagctgtt acgtgacgcg 1140
gccggattga aa 1152 <210> SEQ ID NO 51 <211> LENGTH:
1275 <212> TYPE: DNA <213> ORGANISM: Anabaena
cylindrica PCC 7122 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1275) <223> OTHER
INFORMATION: adenosylhomocysteinase (Anabaena cylindrica PCC 7122)
<400> SEQUENCE: 51 atgacggcta cgacgccacg cctgaaacat
gaagtgaagg accttgcgct tgcgccttta 60 ggtcgtcagc gtattgagtg
ggcggggcgc gaaatgcctg ttttaaagca aatccgcgac 120 cgctttgaaa
aagaaaagcc cttcgcgggc ctgcgtatct cggcttgtgc gcatgttaca 180
acagagacgg ctcatttagc aattgccctg aaggccgggg gagctgatgc cgtattgatc
240 gcaagcaacc cactgtctac gcaggatgac gtagcagcct cgcttgtcgc
tgatcatgag 300 atctctgtgt ttgcacaaaa gggcgaagac gccgcgacgt
actcgcgtca cgtccaaatt 360 gcgttggacc accgccccaa tatcatcgtt
gatgacggtt ccgacgtagt agctgaatta 420 gtacagcacc gtcagaatca
gatcgcggat cttattggat ccactgaaga aactacaact 480 gggattgttc
gccttcgcgc tatgttcaac gagggggttt tgacgtttcc cgcgatgaat 540
gtcaacgacg cagacacaaa acattttttt gacaaccgct acggtacagg acaatctacc
600 ttggacggga tcattcgtgc aaccaacatc ttgcttgccg gcaaaactat
cgtagttgta 660 ggctatggct ggtgcggaaa ggggaccgca ttacgcgccc
gcgggatggg agctaatgtc 720 attgttaccg agatcgatca cattaaggca
attgaggcgg tgatggatgg gtttcgcgtt 780 ctgcccatgg ctgaagccgc
accgcatggt gatatcttta tcactgtaac gggtaataaa 840 cacgtagttc
gtggtgaaca ctttgatgtc atgaaagacg gcgccattgt ttgcaactca 900
ggtcacttcg atttggagtt ggatttaaaa tatttagcag caaatgccaa ggaaatcaaa
960 gatgtgcgcc cattcacaca agaatataaa ttaaccaacg gcaaaagcgt
agtggtatta 1020 ggagaggggc gtttgattaa tcttgcagcg gcagaaggtc
atccgtcggc agttatggac 1080 atgtctttcg ccaatcaagc cttagcagtc
gagtatttag tgaaaaataa aggctccttg 1140 gcggctggat tacattcgat
cccccgcgag gttgatgagg aaatcgctcg tttaaaattg 1200 caagcgatgg
ggatttttat cgattccctg acagcagatc aaatcgatta tattaattct 1260
tggcagtcag ggacg 1275 <210> SEQ ID NO 52 <211> LENGTH:
1374 <212> TYPE: DNA <213> ORGANISM: Klebsiella
quasipneumoniae subsp. Quasipneumoniae <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1374) <223> OTHER INFORMATION:
Cystathionine-beta-synthase (Klebsiella quasipneumoniae subsp.
Quasipneumoniae) <400> SEQUENCE: 52 atggtaatgt cgttattcca
cagtgttagc gatttaatcg gtcacacacc tttattacaa 60 ttgcataagc
ttgatacagg accctgtagt ttgttcttga aacttgagaa tcaaaaccca 120
ggagggtcaa ttaaagatcg tgtagcgctt agcatgatta acgaagcgga acgtcaggga
180 aaacttgcgc caggaggaac tatcatcgag gctacggcgg gaaatactgg
gttggggctt 240 gctttgatcg cagcccagaa aaactaccgt cttatccttg
tagttcccga caagatgtca 300 cgtgaaaaaa ttttccactt gcgtgcctta
ggcgcaaccg tgcttttgac ccgttcagac 360 gtgaacaagg ggcacccggc
atattatcag gactatgctc gccgcttggc agatgagact 420 ccaggggcgt
tctacattga ccaattcaat aatgatgcca atcctttagc acatgcaaca 480
agcacggccc ctgagctgtt ccaacaatta gaaggggaca tcgatgccat tgtggttggt
540 gttgggtcgg gtggaacgtt gggcggcttg caggcctggt tcgcagaaca
ctctcccaaa 600 acagagttca tcttggctga tccagctggg tcgattcttg
ccgaccaggt agacacaggc 660 cgctacgggg aaacgggaag ctggcttgta
gagggtattg gcgaggattt tatcccacca 720 cttgctcgcc tggaaggagt
tcataccgca tatcgtgtat ctgatcgcga agcctttctt 780 acagcccgtc
aactgcttca ggtagagggt gtattagcgg gctcgtcaac gggaacattg 840
ttatctgcgg ccttgcgcta ttgccgtgcc cagtctcgcc caaagcgtgt ggttaccttc
900 gcatgtgact ctggaaataa gtacttgagt aagatgttca atgacgactg
gatgcgccaa 960 cagggactta ttgcgcgccc ggaacaggga gatctgagtg
atttcatcgc cttacgtcac 1020 gacgaggggg ccacggtcac cgccgcgccc
gacgacacac tggcggctgt atttactcgc 1080 atgcgcttgt acgatatctc
ccagcttccg gtcttggaag acggtcgtgt cgttggcatt 1140 gtggacgaat
gggatttaat tcgccatgta cgtggcgacc gtcaacgctt ttccctgcca 1200
gtcagcgagg ctatgtcccg tcacgtagaa acgttagaca aacgcgcccc cgaatccgaa
1260 ttgcaagcta tcttagaccg tggactggta gcagtcattg cagacaatgc
gcgctttctg 1320 ggactggtta cacgttcaga tgtcttaacg gcatggcgca
atcgtgtggc gcaa 1374 <210> SEQ ID NO 53 <211> LENGTH:
1146 <212> TYPE: DNA <213> ORGANISM: Klebsiella
pneumoniae subsp. pneumoniae HS11286 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1146) <223> OTHER INFORMATION:
cystathionine-gamma-lyase (Klebsiella pneumoniae subsp. pneumoniae
HS11286) <400> SEQUENCE: 53 atgtcgtcta ttcacaccct gtctgttcat
agtggcacct tcacggactc acatggcgcg 60 gtgatgcccc caatctatgc
cacctccacg ttcgcgcaac ctgcgcccgg acagcacacc 120 ggatatgaat
actcgcgcag tggaaatcct actcgtcatg ccttagagac tgcgatcgca 180
gacctggaga atggaacgcg cgggtacgca tttgcctcgg gcttggcagc gatctcgact
240 gtccttgaat tgttggataa ggacagccat ttagttgcag tggatgatgt
ctatggtggg 300 acctaccgtt tacttgaaaa cgttcgtcgt cgttctgctg
ggctgcaagt gtcgtgggtc 360 aagccagacg atttagcggg gattgaggcg
gctatccgtc ctgacacccg tatgatctgg 420 gtcgaaacac ctactaatcc
tttgctgaaa ttagccgatt tgagcgccat cgcagctatc 480 gcacgccgtc
acaatcttat ttcagttgcg gataacacgt tcgcttcacc agccatccac 540
cgtcctcttg aacacggttt cgacattgtg gtgcattctg cgacaaaata cttaaatgga
600 cattccgatg tggttgcggg gttagctgtc gtcggagata actccggctt
agccgagaaa 660 ttaggttatt tacaaaatgc agttggcggg gtattagacc
ccttttcctc gttccttaca 720 ttgcgcggca tccgcactct ggcactgcgt
atggaacgtc atagcgcgaa tgcactgcag 780 ttagccgaat ggttggaaca
acagcccgaa gtagagcgtg tatggtttcc ttggctggcc 840 tcccatcctc
atcatcaatt ggcacgtcag cagatggcat tacctggcgg gatgattagc 900
gtagtagtca aaggagatga gggatatgct gagcgcatca tcagtaaact gcgttggttc
960 actcttgccg agtctttagg cggcgtcgag tcgttagttt cccagccgtt
ctcaatgaca 1020 catgcttcga tcccacttga aaagcgtctt gcgaacggca
ttacgcccca gcttattcgc 1080 cttagtgtgg ggatcgaaga cccacatgat
cttatcgcgg attggcaaca agccctgcgt 1140 gccgaa 1146 <210> SEQ
ID NO 54 <211> LENGTH: 483 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(483) <223>
OTHER INFORMATION: cysteinedioxygenase (Bacillus subtilissubsp.
subtilis str. BAB-1) <400> SEQUENCE: 54 atggagttat acgagtgcat
ccaggacatc ttctcggggt tgaaaaaccc ttccgtgaaa 60 gatctggcaa
catccctgaa acaaatcccg aatgcagcta aattatctca gccttacatt 120
aaagagcctg accagtatgc atacggtcgc aatgccatct accgtaacaa cgagttggag
180 attattgtta tcaacattcc tcccaacaaa gagacaaccg tacacgatca
cggacaatcc 240 attggatgcg caatggttct ggaaggtaaa ttacttaata
gcatttatcg ttctgctggt 300 gagcacgccg agctgtccaa ctcttacttt
gttcacgagg gggaatgcct tatctcgact 360 aaaggcttga ttcacaaaat
gagcaacccc acaagcgagc gcatggtatc gttgcatgtt 420 tattcgccac
cgcttgagga catgacagta tttgaggaac agaaagaggt gttaaagaac 480 tct 483
<210> SEQ ID NO 55 <211> LENGTH: 891 <212> TYPE:
DNA <213> ORGANISM: Caenorhabditis elegans <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(891) <223> OTHER INFORMATION: Glutamate Oxaloacetate
Transaminase (Caenorhabditis elegans) <400> SEQUENCE: 55
atgagcgtta gtaaaaaact gttctctacg gctgtgcgtg gtaagagctg gtggtcacac
60 gtcgagatgg gccctcctga tgcgattttg ggggtgactg aagctttcaa
agctgattct 120 aaccccaaga agatcaattt gggcgtggga gcgtaccgtg
atgaccaagg aaaaccgttc 180 gtacttccta gcgtcaagga agccgaacgt
caagttattg cagcaaatct tgacaaggag 240 tacgccggga tcgttggcct
gcctgaattc acgaaactta gtgctcagtt agcattaggg 300 gaaaacagtg
acgtaatcaa aaacaagcgt atttttacga cgcaaagtat ttctgggact 360
ggtgcgctgc gtattggaag tgagttcctg agtaaatatg caaagactaa ggttatctat
420 caacccacgc ctacatgggg aaaccacgtg cctatcttca agttcgcggg
cgtggatgtg 480 aaacagtatc gttattatga caagtctaca tgtggatttg
atgagacggg ggcattggct 540 gatattgcgc aaatccccga aggtagcact
attttgctgc acgcgtgcgc acataaccca 600 acgggggtcg accctagtcg
tgaccaatgg aaaaagattt cagatattgt taagaaacgc 660 aatttgttcg
tgttttttga catggtgaat gagtcagtcc tgagtccgtt actgcctcgc 720
acgcttatgc gcctgcttgt gttgttactg aaatcccgca gtcttttcgc ccactcaaca
780 cccacccatc agtcgatgga attagctctt ttgccggcct cgtcgcgtat
ccaactttct 840 acctccaatg ggtcagaaat gtccagctct tggcttatcg
tcagcagccc t 891 <210> SEQ ID NO 56 <211> LENGTH: 1179
<212> TYPE: DNA <213> ORGANISM: Bacillus halodurans
C-125 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1179) <223> OTHER INFORMATION:
methionine gamma lyase (Bacillus halodurans C-125) <400>
SEQUENCE: 56 atgaaacgcg accaacattt tgaaacacgc gcgatccata ctggttacaa
gccgaacgag 60 cattttgata gcttgactcc ccctatttac caaaccagca
cgttcacatt tgcatcaatg 120 gagcaaggtg gcaaccgttt cgcaggcgag
gaagcaggat atgtttattc acgcctgggg 180 aaccccaccg tgcaaatttt
ggaacaacgc attgctgagt tggagggtgg ggaggcagct 240 cttgcctttg
gatctggcat ggctgctgtc agtgcgattt tggtggggct tacgaaggcc 300
aacgaccaca tcttagtgag caatggagtg tatggttgta cgtttgggtt gttaacgatg
360 ttaaaggaaa aatacaacat cgacgccact ttcagtccga tggacagcgt
agaggaaatc 420 ctggcaaaca tccaggataa taccacgtgc atttatgtgg
aaacacctat caaccccacc 480 atgcagttaa tcgatttgga actggttgtg
cgcgtagcga aggaaaaggg tattaaggta 540 atcgttgata acacgtttgc
cacaccatac ttacaacaac cgattgctct gggatgtgac 600 ttcgttgtcc
attcggccac gaaatacatc gggggtcatg gggacgtggt cgccggagtg 660
ctgattggag acaaggaaac aattcagttg atccgtaaga ccacccagaa ggatatgggg
720 ggcgtaattt ctccatttga tgcgtggctg ctgttgcgcg gattgaaaac
acttgcagta 780 cgtatggatc gccattgcga gaatgctgaa aaattggccg
agaaactgaa agagcatcca 840 aaagtaagta cggttctgta cccgggagac
tttgagcatc ccgatcactc catcgtcgcc 900 aaacagatga aaaagggagg
cggtttatta agctttgaga tcaaggggac tgaggcggac 960 atcgccaaag
ttgtaaatca gttaaaactg attcgtattg ctgttagttt gggtgacgca 1020
gagaccttga ttcagcatcc tgcaaccatg acccatgcag tagtacccga aaagcgccgc
1080 actcaaatgg gtattagtaa aaagttgtta cgcatgtcgg ccgggttaga
ggcctggcaa 1140 gatgtctggg ctgacttaga gcaggcgtta aatcaactg 1179
<210> SEQ ID NO 57 <211> LENGTH: 1269 <212> TYPE:
DNA <213> ORGANISM: Methylobacterium aquaticum <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1269) <223> OTHER INFORMATION: Methionine
aminotransferase (Methylobacterium aquaticum) <400> SEQUENCE:
57 atgaccgcga ttccggcctt ggcagacctg caggctcgtt atgccgactt
acaagggcgt 60 ggtctgaagt tagatatgac gcgcggtaaa ccggcgccag
agcagttgga tttatcggac 120 gatcttttca ctttaccagg taaccgcgat
caccgcacag agagcggaga agacgcgcgt 180 aattacggcg gagtacaggg
cctggctgag gtccgtgcct tattcgcccc tgtgcttggt 240 gcgtcacccg
atcgcattgc cgtaggtaat aactcatcgt tggcattgat gcatgactgc 300
attgcctatg cattgcttaa gggtgtaccc ggcggcgctc gtccttgggc aaaggaagag
360 gagattcgtt ttttatgccc agtcccaggg tacgaccgtc acttcgctct
gtgcgagacc 420 tacgggattg gaatgattcc agtccctatg accgctgacg
ggcctgatat ggaaatggtt 480 gaacgtgagg tacgcgatcc acgcgtcaaa
ggtatgtggg cggtgccgca gtatagtaac 540 ccaggcggtg agacatactc
cgacgcgact gttgagcgcc tggctcgtat ggaaaccggt 600 gcccctgact
tccgtctttt ttgggacaac gcgtatgcac ttcaccattt gaccgaacgt 660
cgcccaaccc ttcgtaatgt gttagatgcc tgtgcggaag ccgggtcacc ggatcgtgct
720 attgtgtttg ctagtacgtc gaaagttaca ctggcggggg caggccttgc
gatgcttgcg 780 tccagcgagg gcaatattcg ctggtattta gctaacgccg
gcaaacgctc aattggtcca 840 gataagctta accagttgcg ccatgttcgc
tttctgcgtg accagggcgg acttgatgca 900 ttaatggacg gccaccgccg
tcttttagct cctaagttcc gcgctgtaac ggaaaccctt 960 gctcgtcatc
tgggcgggac tggagtagcg cgctggagcg agccggaagg ggggtacttt 1020
atcctgctgg aagtccctga gggctgtgcg acacgcgtag ttaagcttgc tgctgcttgc
1080 ggactggctc tgacgcccgc aggggcgacg cacccatacg ggcgtgaccc
tcaagataag 1140 ctgttacgtc ttgccccgtc atacccgaaa ccagcggagg
tcgaggcagc cgctgaggta 1200 gtcgctgtgt gcgttttact tgcggcagct
gaaagccgcg aagctggcgg ttcggggcag 1260 gttgctgca 1269 <210>
SEQ ID NO 58 <211> LENGTH: 1902 <212> TYPE: DNA
<213> ORGANISM: Saccharomyces cerevisiae YJM1615 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1902) <223> OTHER INFORMATION: Aro10p decarboxylase
(Saccharomyces cerevisiae YJM1615) <400> SEQUENCE: 58
atggcacccg tcactattga gaaattcgtg aatcaagaag agcgtcattt agtgagcaat
60 cgttccgcca cgatcccttt tggagaatat attttcaagc gccttctttc
cattgacacc 120 aaaagcgtct tcggggttcc cggcgacttc aatttatctt
tattggaata tttatactcg 180 ccctccgtgg aatctgcggg tcttcgttgg
gttggcacct gtaacgagtt aaatgcagcc 240 tacgctgcag atggatattc
ccgctactct aataaaattg gatgcttaat caccacatac 300 ggcgtaggag
aactgagtgc gcttaatgga atcgcggggt cattcgctga aaatgtaaag 360
gttctgcata tcgtaggggt cgccaagtcc attgattccc gttcgtctaa cttctcggat
420 cgtaacttac atcacttggt cccgcagtta catgattcga actttaaagg
acccaaccat 480 aaggtctatc acgacatggt taaagatcgt gtcgcatgtt
ccgtcgccta cctggaggat 540 attgagacgg cctgtgacca agttgataac
gtgatccgtg acatttataa gtattcaaaa 600 cctggttaca ttttcgtccc
agccgacttt gccgacatgt ccgtaacctg cgacaacttg 660 gtcaatgtac
cgcgtatcag ccaacaagat tgtattgtct accccagcga gaaccaactg 720
tcagacatca ttaataaaat cactagctgg atctactcgt ctaagactcc agcaatcctt
780 ggagacgtct taactgatcg ttatggggta tcaaactttc tgaacaaact
gatctgcaaa 840 accggtatct ggaacttctc caccgtgatg ggaaaatcag
tcattgacga gagtaaccca 900 acttatatgg gtcaatacaa cggcaaagaa
ggtcttaaac aggtctatga acatttcgag 960 ctgtgtgatt tggttttaca
cttcggagta gatattaacg agatcaataa tggtcactac 1020 acgttcactt
acaagccaaa tgcgaaaatt attcaattcc accctaatta tattcgttta 1080
gtagacactc gtcaggggaa tgaacaaatg ttcaaaggca tcaattttgc gccaatcttg
1140 aaagagttgt ataagcgtat cgacgtctct aaattatcgt tgcaatacga
ttccaatgta 1200 acacaataca ccaatgagac tatgcgtctg gaggacccaa
cgaatggtca atcgagcatc 1260 attacccaag tacacctgca aaagaccatg
ccgaaatttt tgaatcccgg cgacgtcgtc 1320 gtgtgtgaga ctggtagttt
ccaattcagt gtacgcgact tcgcattccc cagtcagttg 1380 aaatatatca
gccagggttt ctttttatcc attggtatgg ccttgcctgc cgcgttgggg 1440
gttgggatcg caatgcagga tcattccaac gcgcatatta acggagggaa cgtcaaagaa
1500 gactacaagc cccgcttaat tttgtttgaa ggtgacggcg ccgcgcagat
gaccatccag 1560 gagcttagca cgatccttaa atgcaatatc cctttggagg
tcattatctg gaataacaat 1620 ggatacacta tcgagcgtgc catcatgggt
ccaacacgtt catataacga tgtgatgtcg 1680 tggaaatgga caaagttgtt
cgaagccttt ggggatttcg atggtaagta tacgaattcg 1740 actttaattc
agtgtcctag caaattagcg ttaaaacttg aagaattgaa gaattctaat 1800
aagcgttcgg ggatcgaact gttagaagtg aagctgggtg agcttgactt cccagagcaa
1860 ttgaagtgta tggtagaggc cgcagctctt aaacgtaata ag 1902
<210> SEQ ID NO 59 <211> LENGTH: 666 <212> TYPE:
DNA <213> ORGANISM: Bacillus subtilis <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(666)
<223> OTHER INFORMATION: Methionine import system permease
proteinMetP (Bacillus subtilis) <400> SEQUENCE: 59 atgtttgaga
agtattttcc aaatgttgac ttgaccgagt tatggaatgc cacatatgaa 60
actctgtata tgacattgat ttccttactg tttgccttcg taatcggcgt catcctggga
120 ttgctgttat tcttaacatc taaggggtct ctttggcaaa ataaagcagt
aaattccgtt 180 atcgcagccg ttgtcaacat ctttcgttca attcccttcc
ttattttaat catcctgctt 240 cttggtttca ctaaattctt agtgggaaca
attttgggac caaatgcggc tcttcccgcg 300 ttagtcatcg gtagtgctcc
cttttatgct cgtctggtcg aaatcgcact tcgtgaagtg 360 gacaaaggag
tgattgaggc ggcgaaatcg atgggggcta agacgagcac tattattttt 420
aaggttctta tccccgagtc catgcccgcg ctgatttccg gaattacagt gactgcgatt
480 gcattgatcg ggtcaaccgc catcgcagga gctattggtt ctggtggatt
gggaaactta 540 gcatacgttg aaggctatca atcgaataat gcggatgtga
ccttcgtggc cacagttttc 600 atcctgatta ttgttttcat cattcagatc
attggtgacc ttattaccaa catcatcgat 660 aaacgc 666 <210> SEQ ID
NO 60 <211> LENGTH: 1029 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1029)
<223> OTHER INFORMATION: DL-methionine transporter subunit
MetN (Escherichia coli K-12]) <400> SEQUENCE: 60 atgattaaac
tgagcaacat tactaaggtg ttccaccaag gtacacgtac gatccaggct 60
cttaataatg tgtcactgca cgttcctgct ggtcagattt atggggttat cggtgccagt
120 ggggctggga agagcactct gatccgctgc gtcaatctgt tagagcgccc
tacagagggc 180 tcggtactgg tggacggtca agagttgact actctgtcgg
agtccgagtt gacaaaagca 240 cgccgccaga ttggcatgat tttccaacat
ttcaatttgt tatcgagccg tacagttttc 300 gggaacgtgg ccttaccact
ggagttggac aatactccca aagacgaagt caaacgtcgt 360 gtgaccgaat
tattgtcctt ggtgggtctt ggtgacaaac acgacagtta tcccagtaat 420
ttgagtggcg ggcaaaaaca gcgtgttgcc atcgcacgcg cattagcttc gaatcccaag
480 gtgctgttat gtgatgaagc gaccagcgcc cttgacccag ccacaactcg
tagcatcctg 540 gagcttttga aagatatcaa tcgtcgcctg ggtttgacca
tcttattgat tacgcacgag 600 atggacgttg taaagcgtat ctgtgactgt
gtagcggtga tctccaacgg tgaattaatc 660 gaacaggaca ccgtatcgga
ggtcttctca catcctaaga caccccttgc acaaaaattc 720 atccaaagca
cgctgcattt agatattcct gaagattatc aggaacgcct gcaggctgaa 780
ccgtttactg attgcgttcc aatgcttcgc ttagagttca cagggcaatc ggttgacgct
840 cccttattga gtgaaaccgc ccgccgtttc aatgttaata acaacatcat
ttccgcgcaa 900 atggactacg cggggggtgt taaatttgga atcatgttaa
ccgaaatgca cggcacacag 960 caggatacac aggcggcgat cgcatggctg
caggaacatc atgttaaagt agaagtcctt 1020 gggtatgtg 1029 <210>
SEQ ID NO 61 <211> LENGTH: 654 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(654)
<223> OTHER INFORMATION: metI (Escherichia coli) <400>
SEQUENCE: 61 atgtctgagc cgatgatgtg gctgctggtt cgtggcgtat gggaaacgct
ggcaatgacc 60 ttcgtatccg gtttttttgg ctttgtgatt ggtctgccgg
ttggcgttct gctttatgtc 120 acgcgtccgg ggcaaattat tgctaacgcg
aagctgtatc gtaccgtttc tgcgattgtg 180 aacattttcc gttccatccc
gttcattatc ttgcttgtat ggatgattcc gtttacccgc 240 gttattgtcg
gtacatcgat tggtttgcag gcagcgattg ttccgttaac cgttggtgca 300
gcaccgttta ttgcccgtat ggtcgagaac gctctgctgg agatcccaac cgggttaatt
360 gaagcttccc gcgcaatggg tgccacgccg atgcagatcg tccgtaaggt
gctgttaccg 420 gaagcgctgc cgggtctggt gaatgcggca actatcaccc
tgattaccct ggtcggttat 480 tccgcgatgg gtggtgcagt cggtgccggt
ggtttaggtc agattggcta tcagtatggc 540 tacatcggct ataacgcgac
ggtgatgaat acggtactgg tattgctggt cattctggtt 600 tatttaattc
agttcgcagg cgaccgcatc gtccgggctg tcactcgcaa gtaa 654 <210>
SEQ ID NO 62 <211> LENGTH: 816 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(816)
<223> OTHER INFORMATION: metQ (Escherichia coli) <400>
SEQUENCE: 62 atggcgttca aattcaaaac ctttgcggca gtgggagccc tgatcggatc
actggcactg 60 gtaggctgcg gtcaggatga aaaagatcca aaccacatta
aagtcggcgt gattgttggt 120 gccgaacagc aggttgcaga agtcgcgcag
aaagttgcga aagacaaata tggcctggac 180 gttgagctgg taaccttcaa
cgactatgtt ctgccaaacg aagcattgag caaaggcgat 240 atcgacgcca
acgccttcca gcataaaccg taccttgatc agcaactgaa agatcgtggc 300
tacaaactgg tcgcagtagg caacactttt gtttatccga ttgctggtta ctccaagaaa
360 atcaaatcac tggatgaact gcaggatggt tcgcaggttg ccgtgccaaa
cgacccaact 420 aaccttggtc gttcactgct gctgctgcaa aaagtgggct
tgatcaaact gaaagatggc 480 gttggcctgc tgccgaccgt tcttgatgtt
gttgagaacc ccaaaaatct gaaaattgtt 540 gaactggaag caccgcaact
gccgcgttct ctggacgacg cgcaaatcgc tctggcagtt 600 atcaatacca
cctatgccag ccagattggc ctgactccgg cgaaagacgg tatctttgtt 660
gaagataaag agtccccgta cgtaaacctg atcgtgacgc gtgaagataa caaagacgcc
720 gagaacgtga agaaattcgt ccaggcttat cagtctgacg aagtttacga
agcagcaaac 780 aaagtgttta acggcggagc tgttaaaggc tggtaa 816
<210> SEQ ID NO 63 <211> LENGTH: 2286 <212> TYPE:
DNA <213> ORGANISM: Bacillus atrophaeus UCMB-5137 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(2286) <223> OTHER INFORMATION: MetE (Bacillus
atrophaeus UCMB-5137) <400> SEQUENCE: 63 atgacgacta
tcaaaacatc aaatctgggc ttccctcgca ttggacttaa tcgcgaatgg 60
aaaaaatcac tggaagcgtt ttggaaaggt aacagcgaca aagatacatt tcttaagcag
120 atggatgagt tatttcttac tgccgtaaaa acccagattg atcaaaaaat
cgacatcgtg 180 cccgtgagcg acttcactca ctacgaccac gttcttgaca
cagctatctc ttttaattgg 240 attccagaac gctttaaaca cattacggat
gcgactgata catatttcgc gctggcacgt 300 ggcattaagg atgctgttag
ttcggaaatg actaagtggt ttaataccaa ttaccactat 360 atcgttccgg
aatacaataa agacatcgaa ttccgtttaa cccgcaacaa gcagttagag 420
gactaccgcc gcgtcaaaca agcgtttggc gtcgaaacta aacccgtcat tgtcggtcct
480 tacacattcg tgacgcttgc caagggctac gaacaaagtg aggccaaaga
aatccaaaag 540 cgtttagtcc cattgtatgt gcaattattg aaagaattgg
aacaagaggg cgtgcagtgg 600 gtacaaatcg atgagccagc acttgtgaca
gcctcatccg aggatgttag cgcggccaag 660 gagttatacc aggccattac
gaatgagtta tccggcttga atgtcctttt gcagacttac 720 ttcgattctg
ttgatgctta tgaggagtta atcagctacc cggtacaggg tatcggcttg 780
gattttgtac acgataaagg gcgcaacttg gagcaattaa aagcgcatgg atttccgaag
840 gataaggtat tagcagctgg tgttattgat ggtcgtaaca tttggaagac
ggatttagat 900 gagcgcttgg acgccatcct tgcgctgtta tcttcgacgg
acattgacga attatggatt 960 caaccaagca attcgcttct tcatgtacca
gtagcaaagc acccagacga gcacctggag 1020 aaggatctgt tgaatggctt
gagttacgca aaagaaaagc tggcagaact gtccgcttta 1080 aaagagggtt
tgttatcggg taaagcggca atctcggccg acattcagca ggccaaagcg 1140
gatttacagg ccctgaagca attcgccacc ggggctaaca gtgagcagaa agaggaatta
1200 aatcagttga ccgagaaaga ctttaagcgc ccgatcccct tcgaagagcg
cctgaaaatc 1260 cagaatgaat ccttggggct tcccctgctt cctactacga
ctattggttc ttttcctcaa 1320 agcgccgagg tgcgttcggc gcgccaaaag
tggcgcaaaa gtgagtggag cgacgagcaa 1380 tatcaagaat ttatcaacgc
ggaaacgaag cgctggatcg acattcagga agagcttgat 1440 cttgacgttt
tagtacatgg agagttcgag cgcaccgaca tggtcgaata tttcggtgag 1500
aaactggctg gattcgcgtt tactaaatac gcatgggtcc agagctacgg atcccgctgt
1560 gtacgccctc ccgtcatcta tggggacgtg gagtttattg aacctatgac
tgtcaaggac 1620 acagtgtacg ctcaatcttt aacgagtaag caggttaaag
ggatgttgac tggcccggtc 1680 acaatcttga attggagctt cccgcgtaac
gacattagcc gtaaggagat cgccttccaa 1740 atcgggttag ctcttcgcaa
agaggtcaag gcgttggaag atgctggtat tcaaatcatc 1800 caagttgacg
aaccggccct gcgtgaaggg ctgcctctga aagaaaacga ttgggaagag 1860
tatttaacgt gggccgcgga ggcgttccgc ttaactactt cggctgtgaa aaacgacact
1920 cagattcata cacacatgtg ttattccaat tttgaggaca ttgtcgacac
aattaatgac 1980 ttggatgcgg acgtcattac aatcgaacac tcccgcagtc
acggtgggtt cttggactac 2040 ttgcgcgatc atccgtatct taaaggttta
ggtcttggcg tgtacgatat tcacagccct 2100 cgtgtacccc cgacagagga
aatttataag atcattgacg aagccctgac cgtatgtcct 2160 actgaccgct
tctgggtaaa cccagactgc gggctgaaga cccgtcacca ggaggaaacg 2220
attgccgcgt tgaagaacat ggtcgaggct gctaaacagg ctcgtgccaa acagagtcaa
2280 cttgtc 2286 <210> SEQ ID NO 64 <211> LENGTH: 753
<212> TYPE: DNA <213> ORGANISM: Corynebacterium
glutamicum <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(753) <223> OTHER INFORMATION:
BrnF (Corynebacterium glutamicum) <400> SEQUENCE: 64
atgcagaaaa cacaggagat tcacagctcg ttagaggtta gccccagtaa agctgctctg
60 gagcccgacg ataaggggta tcgtcgttac gaaatcgcac aaggcctgaa
gacctctctt 120 gctgcaggcc tgggaatgta tcctatcgga attgcattcg
gcttactggt gattcaatat 180 ggttatgaat ggtgggccgc tccactgttc
tccggcctga tttttgcggg gtctacggag 240 atgcttgtaa ttgcacttgt
ggtcggcgct gctccgctgg gtgccattgc ccttacgacc 300 ttacttgtta
atttccgtca tgttttctat gccttttcct ttcccttgca cgttgttaaa 360
aaccctattg cgcgcttcta ttctgtattc gctcttattg atgaagcata cgctgttaca
420 gccgctcgtc ccgccggttg gagtgcatgg cgtctgattt caatgcagat
tgcgttccac 480 tcctactggg tatttggagg cttgaccggt gtagcaatcg
cagagttaat tcctttcgag 540 atcaaaggcc tggagttcgc actttgttcg
ttatttgtaa ctcttacttt agacagttgt 600 cgcactaaga aacaaattcc
gagtttgtta ttggctggac tgagctttac tatcgcgtta 660 gtagtgatcc
ccggccaagc tctgttcgct gcgttactta tctttctggg gcttctgaca 720
atccgttatt ttttcttagg gaaggcagcc aaa 753 <210> SEQ ID NO 65
<211> LENGTH: 324 <212> TYPE: DNA <213> ORGANISM:
Corynebacterium glutamicum <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(324) <223>
OTHER INFORMATION: BrnE (Corynebacterium glutamicum) <400>
SEQUENCE: 65 atgacgactg atttctcctg catcctgttg gtggtcgcgg tatgtgcagt
cattacattt 60 gcgcttcgtg ccgtaccttt tctgatcttg aaacccttgc
gtgaatcgca atttgtggga 120 aaaatggcca tgtggatgcc tgcgggcatt
ctggcaatcc tgacggcttc taccttccgt 180 tcaaacgcca tcgatttaaa
gacgttgacg ttcggtctga ttgccgtggc aatcacagtc 240 gtagcccact
tattaggagg ccgtcgcacc ttattatctg ttggcgctgg aacaattgtg 300
tttgtaggtc ttgttaattt gttt 324 <210> SEQ ID NO 66 <211>
LENGTH: 1023 <212> TYPE: DNA <213> ORGANISM: Salmonella
enterica subsp. enterica serovar Typhistr. CT18 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1023) <223> OTHER INFORMATION: threonine
3-dehydrogenase (Salmonella enterica subsp. enterica serovar
Typhistr. CT18) <400> SEQUENCE: 66 atgaaggccc tgagcaaatt
gaaagccgag gaggggatct ggatgaccga tgttcctgaa 60 ccagaagtgg
ggcacaacga ccttttaatc aaaattcgca agactgcaat ctgcgggaca 120
gacgtacata tctataactg ggacgagtgg agtcaaaaaa ctattcccgt ccctatggtg
180 gtcgggcacg agtatgtcgg agaggttgta ggaatcggac aagaagtcaa
aggatttaaa 240 atcggggatc gtgtgagtgg ggagggtcac attacctgtg
ggcattgccg caattgccgt 300 ggaggacgca cacatttgtg ccgtaacact
acaggcgtag gcgtgaatcg tcccggatgt 360 ttcgcggaat accttgtcat
tccagcgttt aacgccttta agatccctga caacatttca 420 gatgatttag
catccatttt tgacccattc ggtaacgcgg tccatactgc gttgagcttc 480
gacttagttg gagaagatgt attagtttcc ggcgccggac cgattggcgt catggcagct
540 gccgttgcga agcacgtggg cgcacgtcat gtggtaatta cggacgtaaa
tgagtatcgt 600 ctggagctgg cacgtaaaat gggggttaca cgtgccgtaa
acgttgcgaa agagtcttta 660 aacgatgtca tggctgaact gggcatgacg
gaagggtttg atgtcggact ggaaatgtcc 720 ggtgccccgc cagccttccg
taccatgttg gacaccatga accatggggg ccgtatcgca 780 atgttgggaa
ttcccccgag cgacatgtct atcgactgga caaaggtaat ttttaaaggc 840
ctgttcatta aggggattta cggtcgtgag atgtttgaga cgtggtacaa gatggctgcc
900 ttgattcaat cggggttgga tctgagccct atcatcacac accgtttttc
agtggatgac 960 tttcaaaaag ggtttgacgc catgtgcagc ggtcaatcag
ggaaagtaat tctttcttgg 1020 gac 1023 <210> SEQ ID NO 67
<211> LENGTH: 999 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(999) <223> OTHER
INFORMATION: threonine aldolase (Escherichia coliO26:H11 str.
CVM10026) <400> SEQUENCE: 67 atgattgacc ttcgttcgga caccgtaacc
cgcccatctc acgcaatgtt ggaagctatg 60 atggccgcgc ctgtggggga
tgacgtttat ggggatgacc cgaccgtcaa cgctttacaa 120 gattacgctg
ctgaattgtc gggcaaagaa gcagcaatct tcttacctac aggtacacaa 180
gctaatcttg tcgccctgct tagtcactgt gagcgtggcg aagaatacat tgttggtcaa
240 gcagcgcata attacctgtt cgaagctgga ggggctgctg ttcttggtag
cattcagccc 300 caacccattg atgctgctgc cgatggtact cttcctctgg
ataaagtcgc tatgaaaatt 360 aagccagacg acattcactt cgcacgcaca
aagctgctgt cgcttgagaa tacacacaat 420 ggaaaagtcc tgccccgtga
gtacctgaaa gaggcttggg aatttacacg cgaacgcaac 480 ctggctctgc
acgtagacgg tgctcgcatc ttcaacgccg ttgtcgccta cggttgcgaa 540
ttgaaagaga ttacgcaata ctgtgactcc ttcacgattt gcttgtccaa aggcttaggc
600 accccggtgg gttcattgtt ggtaggaaac cgtgactata ttaagcgcgc
catccgctgg 660 cgtaaaatgg cagggggtgg aatgcgtcaa tcagggattc
ttgcggcagc tggcatgtac 720 gcgctgaaaa ataatgtggc tcgccttcaa
gaggatcacg ataatgctgc gtggatggct 780 gagcaattac gtgaggcggg
tgcagacgta atgcgccaag ataccaatat gctgttcgta 840 cgtgttgggg
aagaaaacgc tgcggcctta ggagaataca tgaaggcgcg taacgtgttg 900
atcaacgcat cccctattgt tcgccttgta actcaccttg atgtttcacg tgaacaattg
960 gcggaagttg ccgcccactg gcgtgccttt cttgctcgc 999 <210> SEQ
ID NO 68 <211> LENGTH: 1251 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1251)
<223> OTHER INFORMATION: serine hydroxymethyltransferase
(Escherichia coli) <400> SEQUENCE: 68 atgcttaaac gtgagatgaa
tatcgccgac tacgacgccg aattatggca ggcgatggag 60 caggagaaag
tccgccaaga ggaacacatt gagcttattg cgtcggagaa ctatacatcc 120
cctcgcgtta tgcaggcgca aggctcacag ttgacgaaca aatacgctga gggatatccg
180 ggaaagcgtt attatggcgg ttgcgagtac gttgacattg ttgaacagtt
agcgattgat 240 cgtgctaagg agttatttgg agcggattat gccaatgttc
aacctcactc gggcagccag 300 gctaactttg ctgtatacac cgcactttta
gaacctggtg acacggtcct gggtatgaat 360 ttggcccatg gaggccactt
aactcatgga agccctgtga attttagtgg gaagttgtat 420 aacatcgtgc
cctacgggat cgacgccaca ggacacattg attacgcaga tttggagaaa 480
caagccaagg aacataagcc taaaatgatc atcggcggat tttcagcata tagcggagtg
540 gtagactggg ccaaaatgcg cgagattgct gattcgattg gtgcttacct
gtttgtcgat 600 atggcgcatg tcgctggtct ggtcgctgcg ggagtttatc
ctaaccccgt gcctcacgct 660 cacgtcgtga cgactactac acataagact
ttagcgggtc ctcgtggggg tttgattctt 720 gcgaaggggg gctcagagga
actttataag aagcttaact ctgccgtatt tcccggcggt 780 caggggggcc
ctcttatgca cgtcatcgca ggaaaggcgg tggctctgaa ggaagcgatg 840
gaacccgaat tcaagactta ccaacagcaa gtagccaaaa acgccaaagc catggtggag
900 gtattcctgg agcgcggcta caaggtagtt agcgggggga cggacaacca
tttgttctta 960 gtcgatttag tggacaaaaa ccttactggt aaggaggctg
atgctgctct tgggcgtgca 1020 aatatcacag tcaataagaa tagcgtgccc
aatgacccaa agtcgccatt tgtgacttct 1080 ggcatccgcg ttgggactcc
ggcaatcacc cgtcgtggct ttaaggaggc agaggccaag 1140 gagctggcag
ggtggatgtg tgacgtactg gactctatta atgatgaggc agttatcgaa 1200
cgtattaaag gcaaagtgct tgacatttgt gcgcgctacc ccgtgtatgc c 1251
<210> SEQ ID NO 69 <211> LENGTH: 1293 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1293) <223> OTHER INFORMATION: tdcC(Escherichia coli)
<400> SEQUENCE: 69 atgtctactt cggactctat tgtttcatcg
caaacaaaac agtcatcctg gcgtaaatca 60 gataccacct ggactttggg
tctgtttggt accgcgatcg gggctggtgt attgtttttc 120 ccgatccgcg
ctggatttgg tggtttaatt cctatcctgc tgatgcttgt actggcatat 180
cctattgctt tttattgtca tcgcgcagcg cgcttgtgtt taagcggaag caacccctcg
240 ggtaatatca cagagacggt ggaggagcat ttcgggaaaa caggaggggt
cgtaatcaca 300 tttctgtact tttttgctat ttgtcccctg ttgtggattt
atggggttac gatcaccaat 360 acttttatga cgttttggga gaatcaactg
ggctttgcac cgcttaaccg cggattcgtg 420 gcgctgttcc ttttactgtt
gatggcgttt gtcatctggt tcggtaaaga cttaatggtg 480 aaagtcatgt
cttatttggt atggcctttc attgcttcac ttgtcttaat tagtctgtca 540
ttaatccctt attggaactc ggcagtaatc gatcaagtag atctgggtag cctgtctttg
600 accggacatg atgggatctt aattaccgta tggctgggca tttctattat
ggtctttagt 660 tttaactttt cacctatcgt gtcctccttt gtggtgtcca
agcgcgagga atatgagaag 720 gattttggtc gtgattttac ggaacgtaag
tgctcacaaa ttattagccg cgcgtctatg 780 cttatggtgg ctgtcgttat
gttctttgct ttctcctgct tatttacctt gtcaccggcg 840 aacatggcgg
aagcgaaggc gcaaaacatt ccagttttat catatcttgc taatcatttc 900
gcttctatga cagggaccaa aactactttt gccatcacat tggagtatgc ggcgtctatc
960 attgcattag tggccatttt taagtcgttc tttggccatt atttaggtac
tttagaaggg 1020 ttgaatggct tagtcttgaa attcggatac aagggggaca
aaactaaagt ttccttgggt 1080 aagttgaaca caatctcgat gatctttatt
atggggagta catgggtcgt tgcgtatgca 1140 aatccaaaca ttctggattt
aattgaggcg atgggagcac cgattatcgc gtcattgttg 1200 tgccttttgc
cgatgtacgc catccgtaag gcgccttcac tggccaaata tcgtgggcgc 1260
ttggataacg tgttcgtaac cgtcatcgtt tgc 1293 <210> SEQ ID NO 70
<211> LENGTH: 885 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(885) <223> OTHER
INFORMATION: Threonine/ homoserine exporterRhtA, Escherichia coli
(strain K12) <400> SEQUENCE: 70 atgcctggtt ccttgcgtaa
aatgccggtt tggttgccga ttgttattct tctggttgca 60 atggctagca
tccaaggagg cgctagttta gcaaaaagtc tgtttccttt ggtgggggca 120
ccgggtgtga ccgcgctgcg tttggctttg ggcactttaa ttttgattgc cttctttaag
180 ccctggcgcc ttcgttttgc taaagaacaa cgtttgccgc ttttgttcta
cggcgtctca 240 cttggtggca tgaactatct tttttattta agcatccaaa
ccgtacccct gggtattgcg 300 gtggctttgg agttcacggg tccattggca
gttgcccttt tcagctcgcg tcgcccagtc 360 gatttcgtct gggtagtgct
tgcggtactt ggactgtggt tcttactgcc cttaggccaa 420 gacgtgagtc
acgtagacct taccgggtgt gcgctggctt tgggagccgg tgcttgttgg 480
gcaatttaca tcctgtcggg acagcgtgcg ggagcagagc acgggcctgc gacagtagcg
540 attgggtcgc tgatcgcagc cctgattttc gtccccattg gtgccttaca
ggcaggagag 600 gcgttgtggc actggtcagt gattccctta ggtttggcgg
tagcaatcct gtctaccgca 660 cttccttatt ctttagagat gattgcctta
acccgtctgc cgacacgtac gtttggcacc 720 ttaatgtcga tggaaccggc
attggctgcc gtttcaggta tgatcttcct gggagagacg 780 ttaactccca
ttcagttgtt agctcttggg gcaatcatcg ctgcgagtat gggatcgacc 840
cttacggttc gtaaagagtc gaagattaaa gaattggaca tcaat 885 <210>
SEQ ID NO 71 <211> LENGTH: 618 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(618)
<223> OTHER INFORMATION: rhtB(Escherichia coliFVEC1302)
<400> SEQUENCE: 71 atgacgctgg agtggtggtt cgcatacttg
ctgacatcca tcatcctgag tttaagcccc 60 ggatctggtg caatcaacac
gatgactacg tctttgaatc acggctatcg tggtgctgtt 120 gcatccattg
ccggcttgca gacgggatta gccatccata ttgttttagt gggtgtagga 180
cttggaacat tattcagtcg ctcggttatc gcctttgagg tcttaaagtg ggctggtgcc
240 gcttatttga tttggctggg aattcagcaa tggcgtgcag ccggtgcgat
tgacttgaag 300 agccttgcgt ccacacagag ccgccgtcac ttgtttcaac
gtgcagtatt cgtcaatttg 360 accaacccca aaagtatcgt ctttctggcg
gcactgtttc cccagttcat tatgcctcaa 420 cagccgcagt tgatgcagta
catcgtcttg ggcgtcacca ccatcgtagt ggacattatt 480 gtaatgattg
gatacgccac tctggcccaa cgtattgcgc tgtggatcaa gggcccgaaa 540
cagatgaagg cactgaacaa aatttttggt tctttgttta tgttggttgg ggcacttctt
600 gccagtgcac gtcacgcg 618 <210> SEQ ID NO 72 <211>
LENGTH: 618 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(618) <223> OTHER INFORMATION:
RhtCthreonine Rht Transporter (Escherichia coliBL21(DE3))
<400> SEQUENCE: 72 atgctgatgc tttttttaac agtagcaatg
gtgcatatcg tcgcattgat gtcaccggga 60 cctgactttt tttttgtttc
acaaacagca gtatcacgct cacgtaagga ggcaatgatg 120 ggtgtcttag
ggatcacttg cggcgtaatg gtatgggccg gtattgcact tctgggactg 180
catttaatta ttgagaagat ggcctggctt cacacattaa tcatggtagg cggtgggctt
240 tatttatgtt ggatgggcta tcaaatgctg cgtggagctc ttaagaaaga
agccgtgtcc 300 gcaccggctc cccaagtgga acttgcgaaa tcaggtcgct
ccttcttgaa ggggttgttg 360 actaatcttg cgaaccctaa ggccatcatt
tatttcggtt ctgtgtttag tttgttcgtt 420 ggggataatg tgggaaccac
ggaacgctgg ggaatcttcg cattaatcat tatcgagacg 480 ttagcttggt
tcaccgtcgt ggcctccctt tttgctctgc cgcaaatgcg ccgtggttac 540
caacgtttag caaagtggat cgacggtttt gctggagctt tatttgcggg tttcggcatt
600 catctgatta ttagccgt 618 <210> SEQ ID NO 73 <211>
LENGTH: 984 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(984) <223> OTHER INFORMATION:
cysteine desulfhydrase (Escherichia coli) <400> SEQUENCE: 73
atgcccctgc acaacttaac acgttttcca cgcctggaat tcattggtgc accgactccc
60 ttggaatatc tgcctcgctt ttcggactac ttaggccgcg agattttcat
taagcgcgat 120 gatgttacac cgatggctat ggggggtaac aaattgcgta
aattggaatt tcttgcagcg 180 gatgcactgc gtgaaggcgc ggacacttta
attaccgctg gtgcaattca gtcaaatcac 240 gtacgccaaa ctgcggcagt
tgctgcgaag ttaggtcttc attgtgtcgc ccttttggaa 300 aatccaattg
gcacaacggc agaaaattac cttaccaacg ggaaccgttt gttgcttgac 360
ctttttaaca cacagatcga aatgtgcgac gctttaactg atcccaacgc tcaattggag
420 gagcttgcga ctcgcgtgga agctcaaggc ttccgtccgt atgttattcc
ggtcggcggc 480 agcaatgctc ttggggcatt agggtatgta gagtccgctc
tggagatcgc gcaacaatgt 540 gagggcgcgg ttaacatttc gagtgtagtt
gtggcctctg gaagtgcggg cacccacgcc 600 gggctggctg tgggtcttga
gcacttaatg cctgaatctg aactgatcgg ggtcacagtc 660 tcgcgttccg
tcgcagatca gttacctaag gtagtaaact tacagcaagc cattgcgaaa 720
gaattagaat taaccgctag tgcagaaatc ttattatggg atgattactt tgcgcctggg
780 tacggtgtcc ccaatgatga aggtatggaa gcagtcaagc ttttagctcg
tttggagggg 840 atcttgctgg accctgttta caccggcaaa gcaatggcag
gcttaattga cggtatcagt 900 cagaaacgct tcaaagacga gggaccaatt
ctgttcatcc ataccggcgg cgctcctgcc 960 ctttttgcct accaccctca cgtt 984
<210> SEQ ID NO 74 <211> LENGTH: 1413 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1413) <223> OTHER INFORMATION: tnaA (Escherichia
coliDH1) <400> SEQUENCE: 74 atggagaatt tcaagcattt gcccgagccg
ttccgcattc gtgtcattga gcctgtcaag 60 cgtactactc gcgcgtatcg
cgaagaggcg attatcaaat cgggtatgaa tccattttta 120 cttgattcag
aagatgtgtt catcgattta cttacagatt ctgggacagg cgcggtaacg 180
caatcgatgc aagcagcgat gatgcgcggt gacgaagcct attctggctc gcgctcctat
240 tatgctctgg ccgaatcagt caaaaacatt tttggttacc aatatacgat
tcccacgcat 300 cagggacgcg gagcagagca aatctatatc ccagtcttaa
tcaaaaagcg cgagcaagaa 360 aagggattgg accgctcgaa aatggtagcc
ttctcaaatt acttcttcga cactactcag 420 gggcactcgc aaatcaacgg
ctgcactgtt cgcaatgtgt atatcaagga agcctttgat 480 acaggcgtac
gttacgattt caaggggaac tttgacctgg aaggtcttga acgtggcatt 540
gaagaagtag gacccaacaa cgtaccctat atcgtcgcca cgatcacatc taatagcgca
600 ggaggtcagc ctgtgtcttt ggcgaatctg aaagcgatgt attcgatcgc
caaaaagtat 660 gatatccccg tcgtaatgga ttctgcacgt tttgcagaga
acgcctactt cattaaacag 720 cgtgaagcgg agtacaaaga ttggaccatc
gaacagatca ctcgtgagac ttataaatat 780 gctgacatgc tggctatgtc
ggctaagaag gacgctatgg tcccaatggg aggcctttta 840 tgcatgaagg
acgatagttt ttttgacgtt tatacggaat gtcgcaccct ttgtgtagtg 900
caggaaggat tccccactta tggcggcctt gaaggtggag cgatggaacg tttagctgtt
960 ggactgtatg atggtatgaa tctggattgg ctggcatatc gtattgcgca
ggtgcagtac 1020 ctggtagacg ggttagagga gatcggggtt gtgtgccagc
aggccggggg ccatgcggcg 1080 ttcgtggacg caggaaaact gcttccccac
attcccgccg atcagttccc tgcgcaggca 1140 cttgcttgcg agttatacaa
ggtggccggt atccgtgcgg tagagatcgg ctcgtttctt 1200 ttggggcgcg
accctaaaac aggaaaacaa ttgccctgcc ctgccgaact tcttcgcctt 1260
actatccctc gtgcgaccta cactcaaacc cacatggact ttattatcga ggccttcaaa
1320 catgtgaagg agaatgctgc taatatcaag ggcctgacct ttacctacga
gccaaaggtt 1380 ttgcgccact ttacagcaaa acttaaagaa gtt 1413
<210> SEQ ID NO 75 <211> LENGTH: 969 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(969)
<223> OTHER INFORMATION: cysK (Escherichia coliO104:H4 str.
C227-11) <400> SEQUENCE: 75 atgtcaaaaa ttttcgagga taactcgtta
acgatcggcc acactccctt ggttcgtctg 60 aatcgtatcg gtaacgggcg
cattctggca aaggttgaat cacgcaatcc gtccttctca 120 gttaagtgcc
gtattggagc gaatatgatt tgggatgctg agaagcgcgg agtcctgaag 180
cctggggtgg agttggtgga gccaacctct gggaatacag gtatcgcgct ggcttatgta
240 gctgcagcgc gtggctacaa attaacactt accatgcccg agaccatgtc
aatcgaacgt 300 cgtaagttgt tgaaggcatt aggagcgaat ctggtactga
ccgaaggagc taagggaatg 360 aagggcgcta ttcaaaaagc ggaagaaatt
gtcgcaagta accccgaaaa gtatctttta 420 ctgcaacagt tttctaaccc
tgcaaatcct gagatccacg aaaaaacaac aggtcccgaa 480 atctgggaag
acaccgacgg tcaagttgac gtatttatcg ccggggtagg aactggagga 540
accttaacgg gggtcagtcg ttatattaag ggtacgaagg gaaagactga tttgattagc
600 gtagcagtgg agccaacgga tagtcctgtt attgcccaag ccctggcggg
ggaggaaatc 660 aaaccgggac ctcacaaaat ccaagggatt ggtgcgggtt
ttatcccagc caatctggat 720 ctgaaacttg tcgacaaggt cattggaatt
actaatgaag aggcgatctc cactgcgcgc 780 cgtttgatgg aggaagaagg
gattttggca gggatttcaa gcggtgcggc ggtggcagca 840 gctttgaaat
tgcaagaaga cgagtcattc actaataaga atattgttgt tattttacca 900
agcagcggtg agcgctactt atcaaccgct ttgttcgctg atttatttac ggaaaaagag
960 ttacaacaa 969 <210> SEQ ID NO 76 <211> LENGTH: 909
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(909) <223> OTHER INFORMATION: cysM
(Escherichia coliFVEC1412) <400> SEQUENCE: 76 atgtcaacat
tagaacagac aattggtaat acccccctgg tcaaattgca gcgcatgggg 60
ccaaacaatg gaagcgaggt ttggctgaaa ttggaaggca acaacccggc gggatctgtg
120 aaagaccgtg ccgcactgtc catgatcgta gaagctgaga aacgtggcga
gattaaacct 180 ggggatgttt taatcgaggc tacaagtggg aacactggaa
tcgcccttgc catgattgcg 240 gctttaaagg gttatcgtat gaagttactt
atgcccgata acatgagcca ggagcgccgt 300 gccgctatgc gtgcctatgg
tgctgaactt atcttagtta ccaaggagca aggcatggaa 360 ggtgcgcgtg
acttggcatt agaaatggcg aatcgtggcg aagggaagct gcttgaccaa 420
tttaataatc cagataaccc ttatgcacac tataccacga ccggcccgga aatctggcaa
480 caaaccggcg ggcgcatcac ccactttgta tcatccatgg gcacaactgg
tacaattacg 540 ggcgtttctc gtttcatgcg cgagcagagt aaacctgtta
caatcgtggg acttcaacct 600 gaggagggat cttcgatccc aggcattcgt
cgttggcctg ctgagtactt acctggcatt 660 ttcaacgcat ccttagtgga
tgaagttctt gacattcatc agcgcgaagc agagaatacc 720 atgcgcgagt
tggcagtacg tgagggcatt ttctgcgggg tttcttctgg gggggccgtg 780
gcgggtgctt tacgtgtcgc caaagcaaac cccggagcag tagttgttgc cattatttgt
840 gatcgtggtg accgctactt atctacggga gtcttcggag aggaacactt
ttcacaaggg 900 gccggaatt 909 <210> SEQ ID NO 77 <211>
LENGTH: 1170 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1170) <223> OTHER
INFORMATION: malY (Escherichia coli) <400> SEQUENCE: 77
atgttcgatt tttcgaaagt cgtcgatcgt catgggacct ggtgcactca atgggactac
60 gtggcggacc gctttgggac agcagatttg ttaccgttca ctattagcga
catggatttt 120 gccacagcac cttgcattat cgaggcactg aatcagcgct
taatgcatgg ggttttcggt 180 tatagccgtt ggaagaacga tgagttcctt
gcagcaattg cacattggtt cagtacccaa 240 cattataccg ctatcgattc
ccagacggtt gtgtacggcc ccagcgttat ttacatggtg 300 agcgaattga
tccgtcagtg gtctgaaaca ggagaaggtg tagtaatcca tactcccgcc 360
tatgacgcgt tctacaaagc cattgagggg aatcaacgta cagtaatgcc cgttgcctta
420 gaaaaacagg cagacggatg gttttgcgat atgggaaaat tagaggcggt
acttgcaaaa 480 cccgagtgca aaatcatgct tttatgcagt ccgcaaaacc
caacaggcaa ggtctggacc 540 tgtgatgaat tagagattat ggcggatttg
tgcgagcgtc acggagtccg tgtcatctct 600 gacgagattc acatggacat
ggtctggggg gaacagccgc acattccttg gtctaatgtc 660 gcacgtggtg
attgggccct tttgacatcg ggttcgaaaa gctttaacat tccagccctg 720
accggggcat atggaattat cgaaaactcg tcgagccgtg acgcgtattt atctgccctt
780 aagggacgtg atggactttc gagcccgtcg gttcttgcct tgacggcaca
cattgctgct 840 taccaacagg gagcgccgtg gctggacgct cttcgcattt
acctgaagga taaccttact 900 tacattgcgg ataagatgaa tgcggccttc
ccagaactta actggcagat tccccagtca 960 acgtatttag cctggcttga
ccttcgtccc ttaaacattg atgacaacgc actgcaaaag 1020 gcactgatcg
aacaggaaaa ggtagccatc atgcctggct atacctacgg cgaggagggc 1080
cgtgggttcg tccgcctgaa cgcaggatgt ccccgctcga aacttgaaaa aggggtagct
1140 ggtcttatta atgctattcg cgctgtgcgc 1170 <210> SEQ ID NO 78
<211> LENGTH: 1185 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1185)
<223> OTHER INFORMATION: MetC (Escherichia coli) <400>
SEQUENCE: 78 atggccgaca agaagttgga tactcaactg gtgaacgccg ggcgttccaa
aaaatatacc 60 ttgggagctg ttaatagcgt tatccaacgt gcatcaagtt
tagttttcga tagtgtcgaa 120 gcaaagaagc atgcgacacg caatcgcgca
aatggggaat tattttatgg acgccgcggg 180 accttgaccc acttctcttt
acagcaggcc atgtgtgagc tggaaggggg agccggttgt 240 gtattgttcc
cctgcggagc cgcggcggtg gctaacagta tcctggcgtt cgtggagcag 300
ggtgatcacg tcctgatgac gaacaccgcg tacgaaccct cgcaagactt ctgcagtaaa
360 atcttatcca aattaggtgt gactacctcg tggtttgacc cgttgatcgg
ggcggacatt 420 gtgaaacatc tgcagcccaa cacgaaaatt gtttttttgg
agtctcccgg ttcgattact 480 atggaggtac acgacgtgcc agctatcgtt
gcagcagttc gttccgtggc gcccgacgca 540 attatcatga tcgacaatac
atgggccgca ggcgtccttt ttaaagcctt agattttggc 600 attgatgtaa
gtatccaagc ggctaccaag tacttggtcg gacattccga tgcgatgatt 660
ggtacagcag tatgcaatgc acgctgctgg gagcaattgc gtgaaaacgc ttacctgatg
720 gggcaaatgg tagacgcaga taccgcttat attaccagtc gtgggttgcg
tacattagga 780 gtgcgtttgc gtcaacacca cgagtcatcc ctgaaagtgg
ctgaatggct ggctgaacat 840 ccccaggttg ctcgcgtaaa ccaccccgca
cttccgggat caaagggcca tgaattttgg 900 aagcgcgact tcacgggctc
cagtggattg ttttctttcg tacttaagaa aaagttgtct 960 aatgaagaat
tggcgaatta ccttgataac tttagcttgt ttagtatggc atatagttgg 1020
gggggatatg aatcactgat tttggcaaat caaccagaac atattgctgc gattcgtcct
1080 caaggcgaaa ttgattttag cggaacgtta attcgtctgc acatcgggct
tgaggatgtg 1140 gacgatttaa ttgcagattt ggatgcggga tttgcacgta ttgtg
1185 <210> SEQ ID NO 79 <211> LENGTH: 1230 <212>
TYPE: DNA <213> ORGANISM: Trypanosoma grayi <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1230) <223> OTHER INFORMATION: cystathione gamma lyase
(Trypanosoma grayi) <400> SEQUENCE: 79 atgtcaggtg cccagcactt
gttcgcagat ttcagcgaag gatcaggatc gtggcaaccc 60 caggcccaag
ggtttgagac gcttctggta catggtggcg taaagccaga tcccgtcacg 120
ggggcaatcc tgacccccgt ctaccagtct acgacgttcg tgcaagagag tatcgaacgt
180 tatcaagcaa agggctatag ctatacccgt tcagccaatc ctaccgtatc
tgcattggaa 240 gagaaattgt gcgcaatcga gcacggcgaa tatgccactg
tgtatagcac cggcatgtcc 300 gctacgacaa cggccatcag tagttttatg
tctgctggcg accacgctat tgtgaccgaa 360 tgtagctatg gcggaaccaa
tcgtgcctgc cgtgtcttct tcacgcgctt aggtatgtct 420 tttacattcg
tagatatgcg cgacgttaaa aatgtagagg ctgccatcaa acccaatacc 480
aagctggtta tctcagaatc gccagcaaac cctacactga cgcttactga tattgacgca
540 cttagctcgc tttgcaaggc taagggtatt attcacatgt gtgacaacac
tttcgcaacc 600 gctttcatta tgcgtccgct tgatcacgga gcagacgtga
ccctgatctc cacgactaag 660 tttgttgatg gccacaatat gaccgtcgga
ggggccttgg tcactaaatc caaggaatta 720 gacggaaagg tacgtttaac
gcaaaatatc ttaggtaact gtatgagtcc atttgttgcg 780 ttccttcaat
tacaaacggt gaagacgatg agccttcgca tttctcgtca atcagaaaac 840
gcccagaaag tagcggaatt tcttgagacc caccccgcag tggaacgcgt aatgtatcca
900 ggtcttaaat ctttcccaca gaaggcctta gcggatcgtc agcacgcaaa
caatttacat 960 ggcggtatgt tatggtttga agtgcgcgga ggaacagcgg
cagggcgtcg cttgatggac 1020 accgttcagc gcccgtggag cttatgcgag
aatctgggtg cgacggaatc catcattact 1080 tgcccgagtg tcatgaccca
cgcgaacatg actactgagg accgtatgaa ggtcggtatc 1140 accgacggat
ttgtacgtgt cagctgcggg atcgaagatg cagccgatct tatctcagct 1200
ttgaaggccg cactggatgc cttgggcaag 1230 <210> SEQ ID NO 80
<211> LENGTH: 915 <212> TYPE: DNA <213> ORGANISM:
Helicobacter pylori 2017 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(915) <223> OTHER
INFORMATION: Cystathione beta-synthase (Helicobacter pylori 2017)
<400> SEQUENCE: 80 atgatcttaa cagcaatgca agatgcaatc
gggcgtacac ctatcttcaa gtttacacgt 60 aaagattacc caattccatt
gaagtcggca atttacgcga aattggaaca cttaaacccg 120 gggggatccg
tgaaagatcg ccttgggcag tatcttatta aggaggcctt ccgtacacac 180
aagattacct ctactaccac tatcatcgaa cctactgctg ggaatactgg catcgccctt
240 gcccttgtag ctatcaaaca tcatcttaaa acgatctttg ttgttcccga
aaaattttcg 300 gttgagaaac aacagatcat gcgtgctctt ggtgccttag
taatcaatac gcctacctca 360 gagggtatct caggggccat taaaaaaagc
aaagagttag ccgagtctat cccggacagc 420 tacttgcctc ttcaatttga
gaatcccgac aatccggctg cttattacca cactcttgct 480 cctgaaattg
taaaggaact ggggacgaat tttacctctt ttgtagcggg catcggttct 540
ggaggaactt tcgcaggcac cgccaagtac cttaaagaac gtatcccgaa catccgcttg
600 attggagttg aaccagaagg ttctatttta aatgggggtg aaccggggcc
ccacgaaatc 660 gaaggaattg gagtagagtt catcccacca ttcttcgcta
atttggatat tgatgggttt 720 gagacgattt cagacgaaga gggcttcagt
tatacgcgca aattagccaa aaagaacgga 780 ttattagtgg gtagttcgtc
cggagcagcg ttcgccgcgg ctcttaagga agtacaacgt 840 ctgcccgaag
ggtcacaagt gttgacgatt ttcccagata tggctgatcg ctaccttagt 900
aaaggcattt attcc 915 <210> SEQ ID NO 81 <211> LENGTH:
1320 <212> TYPE: DNA <213> ORGANISM: Helicobacter
pylori 2017 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1320) <223> OTHER INFORMATION:
putativeamino transferase (Helicobacter pylori 2017) <400>
SEQUENCE: 81 atgcaagctt tcttgaaccg ttcgttcgcg ccccttttaa acccaaatga
gaacctgctg 60 gatcaagtta agagttcgat tattttgaag aaaggtgtta
gctactttga ctggggtgct 120 agtgggctgg ccagtgcatt ggtcgagaaa
cgtgttaagt ccctgcttcc atattatgcc 180 aatgcccaca gcgtagcaag
taaacatgcc atcttaatgg gcatgttact taaagaatgc 240 caagagaagc
tgaaacgctc gttaaacctt agtactaacc attgcgtgct tagcgccggg 300
tatggcgcga gctcagcgat caagaaattc caagagatcc tgggagtttg catcccctct
360 aaaaccaaaa agaatctgga accttattta aaagacatgg cgctgaaacg
cgtaatcgta 420 ggtccttatg aacatcactc taacgaggtc tcttggcgcg
agtctctttg tgaggtggtg 480 cgcattccac ttaacgaaca tggactgctg
gatttggaga ttttagagca gatcttaaag 540 aaatccccca attctctggt
ctccgtctcg gccgcaagta atgtaacggg gattctgaca 600 cccctgaaag
aaattagctc actgtgcaag gagtatcgcg cgatcctggc gcttgatctg 660
gccaactttt ccgcacacgc gaacccgaaa gactgcgagt accagacggg gttctatgca
720 ccacacaagt tgttgggtgg tattggggga tgcgggcttc ttggaatctc
caaagacttg 780 atcgatacac agatcccacc tagtttttca gccggaggag
tcattaagta cgcaaaccgc 840 acgcgtcacg aatttattga tgagctgccg
ttgcgtgagg agttcggaac tccgggactg 900 ctgcaatttt atcgctcagt
gttagcctac cagttacgtg acgaatgcgg tttggatttc 960 attcataaga
aggagaataa tctgcttcgt gtgttaatgc atggcttgaa agatctgcca 1020
gctatcaaca tttacggcaa tttaaccgca agccgcgtag gagtagtcgc gtttaacatc
1080 ggaggcatta gtccatacga tcttgcccgt gtcctgagtt acgaatatgc
tattgagact 1140 cgcgcagggt gctcttgtgc cggcccgtat ggacatgact
tactgaattt gaatgcacaa 1200 aagtcttccg atttcaatgc aaaacctgga
tggttgcgcg tctcacttca ttttacacac 1260 agtattaatg acattgacta
tctgttggac tctctgaaga aagctgttaa gaaactgcgt 1320 <210> SEQ ID
NO 82 <211> LENGTH: 918 <212> TYPE: DNA <213>
ORGANISM: Bacillus atrophaeus UCMB-5137 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(918)
<223> OTHER INFORMATION: YdeD (Bacillus atrophaeus UCMB-5137)
<400> SEQUENCE: 82 atgaacggtg aacacgccgc gttggcccac
tcccgcacaa aagggattgc tttggtttta 60 acgggcagta tcttatgggg
cgtttcaggg acagttgcgc agtacttatt ccaacaacaa 120 cattttaacg
tagagtggtt gaccgtcgtt cgcttgttgc tgtctggtat cttgctgctt 180
ggccttgcct atcgtaagga aaagcaacgc atctgggctg tctggaaaga caagacagat
240 ggtctgaatc tggttctgtt cgggattttg gggatgttgt ccgtccagta
cacatacttt 300 gcggctatcc agcatggtaa tgcggcgacg gcaactgtac
ttcagtatct ggccccggca 360 cttattacct gctacgtagc cattcgctct
aagcgtcttc caaccgtcaa agagttgatc 420 gcagttttcc tggctattat
tggaacgttt tttttagtca cccatgggga catccacagt 480 cttagtatct
cagggtgggc tttattctgg ggattaagtt cggcgtttgc cctggcgttt 540
tacactttgc accctcataa acttctggcc aagtgggggg cggctatcgt tgttggctgg
600 ggtatgctta tcggagggct tggtctttcc ttaatccatc ctccatggaa
atttgaggga 660 cagtggtcgg tctcggctta tgccgccgtt attttcattg
tcctgtttgg gaccctgact 720 gccttctact gctacctgga atctttaaag
tacttaactg ccagcgaaac ttcattaatc 780 gcctgcgcgg agcccttaag
tgctgcgttc ttaagcgtga tttggttgca tgtgactttt 840 ggtatcagcg
agtggcttgg tacttgttgt attttatcta cgattatgat cttatcgatt 900
aaggagaaga agctgaag 918 <210> SEQ ID NO 83 <211>
LENGTH: 585 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(585) <223> OTHER INFORMATION:
proteinYfiK (Escherichia coli) <400> SEQUENCE: 83 atgacaccca
cgttgcttag cgccttctgg acgtacaccc ttattacagc catgacgcct 60
gggccaaata atatccttgc cttatcatcc gcaacgtcgc atgggttccg ccagtccacc
120 cgtgtgcttg caggtatgtc tcttggcttt ttaatcgtta tgctgttgtg
cgcgggaatc 180 agtttctcct tggcggtaat cgaccccgcc gccgtacatt
tattgtcttg ggctggtgcc 240 gcgtatattg tttggctggc ttggaaaatt
gccacgtctc cgactaagga agatggttta 300 caagcaaaac ccatctcgtt
ttgggcttca tttgcacttc agttcgtgaa tgtcaagatt 360 attctttacg
gggtaacagc cctgtccact ttcgttttac cccagacgca ggcgttgtca 420
tgggtagtcg gagtgtccgt cttattagcc atgatcggta cgtttgggaa tgtgtgctgg
480 gcgctggcgg gccacttgtt tcaacaatta ttccgtcagt acggtcgcca
gttaaatatc 540 gttcttgctt tattactggt gtattgtgca gtccgcatct tctat
585 <210> SEQ ID NO 84 <211> LENGTH: 1188 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1188) <223> OTHER INFORMATION: multidrug efflux
transporterBcr(Escherichia coli) <400> SEQUENCE: 84
atgacgaccc gccagcatag ctcgttcgca atcgtattta ttcttggatt gcttgctatg
60 ttgatgccat tatcaatcga catgtactta ccagccctgc ctgttatttc
ggcccaattt 120 ggagtacccg ctgggtcaac ccaaatgaca ttatcaacat
acattctggg gttcgcttta 180 ggacagttga tttatggtcc aatggctgac
tcgtttgggc gcaaaccagt ggtcttgggc 240 gggacactgg tctttgcggc
cgcagccgtt gcgtgtgcct tggctaacac gatcgaccag 300 cttattgtaa
tgcgtttctt ccatggctta gctgcggcgg ctgccagtgt agtgattaat 360
gcgcttatgc gtgacatcta tccgaaggag gaattcagcc gcatgatgag cttcgtaatg
420 ttggtaacga ccatcgctcc attaatggcc cctattgttg ggggttgggt
cttagtctgg 480 ctttcatggc attacatttt ttggatcctt gccctggcgg
ctattctggc ctcagcgatg 540 attttcttcc tgattaaaga aacccttcct
ccggagcgcc gtcagccttt ccatattcgc 600 actactatcg gtaattttgc
ggccttgttt cgccataaac gcgtgctgtc atacatgttg 660 gcaagcggct
tttctttcgc gggtatgttc tcgtttttaa gtgctggtcc cttcgtgtat 720
atcgaaatca atcacgtagc cccggagaac ttcggctatt acttcgcatt aaatatcgtg
780 tttcttttcg tcatgaccat cttcaactct cgcttcgtcc gtcgtatcgg
tgccttaaat 840 atgtttcgtt cggggctgtg gatccaattt atcatggctg
cgtggatggt gatctccgca 900 ctgttggggc ttgggttttg gtcgcttgtg
gtgggcgtgg ctgcattcgt tggatgtgtc 960 agcatggtat cttctaacgc
gatggctgta attttggatg agttcccaca tatggcaggg 1020 actgcttcct
ctctggctgg cacatttcgc ttcggaattg gtgcaatcgt aggcgcgttg 1080
ctgagcttag cgacattcaa ttcggcgtgg cccatgattt ggtccattgc gttttgtgcg
1140 accagcagca tcctgttctg cctttatgct tcccgtccaa agaagcgt 1188
<210> SEQ ID NO 85 <211> LENGTH: 1356 <212> TYPE:
DNA <213> ORGANISM: Pseudomonas fluorescens R124 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1356) <223> OTHER INFORMATION: TolC (Pseudomonas
fluorescens R124) <400> SEQUENCE: 85 atgaacaaac ttagtatgct
gggagctgcc ttcgcgttgt tggcagggaa ctcagcattg 60 gcagcaatgg
ggcctttcga aatctacgaa caggctcttc gcaatgaccc agttttctta 120
ggggccatta aggagcgtga cgccggattg gaaaaccgca tcatcggccg cgcaggattg
180 ttaccacgct tggggtacaa ctacaatcgt ggccataaca cctctaaagc
gacccagttg 240 acaaatcgtg gctctctgac tgaagaccgt aactataatt
cgtatggttc aactcttaca 300 ttacagcaac ccttattaga ctatgaggcc
tatgccgcct accgtaaggg agtagcgcaa 360 agcttgttcg ccgatgaagc
ctttcgcggt aagtcacagg aattattggt tcgcgtctta 420 gataattaca
cgaaagcgtt gttcgcacaa gaccaaatcg atatcgcaca ggcgaaaaaa 480
aaagcttatg aacaacaatt tcagcagaac gaacatatgt tcaaacaagg cgaggggacg
540 cgcactgaca ttttggaagc tgaaagtcgt tatgaacttg ccacggcaga
agaaatcgag 600 gcgcgtaacg aacaggatgc cgctcttcgc gagcttggtg
cgcttgtcgg tgtcccaact 660 gtcgacattt ctgaacttgc acccttagac
cagaattttc aaacgttcgc gctgatgcct 720 gctaactatg atacgtggca
cgagttagca atttctaata atccgaacct ggcatcacag 780 cgtcaggccg
tggaagtagc aaaatacgaa gttgaacgta accgtgcagg acatttaccc 840
aaggtctcag catatgccag cattcgtcag actgagtctg acagtggtaa tacctacaat
900 caacgttatg atacgaacac cattggcttt gaggtaaacg tccctctgta
tgcaggagga 960 ggagtctcag caagtacacg ccaagcatca cgcacgatgg
agcaggcgga gtatgaatta 1020 gatggaaaga cgcgtgagac gttaattgaa
ttacgtcgtc agttcagcgc gtgccttagt 1080 ggagttaata agttacgcgc
ctatcagaaa gccctggcct cggccgaagc actggtggtc 1140 tcaaccaagc
agagcattct tggcggcgaa cgcaccaact tggacgcgct taacgcggaa 1200
cagcagctgt tcaccacgcg tcgcgacctt gcacaggccc gctatgacta cttgatggcg
1260 tggacgaaac tgcattatta cgcaggaacc ctgaacgaac aagatttagc
gcgtgtggac 1320 gaggcatttg gccaagggcc caaatcaaat cctcgc 1356
<210> SEQ ID NO 86 <211> LENGTH: 1167 <212> TYPE:
DNA <213> ORGANISM: Sinorhizobium meliloti AK83 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1167) <223> OTHER INFORMATION: Tyrosine transaminase
(Sinorhizobium meliloti AK83) <400> SEQUENCE: 86 atgttcgatg
cgctggcgcg tcaagcggat gatccgcttt tggcgctgat cggactgttt 60
cgcaaagacg agcgccccgg taaagtggac ttaggtgtgg gagtttaccg cgacgaaact
120 ggccgcactc cgatctttcg cgcggttaaa gcagccgaaa aacgcttgct
tgagactcag 180 gactcgaagg cctacatcgg cccggaagga gacctggttt
ttcttgaccg tttgtgggaa 240 cttgttgggg gggataccat tgaacgttct
cacgtagctg gtgtacaaac acctggcggg 300 agcggcgcac ttcgtttggc
ggcagattta atcgcccgca tgggcggtcg cgggatttgg 360 ttggggttgc
catcctggcc gaatcacgct cccattttca aagcggctgg actggatatc 420
gcgacttacg atttctttga tatcccgagt caatccgtta tttttgataa cctggtgtct
480 gccctggaag gtgcagcatc tggcgatgcc gtcttattgc atgctagctg
ccacaatcca 540 actggagggg tattatccga ggcacagtgg atggaaattg
ccgcgctggt cgccgaacgc 600 ggactgttac cacttgttga tcttgcgtat
caagggttcg gacgtgggct ggatcaagac 660 gtcgcgggct tacgccattt
attaggtgta gttcccgaag cccttgtcgc cgttagctgc 720 tctaaatcgt
tcggcttgta ccgcgaacgc gctggagcca tcttcgcccg tacatcatct 780
accgcttcag ccgaccgcgt ccgcagtaac ttagctggcc ttgctcgcac atcgtatagt
840 atgccccccg atcacggggc cgcggttgtc cgtacgatct tagacgaccc
agagctgcgt 900 cgtgactgga ccgaggaatt agagacaatg cgcttgcgta
tgacgggtct tcgccgctct 960 cttgcagagg gcttgcgcac ccgttggcag
tctcttggcg ccgtagctga ccaagaaggg 1020 atgttctcga tgctgccgtt
gtccgaagca gaggttatgc gccttcgcac tgagcatgga 1080 atttacatgc
ccgcatcagg acgcattaac attgcggggt taaaaacggc ggaggctgcc 1140
gaaattgcag gtaaatttac gagtttg 1167 <210> SEQ ID NO 87
<211> LENGTH: 1209 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1209)
<223> OTHER INFORMATION: tyrosine transporterTyrP(Escherichia
coliW) <400> SEQUENCE: 87 atgaagaacc gcactcttgg atcagtattc
attgttgcgg ggaccaccat cggtgcaggt 60 atgcttgcca tgcccctggc
tgcagctggc gtcgggttca gcgttaccct gattttactg 120 attggtctgt
gggctctgat gtgttacacg gcattgcttt tgcttgaagt gtaccagcat 180
gtacccgcag acaccggtct tggcactctg gcgaaacgtt atttaggacg ttatggtcaa
240 tggctgaccg gtttctccat gatgtttctg atgtatgcgc tgacggccgc
atacattagt 300 ggtgcaggtg aactgctggc aagttcaatt tctgactgga
cgggcatctc tatgagcgcg 360 actgctgggg ttttattgtt tacatttgtg
gctggcggtg tagtgtgtgt agggacgtca 420 ttagttgatc tgtttaaccg
cttccttttc agtgcaaaaa tcattttcct tgtagtaatg 480 cttgtcttat
tattaccaca tattcataag gtaaatcttt tgacattacc attgcagcag 540
ggattggcgt tatcagccat ccctgtaatc ttcacatcct tcggattcca cgggtccgtc
600 ccatccatcg tgtcctacat ggacggcaat gtacgcaagt tacgttgggt
ctttatcaca 660 gggagcgcca ttccccttgt agcgtatatt ttttggcaag
ttgctactct ggggtcaatc 720 gactctacca ccttcatggg tttacttgcg
aaccacgcgg ggttgaacgg actgttacag 780 gctttgcgtg aaatggttgc
ctcgccacat gttgagttgg cggttcatct ttttgctgac 840 ttagccttag
ctacctcttt ccttggggtt gcgctgggat tattcgacta tctggctgat 900
ctttttcaac gctccaacac cgtaggtgga cgtttacaga ctggagccat tactttcttg
960 ccccctttag cctttgcgct gttttatcca cgtgggtttg ttatggcctt
ggggtatgct 1020 ggagtcgcct tagctgtact tgctcttatt attccatcgt
tattaacgtg gcaatcgcgt 1080 aaacacaacc cccaagcagg gtaccgcgtg
aagggaggac gccccgcgct ggtggttgtt 1140 tttctgtgcg ggattgccgt
catcggcgtg caatttttga ttgcagcagg tttgttgccg 1200 gaggtgggg 1209
<210> SEQ ID NO 88 <211> LENGTH: 1302 <212> TYPE:
DNA <213> ORGANISM: Variovorax paradoxus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1302) <223> OTHER INFORMATION: Beta-phenylalanine
transaminase (Aromatic beta-amino acid aminotransferase;
Beta-phenylalanineaminotransferase; VpAT) <400> SEQUENCE: 88
atgactcatg ctgcaattga ccaggcgttg gcagacgcct atcgtcgttt tactgacgca
60 aaccctgcca gccagcgtca gtttgaagcg caagcccgct atatgcccgg
ggctaactct 120 cgctctgttt tgttttatgc accctttcca ttgacgatcg
cacgtgggga aggcgccgct 180 ctttgggatg cggacggcca ccgttacgct
gactttatcg cggaatacac agctggggtg 240 tatggacaca gtgccccaga
gattcgtgac gcagtaatcg aagctatgca gggtgggatt 300 aatttgacgg
gtcataattt gttggaaggc cgcttagccc gccttatttg tgagcgtttc 360
ccacagatcg aacagttgcg tttcacgaat agcggaacag aggccaatct gatggccctt
420 accgcggcgc ttcattttac tggtcgccgc aaaatcgtcg tatttagtgg
aggttatcat 480 gggggggttc ttgggttcgg tgcccgtcct agccctacca
cagtaccatt tgacttcctt 540 gtgctgcctt acaacgatgc tcagacggct
cgtgctcaga tcgagcgcca cggcccggag 600 atcgcggtcg tgttagtcga
gcccatgcaa ggtgcttctg gctgcatccc aggtcagccc 660 gactttctgc
aagccctgcg cgaatccgct actcaggtag gggcgctgtt agtttttgac 720
gaagtgatga ctagtcgctt agcgccacat ggtttagcta acaaattggg gatccgttcg
780 gatttgacaa ccctgggtaa gtacattggc ggcggtatgt catttggggc
ctttggcggt 840 cgtgctgatg tcatggccct gttcgaccct cgcactggac
ctttggctca ttccggtacg 900 tttaacaaca atgtgatgac gatggctgcc
ggttatgctg gcttaacgaa attattcact 960 ccggaagcgg caggggcatt
ggcagagcgt ggagaagcgc ttcgcgcacg tcttaacgcc 1020 ctgtgtgcta
acgaaggagt agcaatgcag ttcactggca tcggctcgct gatgaatgcc 1080
cacttcgtcc agggagacgt tcgtagctct gaggatctgg ccgcagttga tgggcgttta
1140 cgtcagttgt tgttctttca tttattgaat gaagatattt actcttcacc
gcgtgggttt 1200 gttgtattat cgttgccatt gactgacgct gatattgacc
gctacgttgc tgcgatcggt 1260 tcatttattg gcggtcatgg ggcgttgtta
ccgcgcgcta ac 1302 <210> SEQ ID NO 89 <211> LENGTH:
1401 <212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1401) <223> OTHER INFORMATION: gadA glutamate
decarboxylase (Escherichia coli) <400> SEQUENCE: 89
atggaccaga agctgttaac ggatttccgc tcagaactac tcgattcacg ttttggcgca
60 aaggccattt ctactatcgc ggagtcaaaa cgatttccgc tgcacgaaat
gcgcgatgat 120 gtcgcatttc agattatcaa tgatgaatta tatcttgatg
gcaacgctcg tcagaacctg 180 gccactttct gccagacctg ggacgacgaa
aacgtccata aattgatgga tttgtcgatc 240 aataaaaact ggatcgacaa
agaagaatat ccgcaatccg cagccatcga cctgcgttgc 300 gtaaatatgg
ttgccgatct gtggcatgcg cctgcgccga aaaatggtca ggccgttggc 360
accaacacca ttggttcttc cgaggcctgt atgctcggcg ggatggcgat gaaatggcgt
420 tggcgcaagc gtatggaagc tgcaggcaaa ccaacggata aaccaaacct
ggtgtgcggt 480 ccggtacaaa tctgctggca taaattcgcc cgctactggg
atgtggagct gcgtgagatc 540 cctatgcgcc ccggtcagtt gtttatggac
ccgaaacgca tgattgaagc ctgtgacgaa 600 aacaccatcg gcgtggtgcc
gactttcggc gtgacctaca ccggtaacta tgagttccca 660 caaccgctgc
acgatgcgct ggataaattc caggccgaca ccggtatcga catcgacatg 720
cacatcgacg ctgccagcgg tggcttcctg gcaccgttcg tcgccccgga tatcgtctgg
780 gacttccgcc tgccgcgtgt gaaatcgatc agtgcttcag gccataaatt
cggtctggct 840 ccgctgggct gcggctgggt tatctggcgt gacgaagaag
cgctgccgca ggaactggtg 900 ttcaacgttg actacctggg tggtcaaatt
ggtacttttg ccatcaactt ctcccgcccg 960 gcgggtcagg taattgcaca
gtactatgaa ttcctgcgcc tcggtcgtga aggctatacc 1020 aaagtacaga
acgcctctta ccaggttgcc gcttatctgg cggatgaaat cgccaaactg 1080
gggccgtatg agttcatctg tacgggtcgc ccggacgaag gcatcccggc ggtttgcttc
1140 aaactgaaag atggtgaaga tccgggatac accctgtacg acctctctga
acgtctgcgt 1200 ctgcgcggct ggcaggttcc ggccttcact ctcggcggtg
aagccaccga catcgtggtg 1260 atgcgcatta tgtgtcgtcg cggcttcgaa
atggactttg ctgaactgtt gctggaagac 1320 tacaaagcct ccctgaaata
tctcagcgat cacccgaaac tgcagggtat tgcccagcag 1380 aacagcttta
aacacacctg a 1401 <210> SEQ ID NO 90 <211> LENGTH: 1398
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1398) <223> OTHER INFORMATION: glutamate
decarboxylase (Escherichia coliKO11FL) <400> SEQUENCE: 90
atggataaaa agcaagtgac ggacctgcgc tctgaacttc ttgacagtcg ttttggggca
60 aagagtatta gtaccattgc tgagtcaaag cgttttcctt tgcatgagat
gcgcgatgac 120 gtcgcattcc agattatcaa cgacgagctg tatttggacg
gcaatgcccg ccaaaacttg 180 gccacgtttt gtcagacttg ggatgacgag
aatgttcata aacttatgga cctttcaatt 240 aacaaaaatt ggattgacaa
agaagagtac ccccaatctg ccgcaattga tttacgttgt 300 gttaatatgg
tggccgactt atggcatgca ccagccccta aaaacggcca agcggtggga 360
accaacacga tcgggtctag tgaggcatgt atgttaggcg ggatggccat gaagtggcgt
420 tggcgtaaac gcatggaggc agcagggaaa ccaaccgata aacctaattt
agtctgcgga 480 ccggttcaga tctgttggca taaatttgcg cgctactggg
atgtggaatt acgcgaaatt 540 ccgatgcgtc cgggccaact gttcatggat
cccaaacgta tgatcgaagc atgtgacgaa 600 aacacgattg gggtggtacc
cacctttggg gtcacatata caggtaacta cgagtttcca 660 caaccgttgc
atgatgctct ggacaagttt caagctgaca ccgggatcga cattgatatg 720
cacattgacg ctgcctccgg cggattcttg gccccatttg tagcccctga cattgtctgg
780 gactttcgtc ttccccgtgt gaaatccatc agcgcatccg gtcacaagtt
tgggcttgcc 840 ccattagggt gtggatgggt catctggcgt gatgaggaag
cattacccca agaacttgtc 900 ttcaatgtag attaccttgg gggacagatt
ggcacttttg ccatcaactt ttctcgccca 960 gcgggtcaag tgatcgccca
gtattacgag tttctgcgcc tgggacgtga gggatataca 1020 aaagtgcaga
acgcatcgta ccaggtagcg gcttaccttg cggacgaaat tgcaaagctg 1080
ggaccatacg agtttatctg taccgggcgt ccagatgaag gtattccggc tgtgtgtttt
1140 aagctgaaag acggggaaga tcccggatat acgctgtatg atctgtctga
acgtttacgt 1200 ttgcgcggtt ggcaagttcc agccttcacg ttgggtggcg
aagccactga tattgtagtc 1260 atgcgtatca tgtgtcgtcg cggctttgaa
atggatttcg cagagttact tctggaagac 1320 tacaaagcga gcttaaaata
tttgtctgac catcccaagt tgcaagggat cgcacagcaa 1380 aattcgttta
aacacact 1398 <210> SEQ ID NO 91 <211> LENGTH: 1287
<212> TYPE: DNA <213> ORGANISM: Bacillus atrophaeus
UCMB-5137 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1287) <223> OTHER INFORMATION:
GltT (Bacillus atrophaeus UCMB-5137) <400> SEQUENCE: 91
atgaagaaat tacgcttcgg actggcgact caaatctttg tggggctgat tcttggggta
60 gtagtgggcg ttatctggta cggtaatccg gcggtggtaa cttatttgca
gccagttggg 120 gacctttttt tacgtttgat taaaatgatc gttattccta
tcgtggtgtc ttctttgatc 180 attggcgtcg cgggagctgg gtccggaaaa
caggtcggaa agctgggctt tcgtactatt 240 ctgtacttcg agatcatcac
tacctttgcc atcattctgg gacttgctct ggcgaatctt 300 ttccagcctg
gtacaggagt aaatatcgag agcgcgcaga aaagtgacat ttcccagtac 360
gtggagactg aaaaagagca atccaccaaa tccgtagctg agactttcct gcatatcgtg
420 cccaccaatt tctttcaatc acttgcggaa ggtgatcttc ttgctattat
ctgctttacc 480 gtacttttcg cccttggcat ttcggctatc ggtgaacgtg
gcaaaccggt gcttgctttc 540 tttgacggag tatcccacgc gatgtttcat
gtagtgaacc ttgtgatgaa ggttgctccg 600 ttcggcgtat ttgctctgat
tggagtaaca gtaagcaaat ttggactggg ttctttactg 660 agcctgggta
aacttgtggg gctggtatat gttgctctgg cattttttct tattgtaatc 720
tttggtattg ttggaaagct ggctggcgtg aatatcttca agtttttagc ttacatgaag
780 gatgaaatct tattagcgtt ctcgacctca tcgtccgaga ctgtgttgcc
ccgcatcatg 840 gagaaaatgg agaagatcgg gtgtccaaag ggaattgtaa
gctttgtagt ccccatcggt 900 tacacattca atcttgacgg ctcggtctta
taccaatcta ttgctgcgct gttcttggca 960 caggtttacg gaatcgacct
gactatttgg catcagatta ctctggtgtt agttctgatg 1020 gtcactagca
aaggcatggc agccgttcct ggaactagct ttgtagtcct gctggcaacc 1080
ttaggtacca ttggtgttcc agcggaaggg cttgcattca ttgcgggggt tgaccgcatt
1140 atggacatgg ctcgcactgt ggtcaattta acaggcaatg ctcttgcgag
tgtcgtaatg 1200 agcaagtggg agggtcagta cgacccggtg aaaggtgcag
agattatgag ccgcagcaag 1260 acggaacagg acgctactat ctccgga 1287
<210> SEQ ID NO 92 <211> LENGTH: 858 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(858)
<223> OTHER INFORMATION: mechanosensitive
channelMscS(Escherichia coli) <400> SEQUENCE: 92 atggaggact
tgaacgtagt agatagcatt aatggagcgg gctcatggtt agtagccaac 60
caagccctgt tgttatcgta tgctgtaaat atcgtcgcag ccttagccat cattatcgtt
120 gggttaatca tcgcccgtat gatttctaat gcggtgaatc gcttaatgat
ctcgcgcaag 180 atcgacgcca ctgtcgcgga tttcttgtcc gccctggtgc
gttacggtat catcgcgttc 240 acattgattg cggcattagg gcgcgtagga
gtccagacag cttctgtgat tgcggtatta 300 ggtgcagcag gattagctgt
gggattggcg ttacaggggt ctctttccaa tctggcggcc 360 ggcgtacttc
tggttatgtt tcgccccttt cgcgccggag agtatgtgga tttgggagga 420
gtggccggaa cagtgctgtc agtgcaaatc ttttctacca cgatgcgtac agcagatgga
480 aaaatcatcg tgatccccaa tggcaagatc atcgcgggta acattatcaa
cttctcccgc 540 gaacctgttc gccgcaacga atttatcatc ggtgttgcct
atgattcaga catcgatcag 600 gtcaaacaaa ttcttacgaa catcattcag
tcagaggacc gtattctgaa agaccgcgaa 660 atgacggtgc gtttgaatga
gttaggggct tcaagtatca acttcgtagt ccgcgtgtgg 720 agcaattccg
gtgatttgca aaacgtgtat tgggacgtcc ttgagcgcat taagcgtgaa 780
ttcgatgctg ccgggatctc ctttccgtat cctcagatgg atgtgaattt caagcgtgta
840 aaggaagata aggctgcc 858 <210> SEQ ID NO 93 <211>
LENGTH: 1539 <212> TYPE: DNA <213> ORGANISM: Bacillus
amyloliquefaciens subsp. plantarum str. FZB42 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1539) <223> OTHER INFORMATION: HutH (Bacillus
amyloliquefaciens subsp. plantarum str. FZB42) <400>
SEQUENCE: 93 atgatggtca ccttggatgg gtcttcatta acgacggctg atgcacaacg
tgtacttttc 60 gattttgaag aggtacaggc atcggctgaa tcgatggagc
gcgtaaaaaa gagccgtgcc 120 gccgtggaac gcattgtaca agaagaaaaa
actatctacg gaatcactac ggggtttggt 180 aagttttccg atgtgctgat
ccaaaaagag gacgctgcgg atttacaatt gaatttgatc 240 ttgtcacatg
catgtggagt cggcgatcct ttcccagagt cagtctcccg cgccatgctg 300
cttctgcgtg caaacgcatt gttaaaaggc ttctccggtg ttcgtacgga attaattgac
360 cagcttttag cgtacttaaa ccaccgtatc caccctgtta tcccccaaca
aggttcgctg 420 ggggcctccg gcgatttggc ccctcttagc caccttgcgt
tggcactgat cggacaaggg 480 gaagtgttct acgaaggagc acgtatgccc
actgctcatg cccttgaaca aaccaatctg 540 cagcccgcag tcctgacatc
gaaggaaggg ctggcgttga tcaatgggac tcaggctatg 600 accgcaatgg
gcttaatcgc ataccttgaa gccgaaaagt tggcatatca gagcgagcgc 660
atcgcttcat tgactatcga aggattgcaa ggtattattg acgcgtttga cgaagatatt
720 catgccgctc gtggatacca ggaacaaatg gatgtcgctg agcgcattcg
ctattatctt 780 tcggattcga agctgacaac cgtacaaggc gagctgcgtg
tgcaagatgc ttactccatt 840 cgctgcatcc ctcaagtcca cggagcttct
tggcagaccc tggcgtatgt gaaggagaag 900 ttagaaattg agatgaacgc
tgctactgat aaccctttaa tttttgaaga cggggccaaa 960 attatctcgg
gggggaactt tcacgggcaa ccgatcgcgt ttgcaatgga cttcttgaaa 1020
gtagctgctg ctgagttggc taatatcagc gagcgccgta ttgagcgtct tgtcaatcca
1080 cagctgaatg accttcctcc ttttctttcg ccgcaaccgg gtttacagtc
tggtgccatg 1140 attatgcagt acgccgctgc ctccttggtc tcggaaaaca
aaacacttgc gcatcccgcc 1200 tcagtcgact caatcccctc ctcggctaac
caggaggatc acgtctccat ggggacgatc 1260 gcttcacgtc atgcttacca
gattattgca aacactcgtc gcgtattagc cgtcgaggcc 1320 atttgcgctt
tacaagctgt agagtaccgt ggggaagagc actgcgctag ctacacgaaa 1380
caactttacc atgagatgcg taacatcgtg ccatcgattc aggaggaccg tgttttctcg
1440 tacgacatcg agcacttatc cgactggctt aaaaaggaat ccttcttacc
taatgaacac 1500 caccaaaagt taatgactaa tgagggcggg ttaactcgc 1539
<210> SEQ ID NO 94 <211> LENGTH: 780 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(780)
<223> OTHER INFORMATION: Histidine ABC transporter,
histidine-binding periplasmic protein precursorHisJ (Escherichia
coliO145:H28 str. RM12581]) <400> SEQUENCE: 94 atgaagaaac
ttgtcctttc attgtctctg gtattagcgt tcagttcagc aactgcagca 60
ttcgctgcta ttccgcaaaa tatccgcatc gggacggatc ccacgtatgc gccattcgag
120 tcaaagaatt cacaaggtga attggtcggg ttcgatattg acctggcgaa
agaattgtgt 180 aaacgtatca atacccaatg cacgttcgtg gaaaatccct
tggatgcatt aattccgtct 240 ttgaaagcga aaaaaatcga tgccatcatg
tcatcccttt ctatcacaga aaagcgccag 300 caggagattg ccttcacaga
caagttgtac gctgcagaca gccgcctggt cgttgcaaag 360 aattctgaca
ttcaacctac cgtggaatcg ctgaagggca agcgcgtagg ggtcttgcag 420
ggcactactc aggaaacatt tgggaacgaa cattgggcgc ctaagggaat tgagatcgtg
480 tcttatcagg gtcaggataa catctacagt gatctgacag ccggacgtat
tgacgccgct 540 tttcaggacg aggtggcggc atctgaaggg ttcttaaagc
agccagtcgg caaagactac 600 aaatttggtg ggccgagcgt gaaggacgag
aaattgtttg gggtaggaac agggatgggc 660 ttgcgtaagg aggacaatga
attacgtgaa gctcttaata aagcctttgc tgagatgcgt 720 gcggacggga
cttacgaaaa acttgcaaaa aagtatttcg actttgacgt ctacggcggt 780
<210> SEQ ID NO 95 <211> LENGTH: 684 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(684)
<223> OTHER INFORMATION: Histidine ABC transporter, permease
proteinHisQ (Escherichia coliO145:H28 str. RM12581) <400>
SEQUENCE: 95 atgctgtatg gattcagtgg cgttatcttg cagggggctc ttgtcacttt
agagttagct 60 atctcgtccg ttgtgttagc tgtcattatt ggacttatcg
gggctggtgg caaattgagt 120 cagaaccgtt tgagcggcct tatttttgaa
gggtacacaa ccttaattcg cggagtccca 180 gacttagtgc tgatgttgct
tattttctat ggtttacaga tcgctttgaa tacggttacc 240 gaggcaatgg
gggtcggcca aatcgatatc gatcctatgg tggctggaat cattactttg 300
ggcttcattt acggggcata tttcacggag acgttccgcg gagctttcat ggccgtcccg
360 aagggccaca ttgaagcggc aacagctttt ggattcactc gtgggcaagt
tttccgtcgc 420 atcatgtttc cagcgatgat gcgctatgcg cttcctggga
tcgggaataa ctggcaggta 480 atcttaaaat cgacggcttt agtcagttta
ttggggttgg aagatgtcgt aaaagcgacc 540 cagttggctg ggaaatcgac
ttgggagccc ttttacttcg ctattgtgtg tggcgttatt 600 tacttagttt
tcactacagt atcaaacggt gtgttattgt ttttggaacg tcgctacagc 660
gtgggtgtaa agcgtgctga tttg 684 <210> SEQ ID NO 96 <211>
LENGTH: 771 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(771) <223> OTHER INFORMATION:
hisP (Escherichia coliEPEC C342-62) <400> SEQUENCE: 96
atgtccgaga acaaattaaa tgttatcgat ttgcataagc gttatggaga gcatgaagtg
60 ttgaaaggag tgtctcttca agcaaacgcg ggggacgtaa tttctatcat
cggatcgtct 120 ggttctggta agtcaacctt cctgcgttgt attaacttct
tagagaagcc gtctgagggt 180 tctattgtag ttaatgggca gaccatcaat
cttgtgcgcg ataaggacgg ccagttgaaa 240 gtggcagaca aaaaccaact
tcgtttgctt cgcacccgtc ttaccatggt attccaacac 300 ttcaacctgt
ggtcgcacat gacggtactt gagaacgtga tggaagcgcc aattcaggta 360
cttggattga gcaaacaaga agcccgcgaa cgtgcggtga aatatttggc caaggtgggt
420 atcgacgagc gtgcgcaggg caaatacccc gttcacttgt ccgggggtca
acaacagcgt 480 gtcagtattg cccgcgctct ggctatggaa ccagaggtgc
ttctgtttga cgagccgacg 540 tcagctttgg acccggaatt agtgggcgaa
gtattgcgca tcatgcagca gttagcagaa 600 gaaggcaaga ccatggttgt
tgtcacacac gaaatggggt ttgcgcgtca tgtctcgact 660 catgtaatct
tcttgcatca aggtaaaatc gaggaagaag gagcgccgga acagttattc 720
gggaatcctc aatccccccg tctgcagcag tttcttaaag ggtccttaaa g 771
<210> SEQ ID NO 97 <211> LENGTH: 660 <212> TYPE:
DNA <213> ORGANISM: Clostridium botulinum <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(660) <223> OTHER INFORMATION: proline reductase
(Clostridium botulinum) <400> SEQUENCE: 97 atgtcaatgt
ccgctgagca cgctgaggaa ttaaaaaatg aacctgcggt cgtttgttgt 60
cgcactgagg aggggaccat cttgtcagcc gataatttgg aagacccaaa catttttcca
120 gatatggtgg atagcggttt actgaacatt cctggggact gcttaaaagt
tggggaagta 180 atcggggcca aactgcttaa gacgattgac tctttgaccc
ctcttgccaa ggacatcatt 240 gagggggcca aatccttaga cggagacgta
cgcagtaaat cagagattca gatcgaatca 300 ccagaggaga aggcgatcct
taaaaacaat ttgaaggcgg gagatattat caaggttgag 360 gacctggaga
accctatgca cttcgccaag ttacaagatt cgcttcttat caagctggat 420
gagaaagtgc ttacgcgccg cgaagttgta gacgcgaaac ttacggaaga tgcaccggcg
480 atttcagggg tcactgcatc aatgttggaa ggcttcgagg aaaaggccct
ggagattacc 540 caagatagca aggatgtgga cttcaattca gtaattccac
tgaacggcaa tcgtgaattc 600 cttcgtttga aaatcgagga aggcacaggc
atttatatcg aaattccctt tacccaagtc 660 <210> SEQ ID NO 98
<211> LENGTH: 1347 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1347)
<223> OTHER INFORMATION: ProlineporterII (Escherichia coli
PMV-1) <400> SEQUENCE: 98 atgtcagaaa aacttccggc acctcgcgag
ggtttatccg gtaaagctat gcgtcgtgtt 60 gtcatgggta gctttgccgg
tgcattaatg gaatggtatg atttcttcat ctttgggacg 120 gcggcgggtc
ttgtttttgc accgctgttt tatcctgaca gtgatccgtt tattgggttg 180
atcgcgtcgt tcgctacatt tggagttggt tttttgaccc gcccgttagg aggtatcgtg
240 ttcggtcatt ttggtgacaa gatcgggcgt aagattacct taatctggac
attggcgatt 300 gtggggtgtt ctacattctt aatcggtttc attccaacgt
accaagaaat cggcatttgg 360 gcccctttgg tccttatggt tttgcgcctg
attcagggtt ttggcttggg aggagaatac 420 ggaggggcgg cgttaatgac
catcgaaagt gcccccgaaa gccgccgtgg ttttcttggg 480 tcattgccac
agacggccgc cagcgtcggc atcatgcttg caacgggtat tttcgcgctt 540
tgtaatcatt tccttacttc tgaacagttc ttatcatggg gctggcgtat tcccttctgg
600 ttgtccgcgg ttatgttaat cgtcggactt tttatccgtc tgcatactga
agagacgctt 660 gactttcaga agcaaaaaac gactaacaat aaagaaaagt
cggttccccc ccttatcgag 720 ctttttaaga aacatccacg caatattttg
ttggccttgg gggcccgcct tgcggagtca 780 gtaagcagca acatcattaa
tgcattcggc atcgtctata tcagcagtca acttgcactg 840 agccgtgaca
tccccttgac tgggatgctg attgcaagcg ccatcggaat ttttagttgc 900
ccattggtag gctggctttc ggaccgtatc ggacagaaat cattatattt gtcaggcgct
960 ggattttgtg tcctgtttgc cttcccgttt tttctgctgt tggactcgaa
gagtacactg 1020 attatctggt gctcaatgat tttgggctat aacttgggtc
caactatgat gtttgctgta 1080 caaccaacat tgtttactcg tatgttcggc
accaaggtcc gctacacagg cttatcattt 1140 gcttaccagt tctcggctat
cttaggcggc ctgtccccac tgattgcatc ctcacttctt 1200 gcgttggggg
gcggcaaacc ctggtatgtc gccttgttcc ttttcgctgt gtccgtgtta 1260
tctttcgtct gtgtatggtt aatcgagccc acagacgaac aagagacggc ttcataccgc
1320 tacatccgcg aacaaagtca tgagaac 1347 <210> SEQ ID NO 99
<211> LENGTH: 1377 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1377)
<223> OTHER INFORMATION: Escherichia coli PheP <400>
SEQUENCE: 99 atgaaaaacg cgtcaaccgt atcggaagat actgcgtcga atcaagagcc
gacgcttcat 60 cgcggattac ataaccgtca tattcaactg attgcgttgg
gtggcgcaat tggtactggt 120 ctgtttcttg gcattggccc ggcgattcag
atggcgggtc cggctgtatt gctgggctac 180 ggcgtcgccg ggatcatcgc
tttcctgatt atgcgccagc ttggcgaaat ggtggttgag 240 gagccggtat
ccggttcatt tgcccacttt gcctataaat actggggacc gtttgcgggc 300
ttcctctctg gctggaacta ctgggtaatg ttcgtgctgg tgggaatggc agagctgacc
360 gctgcgggca tctatatgca gtactggttc ccggatgttc caacgtggat
ttgggctgcc 420 gccttcttta ttatcatcaa cgccgttaac ctggtgaacg
tgcgcttata tggcgaaacc 480 gagttctggt ttgcgttgat taaagtgctg
gcaatcatcg gtatgatcgg ctttggcctg 540 tggctgctgt tttctggtca
cggcggcgag aaagccagta tcgacaacct ctggcgctac 600 ggtggtttct
tcgccaccgg ctggaatggg ctgattttgt cgctggcggt aattatgttc 660
tccttcggcg gtctggagct gattgggatt actgccgctg aagcgcgcga tccggaaaaa
720 agcattccaa aagcggtaaa tcaggtggtg tatcgcatcc tgctgtttta
catcggttca 780 ctggtggttt tactggcgct ctatccgtgg gtggaagtga
aatccaacag tagcccgttt 840 gtgatgattt tccataatct cgacagcaac
gtggtagctt ctgcgctgaa cttcgtcatt 900 ctggtagcat cgctgtcagt
gtataacagc ggggtttact ctaacagccg catgctgttt 960 ggcctttctg
tgcagggtaa tgcgccgaag tttttgactc gcgtcagccg tcgcggtgtg 1020
ccgattaact cgctgatgct ttccggagcg atcacttcgc tggtggtgtt aatcaactat
1080 ctgctgccgc aaaaagcgtt tggtctgctg atggcgctgg tggtagcaac
gctgctgttg 1140 aactggatta tgatctgtct ggcgcatctg cgttttcgtg
cagcgatgcg acgtcagggg 1200 cgtgaaacac agtttaaggc gctgctctat
ccgttcggca actatctctg cattgccttc 1260 ctcggcatga ttttgctgct
gatgtgcacg atggatgata tgcgcttgtc agcgatcctg 1320 ctgccggtgt
ggattgtatt cctgtttatg gcatttaaaa cgctgcgtcg gaaataa 1377
<210> SEQ ID NO 100 <211> LENGTH: 1704 <212>
TYPE: DNA <213> ORGANISM: Anabaena variabilis PAL1
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1704) <223> OTHER INFORMATION: Anabaena
variabilis PAL1 <400> SEQUENCE: 100 atgaaaacac tatcacaggc
ccaatctaaa acttcttcac agcaattcag ctttaccggg 60 aactcgtctg
cgaatgtaat tatcggcaat caaaagctga ccattaatga tgtagctcgc 120
gttgcccgga atggcacttt ggtgtcactg acgaacaata ccgacattct gcaaggtatt
180 caagctagct gcgattatat caataacgcc gttgaatctg gcgagccaat
ctacggggta 240 acaagcggtt ttggtgggat ggcgaacgtt gccattagcc
gtgaacaggc gagcgaactt 300 cagaccaacc tcgtttggtt cctaaagaca
ggagctggta ataagttacc tctggctgac 360 gtaagagccg cgatgctgct
tcgcgctaat agtcacatgc gcggcgccag tggtatccgt 420 cttgagctta
tcaagaggat ggaaatcttc ctcaacgcgg gtgtcacacc atatgtttat 480
gagtttggta gtatcggagc cagtggtgat cttgttcccc tgagttatat tacgggttca
540 ttgattggtt tagacccgtc ctttaaagtg gattttaacg ggaaagaaat
ggacgccccg 600 accgctttac gacagcttaa tctgagccca cttactttgc
tccctaaaga aggtcttgcc 660 atgatgaatg gcacctctgt gatgactgga
attgccgcga attgtgtgta tgacacgcag 720 atcctaacgg ccattgccat
gggtgttcac gcgttggaca ttcaagccct gaatggtaca 780 aaccagtcgt
ttcatccgtt tatccataat tcaaaacccc atccgggaca gctttgggct 840
gctgatcaga tgatctcact cctggccaat agtcaactgg ttcgggacga gctcgacggc
900 aaacatgatt atcgcgatca tgagctcatc caggaccggt attcacttcg
ttgtctccca 960 caatacctgg ggcctatcgt tgatggtata tctcaaattg
cgaagcaaat tgaaattgag 1020 atcaatagcg taaccgacaa cccgcttatc
gatgttgata atcaggcctc ttatcacggt 1080 ggcaattttc tgggccagta
tgttggtatg gggatggatc acctgcggta ctatattggg 1140 cttctggcta
aacatcttga tgtgcagatt gccttattag cttcaccaga attttcaaat 1200
ggactgccgc catcattgct cggtaacaga gaaaggaaag taaatatggg ccttaagggc
1260 cttcagatat gtggtaactc aatcatgccc ctcctgacct tttatgggaa
ctcaattgct 1320 gatcgttttc cgacacatgc tgaacagttt aaccaaaaca
ttaactcaca gggctataca 1380 tccgcgacgt tagcgcgtcg gtccgtggat
atcttccaga attatgttgc tatcgctctg 1440 atgttcggcg tacaggccgt
tgatttgcgc acttataaaa aaaccggtca ctacgatgct 1500 cgggcttgcc
tgtcgcctgc caccgagcgg ctttatagcg ccgtacgtca tgttgtgggt 1560
cagaaaccga cgtcggaccg cccctatatt tggaatgata atgaacaagg gctggatgaa
1620 cacatcgccc ggatatctgc cgatattgcc gccggaggtg tcatcgtcca
ggcggtacaa 1680 gacatacttc cttgcctgca ttaa 1704 <210> SEQ ID
NO 101 <211> LENGTH: 1599 <212> TYPE: DNA <213>
ORGANISM: Photorhabdus luminescens PAL3 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1599) <223> OTHER INFORMATION: Photorhabdus luminescens
PAL3 <400> SEQUENCE: 101 atgaaagcta aagatgttca gccaaccatt
attattaata aaaatggcct tatctctttg 60 gaagatatct atgacattgc
gataaaacaa aaaaaagtag aaatatcaac ggagatcact 120 gaacttttga
cgcatggtcg tgaaaaatta gaggaaaaat taaattcagg agaggttata 180
tatggaatca atacaggatt tggagggaat gccaatttag ttgtgccatt tgagaaaatc
240 gcagagcatc agcaaaatct gttaactttt ctttctgctg gtactgggga
ctatatgtcc 300 aaaccttgta ttaaagcgtc acaatttact atgttacttt
ctgtttgcaa aggttggtct 360 gcaaccagac caattgtcgc tcaagcaatt
gttgatcata ttaatcatga cattgttcct 420 ctggttcctc gctatggctc
agtgggtgca agcggtgatt taattccttt atcttatatt 480 gcacgagcat
tatgtggtat cggcaaagtt tattatatgg gcgcagaaat tgacgctgct 540
gaagcaatta aacgtgcagg gttgacacca ttatcgttaa aagccaaaga aggtcttgct
600 ctgattaacg gcacccgggt aatgtcagga atcagtgcaa tcaccgtcat
taaactggaa 660 aaactattta aagcctcaat ttctgcgatt gcccttgctg
ttgaagcatt acttgcatct 720 catgaacatt atgatgcccg gattcaacaa
gtaaaaaatc atcctggtca aaacgcggtg 780 gcaagtgcat tgcgtaattt
attggcaggt tcaacgcagg ttaatctatt atctggggtt 840 aaagaacaag
ccaataaagc ttgtcgtcat caagaaatta cccaactaaa tgatacctta 900
caggaagttt attcaattcg ctgtgcacca caagtattag gtatagtgcc agaatcttta
960 gctaccgctc ggaaaatatt ggaacgggaa gttatctcag ctaatgataa
tccattgata 1020 gatccagaaa atggcgatgt tctacacggt ggaaatttta
tggggcaata tgtcgcccga 1080 acaatggatg cattaaaact ggatattgct
ttaattgcca atcatcttca cgccattgtg 1140 gctcttatga tggataaccg
tttctctcgt ggattaccta attcactgag tccgacaccc 1200 ggcatgtatc
aaggttttaa aggcgtccaa ctttctcaaa ccgctttagt tgctgcaatt 1260
cgccatgatt gtgctgcatc aggtattcat accctcgcca cagaacaata caatcaagat
1320 attgtcagtt taggtctgca tgccgctcaa gatgttttag agatggagca
gaaattacgc 1380 aatattgttt caatgacaat tctggtagtt tgtcaggcca
ttcatcttcg cggcaatatt 1440 agtgaaattg cgcctgaaac tgctaaattt
taccatgcag tacgcgaaat cagttctcct 1500 ttgatcactg atcgtgcgtt
ggatgaagat ataatccgca ttgcggatgc aattattaat 1560 gatcaacttc
ctctgccaga aatcatgctg gaagaataa 1599 <210> SEQ ID NO 102
<211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM:
Legionella pneumophila phhA <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(819) <223>
OTHER INFORMATION: Legionella pneumophila phhA <400>
SEQUENCE: 102 atggagttta gtagccggta tgtcgcacat gtccctgatg
ctcagggttt agtcgattat 60 tcggcacaag aaaatagaat ttggaatatt
ttatttgaga ggcaactcaa gttattgcca 120 ggaagagctt gtgatgaatt
tctgtctgga ttacagactt taggacttaa ctcctcgact 180 attccacaac
ttccagaagt aagtgagcga ttaaaggcca aaacgggatg gcaagtagcg 240
ccagttgctg ctttaatttc agccagggaa ttttttgaat tattagcaga aaaatatttt
300 cctgcggcga cttttattcg aagtgaagaa gaattggatt atgttcaaga
acctgatatt 360 tttcatgagc tttttggtca ttgtcctatg ttaaccgata
gagtctatgc tgaatttgtc 420 catgattacg catgtaaggt attaactttt
cctgaacagg attggccttt attgcaaaga 480 atgttttggt ttactgtaga
gtttggattg attaaaacgc ctaaagggct tagagcatac 540 ggcgggggaa
ttttatcttc tatcagtgaa acggtatatt gtgtggaaag tgatattcct 600
gtgcgaattt tatttgatcc agtggtggct tttcgaatgc cttatcggat tgaccagcta
660 caacctgttt atttcgttat tgacagctat caaaatttat atgatttcgt
gctttctgac 720 atgggtaaat tcatggatcg tgcgcgagag ttaggtgaat
ttccaccgta ttttgatgtg 780 gatccggata atccaaatat tcatataagg
gcttgttaa 819 <210> SEQ ID NO 103 <211> LENGTH: 717
<212> TYPE: DNA <213> ORGANISM: Escherichia coli hisM
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(717) <223> OTHER INFORMATION: Escherichia
coli hisM <400> SEQUENCE: 103 gtgatcgaaa tcttacatga
atactggaaa ccgctgctgt ggaccgacgg ttatcgcttt 60 actggtgtgg
cgatcactct gtggctgctt attttgtcgg tagtgatagg cggagtcctg 120
gcgctgtttc tggcgattgg tcgtgtctcc agtaataaat acatccagtt tccaatctgg
180 ttatttacct atatttttcg cggtacgccg ctgtatgttc agttgctggt
gttctattcc 240 ggcatgtaca cgcttgagat tgttaaggga accgaattcc
ttaacgcttt cttccgcagt 300 ggcctgaact gtaccgtgct ggcgctgacg
cttaacacct gcgcttacac taccgagatt 360 tttgctgggg caatccgttc
ggttccgcat ggggaaattg aagccgccag agcctatggc 420 ttctcgactt
ttaaaatgta tcgctgcatt attttgcctt ctgcgctgcg tattgcgtta 480
ccggcataca gcaacgaagt gatcctgatg ctgcactcta ctgcgttggc atttactgcc
540 acggtgccgg atctgctgaa aatagcccgc gatattaacg ccgccacgta
tcaacctttt 600 accgccttcg gcattgccgc ggtgctctat ttaatcatct
cttatgtcct gatcagcctc 660 tttcgcagag cggaaaaacg ctggttgcag
catgtgaaac cttcttcaac gcactga 717 <210> SEQ ID NO 104
<211> LENGTH: 967 <212> TYPE: DNA <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION: clbA
(wild-type) <400> SEQUENCE: 104 caaatatcac ataatcttaa
catatcaata aacacagtaa agtttcatgt gaaaaacatc 60 aaacataaaa
tacaagctcg gaatacgaat cacgctatac acattgctaa caggaatgag 120
attatctaaa tgaggattga tatattaatt ggacatacta gtttttttca tcaaaccagt
180 agagataact tccttcacta tctcaatgag gaagaaataa aacgctatga
tcagtttcat 240 tttgtgagtg ataaagaact ctatatttta agccgtatcc
tgctcaaaac agcactaaaa 300 agatatcaac ctgatgtctc attacaatca
tggcaattta gtacgtgcaa atatggcaaa 360 ccatttatag tttttcctca
gttggcaaaa aagatttttt ttaacctttc ccatactata 420 gatacagtag
ccgttgctat tagttctcac tgcgagcttg gtgtcgatat tgaacaaata 480
agagatttag acaactctta tctgaatatc agtcagcatt tttttactcc acaggaagct
540 actaacatag tttcacttcc tcgttatgaa ggtcaattac ttttttggaa
aatgtggacg 600 ctcaaagaag cttacatcaa atatcgaggt aaaggcctat
ctttaggact ggattgtatt 660 gaatttcatt taacaaataa aaaactaact
tcaaaatata gaggttcacc tgtttatttc 720 tctcaatgga aaatatgtaa
ctcatttctc gcattagcct ctccactcat cacccctaaa 780 ataactattg
agctatttcc tatgcagtcc caactttatc accacgacta tcagctaatt 840
cattcgtcaa atgggcagaa ttgaatcgcc acggataatc tagacacttc tgagccgtcg
900 ataatattga ttttcatatt ccgtcggtgg tgtaagtatc ccgcataatc
gtgccattca 960 catttag 967 <210> SEQ ID NO 105 <211>
LENGTH: 424 <212> TYPE: DNA <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: clbA knock-out
<400> SEQUENCE: 105 ggatgggggg aaacatggat aagttcaaag
aaaaaaaccc gttatctctg cgtgaaagac 60 aagtattgcg catgctggca
caaggtgatg agtactctca aatatcacat aatcttaaca 120 tatcaataaa
cacagtaaag tttcatgtga aaaacatcaa acataaaata caagctcgga 180
atacgaatca cgctatacac attgctaaca ggaatgagat tatctaaatg aggattgatg
240 tgtaggctgg agctgcttcg aagttcctat actttctaga gaataggaac
ttcggaatag 300 gaacttcgga ataggaacta aggaggatat tcatatgtcg
tcaaatgggc agaattgaat 360 cgccacggat aatctagaca cttctgagcc
gtcgataata ttgattttca tattccgtcg 420 gtgg 424 <210> SEQ ID NO
106 <211> LENGTH: 1921 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic: Prp promoter <400> SEQUENCE:
106 ttacccgtct ggattttcag tacgcgcttt taaacgacgc cacagcgtgg
tacggctgat 60 ccccaaataa cgtgcggcgg cgcgcttatc gccattaaag
cgtgcgagca cctcctgcaa 120 tggaagcgct tctgctgacg agggcgtgat
ttctgctgtg gtccccacca gttcaggtaa 180 taattgccgc ataaattgtc
tgtccagtgt tggtgcggga tcgacgctta aaaaaagcgc 240 caggcgttcc
atcatattcc gcagttcgcg aatattaccg ggccaatgat agttcagtag 300
aagcggctga cactgcgtca gcccatgacg caccgattcg gtaaaaggga tctccatcgc
360 ggccagcgat tgttttaaaa agttttccgc cagaggcaga atatcaggct
gtcgctcgcg 420 caagggggga agcggcagac gcagaatgct caaacggtaa
aacagatcgg tacgaaaacg 480 tccttgcgtt atctcccgat ccagatcgca
atgcgtggcg ctgatcaccc ggacatctac 540 cgggatcggc tgatgcccgc
caacgcgggt gacggctttt tcctccagta cgcgtagaag 600 gcgggtttgt
aacggcagcg gcatttcgcc aatttcgtca agaaacagcg tgccgccgtg 660
ggcgacctca aacagccccg cacgtccacc tcgtcttgag ccggtaaacg ctccctcctc
720 atagccaaac agttcagcct ccagcaacga ctcggtaatc gcgccgcaat
taacggcgac 780 aaagggcgga gaaggcttgt tctgacggtg gggctgacgg
ttaaacaacg cctgatgaat 840 cgcttgcgcc gccagctctt tcccggtccc
tgtttccccc tgaatcagca ctgccgcgcg 900 ggaacgggca tagagtgtaa
tcgtatggcg aacctgctcc atttgtggtg aatcgccgag 960 gatatcgctc
agcgcataac gggtctgtaa tcccttgctg gaggtatgct ggctatactg 1020
acgccgtgtc aggcgggtca tatccagcgc atcatggaaa gcctgacgta cggtggccgc
1080 tgaataaata aagatggcgg tcattcctgc ctcttccgcc aggtcggtaa
ttagtcctgc 1140 cccaattaca gcctcaatgc cgttagcttt gagctcgtta
atttgcccgc gagcatcctc 1200 ttcagtgata tagcttcgct gttcaagacg
gaggtgaaac gttttctgaa aggcgaccag 1260 agccggaatg gtctcctgat
aggtcacgat tcccattgag gaagtcagct ttcccgcttt 1320 tgccagagcc
tgtaatacat cgaatccgct gggtttgatg aggatgacag gtaccgacag 1380
tcggcttttt aaataagcgc cgttggaacc tgccgcgata atcgcgtcgc agcgttcggt
1440 tgccagtttt ttgcgaatgt aggctactgc cttttcaaaa ccgagctgaa
taggcgtgat 1500 cgtcgccaga tgatcaaact ccaggctgat atcccgaaat
agttcgaaca ggcgcgttac 1560 cgagaccgtc cagatcaccg gtttatcgct
attatcgcgc gaagcgctat gcacagtaac 1620 catcgtcgta gattcatgtt
taaggaacga attcttgttt tatagatgtt tcgttaatgt 1680 tgcaatgaaa
cacaggcctc cgtttcatga aacgttagct gactcgtttt tcttgtgact 1740
cgtctgtcag tattaaaaaa gatttttcat ttaactgatt gtttttaaat tgaattttat
1800 ttaatggttt ctcggttttt gggtctggca tatcccttgc tttaatgagt
gcatcttaat 1860 taacaattca ataacaagag ggctgaatag taatttcaac
aaaataacga gcattcgaat 1920 g 1921 <210> SEQ ID NO 107
<211> LENGTH: 864 <212> TYPE: DNA <213> ORGANISM:
Salmonella enterica subsp. enterica serovar Typhimurium LT2
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(864) <223> OTHER INFORMATION: Tsx -
Salmonella enterica subsp. enterica serovar Typhimurium LT2
(STM0413) <400> SEQUENCE: 107 atgaaaaaaa ctttactcgc
agtcagcgca gcgctggcgc tcacctcatc ttttactgct 60 aacgcagcag
aaaatgatca gccgcagtat ttgtccgact ggtggcacca gagcgtaaac 120
gtggtaggca gctaccatac ccgtttctcg ccgaaattga acaacgacgt ctatctggaa
180 tatgaagcat ttgccaaaaa agactggttt gatttctacg gctatatcga
tattcccaaa 240 acctttgatt ggggtaacgg caacgataaa ggtatctggt
ccgacggttc tccgctgttc 300 atggaaatcg aaccgcgttt ctcaattgat
aagctgaccg gcgcagacct gagcttcggc 360 ccgtttaaag agtggtattt
cgccaacaac tacatctacg atatgggcga taacaaagcc 420 agccgccaga
gcacgtggta tatgggtctg gggaccgata tcgacaccgg cctgccgatg 480
ggtctgtcgc tgaacgtgta tgcgaaatat cagtggcaaa actacggcgc gtccaatgaa
540 aacgaatggg acggctaccg tttcaaagtg aaatacttcg tccccatcac
cgatctgtgg 600 ggcggtaaac tgagctatat cggctttacc aactttgact
ggggatctga tttaggcgac 660 gatccgaacc gtaccagcaa ctccatcgct
tccagccata tcctggcgct gaactacgat 720 cactggcact actcggtcgt
tgcgcgttac ttccataacg gcggacagtg gcagaatggc 780 gcaaaactga
actggggcga cggcgatttc agcgcgaaat ctaccggctg gggcggctac 840
ctggtcgtgg gttacaactt ctaa 864 <210> SEQ ID NO 108
<211> LENGTH: 885 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(885) <223> OTHER
INFORMATION: Tsx - Escherichia coli K-12 MG1655 (b0411) <400>
SEQUENCE: 108 atgaaaaaaa cattactggc agccggtgcg gtactggcgc
tctcttcgtc ttttactgtc 60 aacgcagctg aaaacgacaa accgcagtat
ctttccgact ggtggcacca gagcgttaac 120 gttgtcggaa gctatcacac
ccgtttcgga ccgcagatcc gcaacgatac ctaccttgag 180 tacgaagcat
tcgctaaaaa agactggttc gacttctatg gttatgcgga tgcgccggta 240
ttcttcggcg gtaactccga tgctaaaggt atctggaacc acggttctcc gctgtttatg
300 gaaatcgaac cacgtttctc catcgacaag ctgaccaata ctgaccttag
cttcggtccg 360 ttcaaagagt ggtacttcgc gaacaactac atttacgaca
tgggtcgtaa taaagatggt 420 cgccagagca cctggtacat gggtctgggt
accgatatcg acactggcct gccgatgagc 480 ctgtccatga acgtctatgc
gaaataccag tggcagaact atggcgcagc gaacgaaaac 540 gagtgggacg
gttaccgttt caaaattaaa tactttgtgc cgattaccga tctgtggggc 600
ggtcagctga gctacatcgg cttcaccaac ttcgactggg gttccgattt aggggatgac
660 agcggtaacg caatcaacgg tattaagacc cgtactaata actctatcgc
ttccagccat 720 attctggctc tgaactacga tcactggcac tactctgtcg
tagctcgtta ctggcacgac 780 ggtggtcagt ggaacgacga tgcagaactg
aacttcggca acggcaactt caacgttcgc 840 tctaccggct ggggtggtta
cctggtagta ggttacaact tctga 885 <210> SEQ ID NO 109
<211> LENGTH: 1221 <212> TYPE: DNA <213>
ORGANISM: Bacillus halodurans <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1221)
<223> OTHER INFORMATION: BH1446 - Bacillus halodurans
(BAB05165) <400> SEQUENCE: 109 atgaatattt tgtggggttt
attaggaatc gtcgttgttt ttctaatcgc ttttgcattt 60 tccacaaatc
gtcgtgcaat taaaccacga acgatattag gtggtctcgc gattcagcta 120
ttatttgcga ttattgtatt aaaaattcca gctggacaag cgttacttga gagcttaacc
180 aatgtagttt tgaacattat tagttatgcg aatgaaggga tcgacttcgt
atttggtgga 240 tttttcgaag aaggttcagg cgtaggcttc gtttttgcaa
ttaacgtttt gtctgtcgtc 300 attttcttct cagcactaat ctcgatcctt
tattatttag ggatcatgca atttgtcatt 360 aaaattatcg gtggtgcgct
gtcctggcta ctcggaacat caaaggcaga atcaatgtca 420 gcagcagcta
acattttcgt tgggcaaacg gaagcgccac tcgttgttaa gccatactta 480
ccaaaaatga cgcaatccga gctctttgcg gttatgaccg ggggacttgc ttctgttgct
540 ggttctgttt taatcggtta ttctctttta ggagtaccgc tacaatattt
attagcggca 600 agctttatgg ctgctcctgc gggcttgatt atggcgaaaa
tgatcatgcc tgaaacggag 660 aaaacaaccg atgcagaaga tgactttaag
ctcgcaaagg atgaagagtc cacgaacttg 720 attgacgcgg ccgccaatgg
ggcgagcact gggttaatgc tcgttctaaa tattgcggcg 780 atgttactag
cgttcgttgc attgattgca ttaattaatg gaattcttgg atggatcgga 840
ggattgtttg gggcgtcgca attgtcttta gagttaatcc tcggatacgt gtttgctccg
900 cttgcgtttg tcatcggaat tccttgggct gaagcgcttc aagcgggaag
ctacatcgga 960 cagaaactcg tagtgaacga atttgttgcc tacttaagct
ttgcaccaga aattgaaaac 1020 ctttcagata aagcggtgat ggtgattagt
tttgcccttt gcggatttgc taacttctca 1080 tccctcggaa tccttttagg
aggattgggt aagcttgctc cgagccgtcg ccctgatatt 1140 gcccgtctcg
gattacgcgc gatccttgca ggtacgctag cttctttact cagcgcctcc 1200
attgcgggaa tgttattcta a 1221 <210> SEQ ID NO 110 <211>
LENGTH: 1182 <212> TYPE: DNA <213> ORGANISM: Bacillus
subtilis <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1182) <223> OTHER INFORMATION:
nupC Bacillus subtilis subsp. subtilis 168 (BSU39410; CAA57663)
<400> SEQUENCE: 110 atgaagtatt tgattgggat tatcggttta
atcgtgtttt taggcctcgc gtggatcgcg 60 agcagcggca aaaaaagaat
taagatccgc ccaattgttg ttatgctcat tttgcaattt 120 attcttggct
acattctcct caataccgga atagggaatt tcctcgtggg aggatttgca 180
aaaggattcg gttacctgct tgaatacgcg gcagagggaa ttaactttgt gtttggcggc
240 ttggtgaatg cggaccaaac gacattcttt atgaatgttc tcttgccaat
cgtgtttatt 300 tccgctctga tcgggattct gcaaaagtgg aaagtcctcc
cgtttatcat tagatatatc 360 ggccttgccc tcagcaaggt aaacggtatg
ggaagattgg aatcgtataa cgcagtggct 420 tctgcgattt tagggcagtc
agaagtattt atctccttga agaaagaact cggtctttta 480 aatcagcagc
gcttgtacac gctttgcgca tctgcgatgt caactgtatc aatgtcgatt 540
gtcggtgcgt atatgacaat gctgaaaccg gaatatgttg taacagcgct tgttttgaac
600 ttatttggcg gtttcattat cgcttctatt atcaatccgt acgaggttgc
aaaagaagag 660 gatatgcttc gtgttgagga agaagaaaaa caatccttct
tcgaagtgct cggagaatac 720 attcttgacg gtttcaaagt agcggttgtc
gtcgctgcga tgctgattgg atttgtcgcg 780 attattgcat tgatcaatgg
catttttaat gcagtattcg gtatttcgtt ccaaggcatt 840 cttggatatg
tgtttgctcc attcgctttt cttgtcggta tcccatggaa tgaagctgtt 900
aatgcgggaa gcattatggc aacaaaaatg gtatcgaatg aatttgtcgc catgacgtcg
960 cttacgcaaa acggtttcca tttcagcggc cgtacaacag cgatcgtatc
ggtattcctt 1020 gtgtcatttg cgaacttctc ctcaatcgga atcattgccg
gtgccgtaaa aggactgaat 1080 gaaaagcaag gaaatgtcgt cgctcgtttc
ggcttgaaat tattatacgg tgctacgctt 1140 gtcagctttt tatcagcagc
aattgtgggc ttgatttact ga 1182 <210> SEQ ID NO 111 <211>
LENGTH: 1215 <212> TYPE: DNA <213> ORGANISM: Bacillus
subtilis <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1215) <223> OTHER INFORMATION:
yutK - Bacillus subtilis subsp. subtilis 168: BSU32180 <400>
SEQUENCE: 111 atgaatgttc tgtgggggct gctgggcgca gttgcgatca
ttgctatcgc gtttttattt 60 tcagaaaaga aaagcaatat taagataaga
accgtcatcg ttggtttatg cacacaggtg 120 gcgtttggat acatcgtgtt
gaaatgggaa gcgggacgcg ctgttttttt atggttttca 180 agccgtgtac
agcttctgat tgactatgcg aatgaaggca tcagttttat ttttggaccg 240
cttctaaagg tcggagacag tccggcattt gcattaagtg tactgcccgt tatcattttc
300 ttctcagcac tgattgcagt tttatatcat ttgaaaatca tgcagctcgt
tttccgtgtc 360 attggcggcg gattgtcgaa gctccttgga acaagcaaaa
cggaatctct ggcggctgct 420 gccaatattt ttgtaggaca atcagaatct
ccgttagtga tcaaacccct gattgccggg 480 ctgacgcgct ctgagttgtt
tacgattatg acgagcggtc tatcggcagt tgcgggatct 540 accttgtttg
ggtacgcgct tctcggtatt ccgattgagt acttgctggc ggccagcttt 600
atggctgctc cagctggact agtctttggt aaattgatta tacccgaaac ggaaaaaacg
660 caaaccgtaa aaagcgattt caaaatggat gaaggcgaag gcgcagccaa
tgtcattgac 720 gcagctgcaa agggagcgtc aacaggactg caaattgcgt
taaatgttgg ggcgatgctg 780 cttgcgtttg ttgcgttaat cgctgtagta
aacggtattc tcggcggggc tttcggcttg 840 ttcggtttaa aaggcgtaac
attagaatcc attctcggct atgtgttttc tcctatcgcc 900 tttttgattg
gcgtgccttg gcatgaagca ttgcaggcgg gaagctatat cggccagaaa 960
ttggtgctga atgagtttgt cgcttattct aacttcggtt cgcacatcgg cgagttttct
1020 aagaaaactg ctaccattat cagtttcgcg ttatgcggat tcgccaattt
ttcatcaatt 1080 gcgattatgc ttggtacgct tggcggttta gcgcccagcc
gccgttcaga tatcgcacgt 1140 ctcggcctga aggctgttct tgcaggaaca
ttagccaatc tgctcagcgc agccattgcc 1200 ggcatgttta tataa 1215
<210> SEQ ID NO 112 <211> LENGTH: 1194 <212>
TYPE: DNA <213> ORGANISM: Bacillus subtilis <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1194) <223> OTHER INFORMATION: yxjA - Bacillus subtilis
subsp. spizizenii W23 (BSUW23_19355) <400> SEQUENCE: 112
atgtactttt tattaaacct tgtcggtctc attgtgatta tggcagttgt gttcctatgc
60 tccccgcaga aaaagaaaat ccagtggcgt ccgatcatta cgttaattgt
tctggaattg 120 ctgattactt ggtttatgct gggaacaaag gtcgggagct
gggccatcgg taaaattggt 180 gatttcttca cttggctgat tgcttgcgcc
agtgacggta tcgcgtttgc cttcccgtca 240 gtcatggcga atgaaacagt
agactttttc tttagtgcac ttcttccaat tatctttatc 300 gtcacattct
ttgatatttt aacatatttc ggcattttgc cttggctgat tgataaaatc 360
ggatgggtga tttcaaaggc ttcccgcttg ccgaaattag aaagcttttt ctctattcaa
420 atgatgttct tgggaaatac tgaagcactt gcggtcatcc gccagcagct
tacggtatta 480 aataacaacc gcttgcttac atttggctta atgagcatga
gcagcatcag cggctccatt 540 attggatctt acctgtcaat ggtgccggcg
acatacgtgt ttacagcgat tccattgaac 600 tgcttaaacg cgctgattat
tgcaaacctg ctgaaccctg ttcatgtgcc ggaggatgaa 660 gatatcatct
atacaccgcc taaagaagag aagaaagact ttttctctac gatttctaac 720
agtatgcttg tcggcatgaa catggttatc gttattttgg caatggtgat cggatatgta
780 gcattaacgt ctgcagtcaa tggcattctt ggtgttttcg tacacggcct
gaccatccag 840 acaatttttg cttatctctt cagtccgttc gcattcctgc
ttggtctgcc agtacatgat 900 gcaatgtatg tcgctcagct aatgggaatg
aaattggcaa cgaacgagtt tgttgcgatg 960 cttgacttga aaaacaatct
tacaacactt ccgcctcaca cagttgcggt ggcgacgaca 1020 ttcctgacgt
catttgccaa cttcagtact gtcggcatga tttacggaac gtacaactcg 1080
atccttgacg gcgaaaagtc aacggtcatc gggaaaaacg tgtggaaatt gctcgtcagc
1140 ggcattgcgg tatctttact aagtgctgcg attgtcggcc tgtttgtgtg gtag
1194 <210> SEQ ID NO 113 <211> LENGTH: 1281 <212>
TYPE: DNA <213> ORGANISM: Caulobacter crescentus CB15
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1281) <223> OTHER INFORMATION: ccCNT (CC2089)
- Caulobacter crescentus CB15 (AAK24060) <400> SEQUENCE: 113
atgttccgtc ccgagaacgt tcaggccctc gcgggtctgg cgctcaccct gggcctgtgc
60 tggctcgttt ccgagaatcg caagcggttc ccctggggcc tggccatcgg
cgcggtcgtc 120 attcaggtcc tgctggtcct ggtcctgttc ggcctgccgc
aagcccagca gatgctgcgc 180 ggcgtcaacg gcgcggtgga gggccttgcc
gcctcgaccc aggccggcac cgccttcgtg 240 ttcggctttc tggccggcgg
cgaccagccc tatccggtca gcaatccggg cgcgggcttc 300 atcttcgcct
tccgcgtgct gccggtgatc ctggtggtct gcgccctgtc ggcgctgctg 360
tggcactgga agattctcaa gtggctggct cagggcttcg gctttgtgtt ccagaagacg
420 ctgggcctgc gcggcccgcc ggccctggcc accgccgcga ccatcttcat
gggtcaggtc 480 gaggggccga tcttcatccg cgcctatctc gacaagctga
gccgctcgga actcttcatg 540 ctgatcgcgg tcggcatggc ctgcgtgtcg
ggctcgacca tggtcgccta cgccaccatc 600 ctggccgacg tcctgcccaa
cgccgccgcc cacgtgctga ccgcctcgat catctcggct 660 ccggccggcg
tgctgctggc ccggatcatt gtgccgtccg atccgatgga gaagagcgcc 720
gatcttgatc tgtcgaccga ggacaagacc tatggcagct cgatcgacgc cgtgatgaag
780 ggcaccaccg acggcctgca gatcgcgctg aacgtcggcg ccaccctgat
cgtcttcgtg 840 gccctggcca ccatggtcga caaggtcctg ggcgccttcc
cgccggtggg cggcgagccg 900 ctgagcatcg cgcgcggcct gggcgtggtc
ttcgcgccgc tggcctggtc gatgggcatc 960 ccgtggaaag aagcgggcac
ggccggcggt ctgctgggcg tgaagctgat cctgaccgag 1020 ttcaccgcct
tcatccagct gtccaaggtg ggcgaagccc tgctggacga acgcacccgg 1080
atgatcatga cctacgctct gtgcggtttc gccaatatcg gctcggtcgg catgaacgtc
1140 gccggcttct cggtgctggt gccccagcgc cggcaggaag tgctgggcct
ggtctggaag 1200 gcgatgatgg ccggcttcct ggccacctgc ctgaccgcct
cgctggtcgg cctgatgccg 1260 cgaagcctgt ttgggctgta a 1281 <210>
SEQ ID NO 114 <211> LENGTH: 1251 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1251) <223> OTHER INFORMATION: yeiJ - Escherichia coli
K-12 W3110 (AAC75222; JW2148) <400> SEQUENCE: 114 atggatgtca
tgagaagtgt tctgggaatg gtggtattgc tgacgattgc gtttttactg 60
tcagtaaaca agaagaagat cagcctgcgt accgttggcg cggcgttagt gttacaggtc
120 gtgattggcg gcattatgct ttggttaccg ccagggcgtt gggtcgctga
aaaagtcgct 180 tttggcgtgc ataaagtgat ggcgtacagc gacgcgggta
gcgcatttat cttcggttct 240 ctggtcggac cgaaaatgga taccttattt
gatggtgcag gatttatctt tggtttcagg 300 gtgttaccgg caattatctt
cgtcaccgcg ctggtgagta ttctctacta catcggtgtg 360 atggggattt
taattcgaat tctcggcggt atcttccaga aagcattaaa tatcagcaag 420
atcgagtcat tcgtcgcggt caccaccatt ttcctcgggc aaaacgaaat tccggcaatc
480 gtcaaaccct ttatcgatcg tctgaatcgc aatgaattat ttacagcgat
ttgtagtggc 540 atggcctcga ttgctggttc gacaatgatt ggttacgccg
cactgggcgt gcctgtggaa 600 tatctgctgg cggcatcatt aatggcgatc
cctggcggga tcttgtttgc ccgcctgtta 660 agcccggcaa cggaatcttc
gcaggtttcc tttaataacc tctctttcac cgaaacaccg 720 ccaaaaagca
ttattgaagc cgctgcgaca ggggcaatga ccgggctgaa aatcgccgca 780
ggtgtggcaa cagtggtgat ggcatttgtt gcaataattg cgttgattaa cggtattatc
840 ggcggcgttg gtggctggtt tggttttgaa catgcctcgc tggagtccat
tttaggttac 900 ctgctggctc cactggcgtg ggtgatgggt gtggactgga
gtgatgcgaa tcttgccggg 960 agtttgattg gacagaaact ggcaataaat
gaatttgtcg cttatctcaa tttctcaccc 1020 tatctgcaaa cggctggcac
tctcgatgct aaaactgtgg cgattatttc cttcgcgttg 1080 tgcggtttcg
ctaactttgg ttctatcggg gtggtggtgg gggcgttttc tgcggttgcg 1140
ccacaccgtg cgccggaaat cgcccagctt ggtttacggg cgctggcggc ggcgacgctt
1200 tccaacttga tgagtgcgac cattgccggg ttctttattg gtttagcttg a 1251
<210> SEQ ID NO 115 <211> LENGTH: 1251 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1251) <223> OTHER INFORMATION: yeiM - Escherichia coli
K-12 W3110 (AAC75225; JW2151) <400> SEQUENCE: 115 atggatataa
tgagaagtgt tgtggggatg gtggtgttac tggcaatagc atttctgttg 60
tcagtgaata aaaagagcat cagtttgcgc acggttggag ccgcactgct gctgcaaatc
120 gctattggtg gcatcatgct ctacttccca ccgggaaaat gggcagtaga
acaggcggca 180 ttaggcgttc ataaagtgat gtcttacagt gatgccggta
gcgccttcat ttttggttcg 240 ctggttgggc cgaaaatgga tgtcctgttt
gacggtgcgg gttttatctt cgcctttcgc 300 gtacttccgg cgattatttt
cgttactgcg ctcatcagtc tgctgtacta cattggcgtg 360 atggggctgc
tgattcgcat ccttggcagc attttccaga aagccctcaa catcagcaaa 420
atcgaatctt ttgttgcggt tactactatt ttcctcgggc aaaatgagat cccggcgatc
480 gttaaaccgt ttatcgatcg catgaatcgc aacgagttgt ttaccgcaat
ttgtagcggg 540 atggcgtcca ttgctggttc gatgatgatt ggttatgccg
gaatgggcgt accaattgac 600 tacctgttag cggcatcgct gatggcgatc
cctggcggga ttttgtttgc acgtattctt 660 agcccggcaa ccgagccttc
gcaggtcaca tttgaaaatc tgtcgttcag cgaaacgccg 720 ccaaaaagct
ttatcgaagc ggcggcgagc ggtgcgatga ccgggctaaa aatcgccgct 780
ggtgtggcga cggtggtaat ggcgtttgtc gcaattattg cgctgatcaa cggcattatc
840 ggcggaattg gcggctggtt tggtttcgcc aatgcctctc tggaaagtat
ttttggctat 900 gtgctggcac cgctggcgtg gatcatgggt gtggactgga
gtgatgccaa tcttgcgggt 960 agcctgattg ggcagaaact ggcgattaac
gaattcgtcg cttacctgag tttctcccca 1020 tacctgcaaa cgggcggcac
gctggaagtg aaaaccattg cgattatctc ctttgcgctt 1080 tgtggttttg
ctaactttgg ttctatcggt gttgtcgttg gcgcattttc ggctatttcg 1140
ccaaaacgcg cgccggaaat cgcccagctt ggtttacggg cgctggcagc agcaacgctt
1200 tccaacctga tgagtgcgac tattgccggg ttctttattg gtctggcgta a 1251
<210> SEQ ID NO 116 <211> LENGTH: 1254 <212>
TYPE: DNA <213> ORGANISM: Haemophilus influenzae <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1254) <223> OTHER INFORMATION: HI0519 - Haemophilus
influenzae Rd KW20 serotype d(AAC22177) <400> SEQUENCE: 116
atgagtgtgt taagcagcat tttgggaatg gtcgtattaa tcgctattgc cgtgttactt
60 tctaataatc gtaaagcgat tagtattcga accgtagtag gggcgttagc
aatccaagta 120 ggatttgccg cccttatttt atatgtgcca gcaggtaaac
aagcgttggg tgccgctgcg 180 gatatggtat ccaatgttat tgcctatggt
aatgacggga ttaatttcgt tttcggcgga 240 ttggcagatc caagtaaacc
atccggtttc atttttgcag tgaaagtatt accgattatc 300 gtgttcttct
ctggcttaat ttctgtgctt tactatctcg gcattatgca agtcgtgatt 360
aaagtattag gtggcgcatt acaaaaagca ttgggtacgt caaaagcgga atcaatgtca
420 gcggcggcga atatcttcgt cggtcaaact gaagcaccat tagttgttcg
cccttacatt 480 aaaaatatga cccaatctga attatttgcc attatggtgg
gtggtacagc gtctatcgcg 540 ggttcagtaa tggcaggtta tgctggaatg
ggcgtgccat tgacatactt aatcgctgcg 600 tcatttatgg cggcaccagc
aggtttatta tttgcgaaat taatgttccc acaaaccgaa 660 caattcacag
ataaacaacc agaagacaat gattcagaaa aaccaactaa cgtacttgaa 720
gcaatggcgg gcggtgcgag tgcaggtatg caacttgcgt taaacgtagg tgcaatgtta
780 atcgcattcg ttggtttaat tgcattaatt aatggtattt taagtggcgt
aggcggatgg 840 ttcggctatg gcgacttaac cttacaatct atctttggtt
taatttttaa accattagca 900 tacttaatcg gtgtaactga tggtgctgaa
gcaggtattg caggacaaat gatcgggatg 960 aaattagcgg ttaatgaatt
tgtgggttat cttgaatttg caaaatattt acaaccagat 1020 tctgcaattg
tattaactga aaaaaccaaa gcgattatta ctttcgcact ttgtggtttt 1080
gctaacttca gctcaattgc aatcttaatt ggtggtttag gtggtatggc accaagccgt
1140 cgtagtgatg ttgctcgttt aggtatcaaa gccgttatcg ctggtactct
cgctaactta 1200 atgagtgcaa ctattgctgg tttatttatc ggcttaggtg
ctgcagcact ttaa 1254 <210> SEQ ID NO 117 <211> LENGTH:
1257 <212> TYPE: DNA <213> ORGANISM: Helicobacter
pylori 26695 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1257) <223> OTHER
INFORMATION: nupC (HP1180) - Helicobacter pylori 26695 (AAD08224)
<400> SEQUENCE: 117 atgattttta gctctctttt tagtgttgta
gggatggcgg tgctttttct tattgcttgg 60 gtgttttcta gcaataaaag
ggctattaat tatcgcacga ttgtcagtgc ctttgtgatt 120 caagtggctt
taggggcgtt ggctttatat gtgcctttgg gtagggaaat gctgcaaggc 180
ttagccagcg gcatacaaag cgtgatttct tacggctatg agggggtgcg ttttttattt
240 ggcaatctcg ctccaaacgc taagggcgat caagggatag gggggtttgt
ctttgcgatc 300 aatgttttag cgatcattat cttttttgct agcttgattt
cacttctata ttatttaaaa 360 atcatgcctt tatttatcaa tctcatcggt
ggggcgttgc aaaaatgctt aggcacttct 420 agagcagaaa gcatgagtgc
agcggctaat atttttgtag cgcacaccga agcgccctta 480 gtcattaaac
cttatttgaa aagcatgagc gattcagaga tttttgcggt catgtgcgtg 540
ggcatggcta gcgttgcggg gcctgtgtta gccgggtatg cgagcatggg cattcctttg
600 ccttatttga tcgccgcttc gtttatgtcc gctcctgggg ggttgttgtt
cgctaaaatc 660 atttacccac aaaacgaaac catttctagc catgcagatg
tttctataga aaagcatgtc 720 aatgccatag aagctatcgc taatggggca
agcacagggc taaatttagc cttgcatgtg 780 ggagcgatgc ttttagcctt
tgtggggatg ctcgcgctca ttaacgggct tttaggggtt 840 gtagggggtt
ttttaggcat ggagcatttg tctttagggt tgattttagg cacgctctta 900
aaacccttag cctttatgtt aggcattcct tggagccagg ccgggattgc cggagaaatc
960 ataggcatta aaatcgcgct caatgaattt gtgggctata tgcagttatt
gccttatttg 1020 ggcgataacc ctcctttaat cttgagcgag aaaactaaag
cgatcatcac ttttgcgttg 1080 tgcgggtttg ctaatttaag ctcagtcgct
atgctcattg gagggcttgg cagtttagtg 1140 cctaaaaaga aggatctcat
tgtaaggctt gctttaaaag cggtgcttgt aggcacgctt 1200 tctaatttca
tgagcgcgac tatcgccggg ttattcatag ggctaaacgc tcattaa 1257
<210> SEQ ID NO 118 <211> LENGTH: 1230 <212>
TYPE: DNA <213> ORGANISM: Staphylococcus aureus <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1230) <223> OTHER INFORMATION: nupC (SA0600) -
Staphylococcus aureus subsp. aureus N315 (BAB41833) <400>
SEQUENCE: 118 atgtttttat taatcaacat tattggtcta attgtatttc
ttggtattgc ggtattattt 60 tcaagagatc gcaaaaatat ccaatggcaa
tcaattggga tcttagttgt tttaaacctg 120 tttttagcat ggttctttat
ttattttgat tggggtcaaa aagcagtaag aggagcagcc 180 aatggtatcg
cttgggtagt tcagtcagcg catgctggta caggttttgc atttgcaagt 240
ttgacaaatg ttaaaatgat ggatatggct gttgcagcct tattcccaat attattaata
300 gtgccattat ttgatatctt aatgtacttt aatattttac cgaaaattat
tggaggtatt 360 ggttggttac tagctaaagt aacaagacaa cctaaattcg
agtcattctt tgggatagaa 420 atgatgttct taggaaatac tgaagcatta
gccgtatcaa gtgagcaact aaaacgtatg 480 aatgaaatgc gtgtattaac
aatcgcaatg atgtcaatga gctctgtatc cggagctatt 540 gtaggtgcgt
atgtacaaat ggtaccagga gaactggtac taacggcaat tccactaaat 600
atcgttaacg cgattattgt gtcatgcttg ttgaatccag taagtgttga agagaaagaa
660 gatattattt acagtcttaa aaacaatgaa gttgaacgtc aaccattctt
ctcattcctt 720 ggagattctg tattagcagc aggtaaatta gtattaatca
tcatcgcatt tgttattagt 780 tttgtagcgt tagctgatct atttgatcgt
tttatcaatt tgattacagg attgatagca 840 ggatggatag gcataaaagg
tagtttcggt ttaaaccaaa ttttaggtgt gtttatgtat 900 ccatttgcgc
tattactcgg tttaccttat gatgaagcgt ggttggtagc acaacaaatg 960
gctaagaaaa ttgttacaaa tgaatttgtt gttatgggtg aaatttctaa agatattgca
1020 tcttatacac cacaccatcg tgcggttatt acaacattct taatttcatt
tgcaaacttc 1080 tcaacgattg gtatgattat cggtacattg aaaggcattg
ttgataaaaa gacatcagac 1140 tttgtatcta aatatgtacc tatgatgcta
ttatcaggta tcctagtttc attattaaca 1200 gcagctttcg ttggtttatt
tgcatggtaa 1230 <210> SEQ ID NO 119 <211> LENGTH: 1230
<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1230) <223> OTHER INFORMATION: nupC (SAV0645)
-Staphylococcus aureus subsp. aureus Mu50 (BAB56807) <400>
SEQUENCE: 119 atgtttttat taatcaacat tattggtcta attgtatttc
ttggtattgc ggtattattt 60 tcaagagatc gcaaaaatat ccaatggcaa
tcaattggga tcttagttgt tttaaacctg 120 tttttagcat ggttctttat
ttattttgat tggggtcaaa aagcagtaag aggagcagcc 180 aatggtatcg
cttgggtagt tcagtcagcg catgctggta caggttttgc atttgcaagt 240
ttgacaaatg ttaaaatgat ggatatggct gttgcagcct tattcccaat attattaata
300 gtgccattat ttgatatctt aatgtacttt aatattttac cgaaaattat
tggaggtatt 360 ggttggttac tagctaaagt aacaagacaa cctaaattcg
agtcattctt tgggatagaa 420 atgatgttct taggaaatac tgaagcatta
gccgtatcaa gtgagcaact aaaacgtatg 480 aatgaaatgc gtgtattaac
aatcgcaatg atgtcaatga gctctgtatc cggagctatt 540 gtaggtgcgt
atgtacaaat ggtaccagga gaactggtac taacggcaat tccactaaat 600
atcgttaacg cgattattgt gtcatgcttg ttgaatccag taagtgttga agagaaagaa
660 gatattattt acagtcttaa aaacaatgaa gttgaacgtc aaccattctt
ctcattcctt 720 ggagattctg tattagcagc aggtaaatta gtattaatca
tcatcgcatt tgttattagt 780 tttgtagcgt tagctgatct atttgatcgt
tttatcaatt tgattacagg attgatagca 840 ggatggatag gcataaaagg
tagtttcggt ttaaaccaaa ttttaggtgt gtttatgtat 900 ccatttgcgc
tattactcgg tttaccttat gatgaagcgt ggttggtagc acaacaaatg 960
gctaagaaaa ttgttacaaa tgaatttgtt gttatgggtg aaatttctaa agatattgca
1020 tcttatacac cacaccatcg tgcggttatt acaacattct taatttcatt
tgcaaacttc 1080 tcaacgattg gtatgattat cggtacattg aaaggcattg
ttgataaaaa gacatcagac 1140 tttgtatcta aatatgtacc tatgatgcta
ttatcaggta tcctagtttc attattaaca 1200 gcagctttcg ttggtttatt
tgcatggtaa 1230 <210> SEQ ID NO 120 <211> LENGTH: 1203
<212> TYPE: DNA <213> ORGANISM: Streptococcus pyogenes
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1203) <223> OTHER INFORMATION: nupC (SpNupC)
- Streptococcus pyogenes SF370 serotype M1 (AAK34582) <400>
SEQUENCE: 120 atgcaattta tttatagtat tattggtatt ttattggtat
taggaattgt gtatgcaatt 60 tctttcaatc gtaagagtgt ttctctaagt
ttaattggaa aagctcttat cgttcaattc 120 attattgcgc taatcttagt
acgtatccca ctaggccaac aaattgttag tgttgtttca 180 actggagtta
ctagcgtaat caactgtggt caagctggtt taaattttgt gtttgggtca 240
ttagcagata gtggcgcaaa aactggtttt attttcgcta ttcaaacgct tggtaatatt
300 gttttcttat ctgccctagt tagtctactt tattatgtag gaatccttgg
atttgtagta 360 aaatggatag gtaagggcgt tggtaaaatt atgaaatcct
cagaggttga gagttttgtt 420 gctgtagcta atatgtttct tggtcaaaca
gacagtccaa tcttggttag caaataccta 480 ggtcgtatga ctgatagtga
gataatggtt gtgttggtat caggtatggg aagtatgtca 540 gtttctattc
ttggtggcta tattgcatta ggcattccaa tggaatatct cttgattgct 600
tcaacaatgg ttcctattgg cagtattctc attgctaaaa tcttattgcc tcaaacagaa
660 cctgttcaaa aaattgatga cattaagatg gataataaag gtaataacgc
caatgtgatt 720 gatgcaatcg ctgagggtgc aagcacaggt gcacaaatgg
ctttctcaat tggtgctagt 780 ttgattgcct ttgttggttt agtttctttg
attaatatga tgttaagtgg attgggaatc 840 cgcttagaac aaatcttttc
atatgttttt gctccatttg gttttcttat gggatttgac 900 cacaaaaaca
ttcttctaga aggaaacctt cttggaagta agttgatttt aaatgagttt 960
gtttcgttcc aacaattggg tcacctaatc aaatctttag attatcgtac agcattggta
1020 gcaactattt cactctgtgg ttttgctaat ttatcaagtt taggtatttg
tgtttcaggt 1080 attgctgttc tttgcccgga gaaacgtagc accctagctc
gacttgtttt ccgtgcaatg 1140 attggtggta ttgctgtaag tatgcttagc
gcctttatcg tcggtattgt aactctattc 1200 taa 1203 <210> SEQ ID
NO 121 <211> LENGTH: 1257 <212> TYPE: DNA <213>
ORGANISM: Vibrio cholerae O1 biovar El Tor N16961 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1257) <223> OTHER INFORMATION: nupC (VC2352) - Vibrio
cholerae O1 biovar El Tor N16961 (AAF95495) <400> SEQUENCE:
121 atgagcctgt ttatgagcct catcggcatg gcagttctgc taggaatcgc
agttctactg 60 tcaagtaacc gtaaagctat caatctaaga actgtgggtg
gcgcttttgc tatccaattt 120 tcactgggtg catttattct gtatgtgcct
tggggccaag agctacttcg tggcttttcg 180 gatgccgtat cgaatgttat
taactacggt aacgatggta cttcattcct cttcggtgga 240 ctggtatcag
gcaaaatgtt tgaagtgttt ggcggcggcg gtttcatttt cgcattccgc 300
gtactaccaa cactgatctt cttctcagca ctgatttctg tactgtacta cttgggtgtt
360 atgcaatggg ttatccgcat tcttggcggt ggtctgcaaa aagcactggg
tacatcacgc 420 gcggaatcta tgtctgcggc tgcaaacatt ttcgtgggtc
aaactgaagc accattagtt 480 gttcgtccat tcgttccaaa aatgactcaa
tctgagctgt ttgcggtaat gtgtggtggc 540 ttggcttcta tcgcaggtgg
tgtacttgcg ggttacgctt caatgggcgt taagatcgaa 600 tacttggtag
cggcgtcatt catggcggca ccgggtggtc tgctgttcgc aaaactgatg 660
atgcctgaaa ctgaaaaacc acaagacaat gaagacatta ctcttgatgg tggtgacgac
720 aaaccggcta acgttatcga tgcggctgct ggcggtgctt ctgctggtct
gcaacttgct 780 ctgaacgttg gtgcaatgtt gattgccttt atcggtttga
ttgctctgat caacggtatg 840 ttgggtggca tcggtggttg gttcggtatg
cctgaactga aactggaaat gctactgggc 900 tggttgtttg cgcctctggc
tttcctgatc ggtgttcctt ggaacgaagc aactgttgcg 960 ggtgagttca
tcggtctaaa aaccgttgct aacgaattcg ttgcttactc tcagtttgcg 1020
ccttacctga ctgaagcggc accagtggtt ctgtctgaga aaaccaaagc gatcatctct
1080 ttcgctctgt gtggttttgc gaacctttct tctatcgcaa ttctgcttgg
tggtttgggt 1140 agcttggcac ctaagcgtcg tggcgacatc gctcgtatgg
gggtcaaagc ggttatcgca 1200 ggtactctat ctaacctgat ggcagcgacc
atcgctggct tcttcctctc tttctaa 1257 <210> SEQ ID NO 122
<211> LENGTH: 1218 <212> TYPE: DNA <213>
ORGANISM: Vibrio cholerae O1 biovar El Tor N16961 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1218) <223> OTHER INFORMATION: nupC (VC1953) - Vibrio
cholerae O1 biovar El Tor N16961 (AAF95101) <400> SEQUENCE:
122 ttgggcggcg ttatgtcatc actcctcggt atgggcgcaa ttttgctggt
tgcgtggcta 60 ttttctacca atagaaaaaa tatcaacttg cgtacagttt
ctttagcgtt actgctgcaa 120 atcttcttcg ccttactggt gctgtatgta
cctgcgggta aagaggcact caatcgtgtg 180 acgggcgcgg tgtcacaact
gatcaactat gggcaagatg gtatcggttt tgtgtttggt 240 ggcctcgcca
atggcagcgt aggttttgtg tttgcgatta atgtccttgg catcatcatt 300
ttcttctctg cactgatttc tggcctttac catttaggca tcatgccgaa agtgattaac
360 ctcatcggtg gtggtttaca gaaattgctt ggcacaggcc gtgcagaatc
cctttctgct 420 accgcaaaca ttttcgtggg tatgattgaa gcgccgctgg
tggtgaaacc ttatcttcat 480 aaaatgaccg attcgcaatt ctttgcagtg
atgacgggcg gcttagcgtc ggttgctggc 540 ggtactttgg ttggttatgc
ctctttaggt gtggaattga actatctgat cgcggcggct 600 ttcatgtctg
cccctgcggg tcttttgatg gcaaaaatca tgttgccaga aaccgaacac 660
gtcgatgccg cgattgcgca agatgagttg gatctgccga aatccactaa cgtcgtcgaa
720 gcgattgcgg atggcgcgat gtcgggtgtg aaaattgctg ttgcggtagg
ggcgactttg 780 ctcgctttcg tgagtgtgat tgctctgtta aacggcttgc
tcggttggtt tggtggctgg 840 tttggcatcg agctaagctt tgaactgatc
atggggtatg ttttcgctcc ggtagcttgg 900 ctgattggta ttccatggca
tgaggcgatc acggcaggct cgctgattgg taacaaagtg 960 gtggtgaacg
agtttgtcgc tttcattcaa ctgattgaag tgaaagagca attgagtgcg 1020
cattcacaag cgatcgtgac tttcgcgctg tgcggttttg cgaatatttc taccatggcg
1080 attttgattg gtggtttggg tagccttgta cctgaacgtc gctcttttat
ctcccaatac 1140 ggcttccgtg cgattggcgc aggcgtatta gctaacctaa
tgagtgcatc gatcgctgga 1200 gtgattttgt ctttgtga 1218 <210> SEQ
ID NO 123 <211> LENGTH: 1209 <212> TYPE: DNA
<213> ORGANISM: Vibrio cholerae O1 biovar El Tor N16961
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1209) <223> OTHER INFORMATION: nupC (VCA0179)
- Vibrio cholerae O1 biovar El Tor N16961 (AAF96092) <400>
SEQUENCE: 123 atggcgattt tgtttggaat catcggtgtt acggtactga
tcttatgcgc gtatctgctc 60 tctgaaagcc gcagtgcgat taattggaaa
accatttccc gagccttgtt gttgcaaatt 120 ggttttgcgg ctcttgtgct
ttatttccca ttggggcaaa ccgcgctaag cagcttgagt 180 aatggggttt
ctggtttgct tggttttgcc gatgtcggca ttcgctttct gtttggtgat 240
cttgccgata cgggctttat ttttgctgtt cgtgtattac ctatcatcat cttcttcagt
300 gcgctgattt ctgcccttta ttaccttggt gtgatgcaaa aagtgatcgc
cctgatcggc 360 ggtggcattc aacgcttctt aggcaccagt aaggcggaat
cactggtcgc gacaggcaat 420 attttcctat cacaaggcga atcgccactt
ttggtgcgcc ccttccttgc caatatgaca 480 cgctctgaac tgtttgcggt
catggcgggc ggtatggcat cggtagcagg ctctgtgctg 540 ggtggttacg
caggtttagg ggttgagctg aaatacctga ttgcagcgag tttcatggcg 600
gcgccgggca gtttaatgat ggcgaaaatc atcgttcctg agcgtggtgt gccaatcgat
660 caaagccaag tcgagttaga taaagcgcaa gacagcaact tgattgatgc
tctcgctagc 720 ggtgcgatga atggtatgaa agtcgccgtt gcagtgggca
ctatgttgat tgcgttcgtc 780 agcgtgatcg ctatggtcaa cactggcctt
gaaaatctgg gcgatctggt tgggtttagc 840 ggcattacct tacaagccat
gttcggttat ctgtttgctc ctctggcatg ggtgattggc 900 attccaagtc
acgaagtgct ggcggcaggt tcctacatcg gtcagaaagt ggtgatgaac 960
gaatttgtgg ctttcattga ctttgttgag cataaagcgc tgctttctga gcatagccaa
1020 gtcatcatca cgtttgcatt gtgtggcttt gccaacattg gctctatcgc
gatccaatta 1080 ggctccattg gcgtgatagc ccctgagcgc cgctcggaag
tggcgaacct aggcataaaa 1140 gcggtcattg ctggcacttt agccaaccta
atgagcgctt gcttagcggg gattttcatc 1200 tcgctataa 1209 <210>
SEQ ID NO 124 <211> LENGTH: 1278 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1278) <223> OTHER INFORMATION: yegT - Escherichia coli
K-12 W3110 (P76417; JW2085) <400> SEQUENCE: 124 atgaaaacaa
cagcaaagct gtcgttcatg atgtttgttg aatggtttat ctggggcgcg 60
tggtttgtgc cattgtggtt gtggttaagt aaaagcggtt ttagtgccgg agaaattggc
120 tggtcgtatg cctgtaccgc cattgcggcg atcctgtcgc caattctggt
tggctccatc 180 actgaccgct ttttctcggc gcaaaaagtg ctggcggtat
tgatgttcgc aggcgcgctg 240 ctgatgtatt tcgctgcgca acagaccact
tttgccgggt tcttcccgtt actgctggcc 300 tactcgctaa cctatatgcc
gaccattgcg ctgactaaca gcatcgcttt tgccaacgtg 360 ccggatgttg
agcgtgattt cccgcgcatt cgtgtgatgg gcactatcgg ctggattgcc 420
tccggtctgg catgtggttt cttgccgcaa atactggggt atgccgatat ctcaccgact
480 aacatcccgc tgctgattac cgccggaagt tctgctctgc tcggtgtgtt
tgcgtttttc 540 ctgcccgaca cgccaccaaa aagcaccggc aaaatggata
ttaaagtcat gctcggcctg 600 gatgcgctga tcctgctgcg cgataaaaac
ttcctcgtct ttttcttctg ttcattcctg 660 tttgcgatgc cactagcgtt
ctattacatc tttgccaacg gttatctgac cgaagttggc 720 atgaaaaacg
ccaccggctg gatgacgctc ggccagttct ctgaaatctt ctttatgctg 780
gcattgccgt ttttcactaa acgctttggt atcaaaaagg tattattgct tggtctggtc
840 accgctgcga tccgctatgg cttctttatt tacggtagtg cggatgaata
tttcacctac 900 gcgttactgt tcctcggtat tttgcttcac ggcgtaagtt
acgattttta ctacgttacc 960 gcttacatct atgtcgataa aaaagccccc
gtgcatatgc gtaccgctgc gcaggggctg 1020 atcacgctct gctgccaggg
cttcggcagt ttgctcggct atcgtcttgg cggtgtgatg 1080 atggaaaaga
tgttcgctta tcaggaaccg gtaaacggac tgactttcaa ctggtccggg 1140
atgtggactt tcggcgcggt gatgattgcc attatcgccg tgctgttcat gatttttttc
1200 cgcgaatccg acaacgaaat tacggctatc aaggtcgatg atcgcgatat
tgcgttgaca 1260 caaggggaag ttaaatga 1278 <210> SEQ ID NO 125
<211> LENGTH: 1257 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1257)
<223> OTHER INFORMATION: nupG - Escherichia coli K-12 W3110
(P09452; JW2932) <400> SEQUENCE: 125 atgaatctta agctgcagct
gaaaatcctc tcttttctgc agttctgtct gtggggaagt 60 tggctgacga
ccctcggctc ctatatgttt gttaccctga agtttgacgg tgcttctatt 120
ggcgcagttt atagctcact gggtatcgca gcggtcttta tgcctgcgct gctggggatt
180 gtggccgaca aatggttaag tgcgaaatgg gtatatgcca tttgccacac
cattggcgct 240 atcacgctgt tcatggcggc acaggtcacg acaccggaag
cgatgttcct tgtgatattg 300 attaactcgt ttgcttatat gccaacgctt
gggttaatca acaccatctc ttactatcgc 360 ctgcaaaatg ccgggatgga
tatcgttact gacttcccgc caatccgtat ctggggcacc 420 atcggcttta
tcatggcaat gtgggtggtg agcctgtctg gcttcgaatt aagccacatg 480
cagctgtata ttggcgcagc actttccgcc attctggttc tgtttaccct gactctgccg
540 catattccgg ttgctaaaca gcaagcgaat cagagctgga caaccctgct
gggcctcgat 600 gcattcgcgc tgtttaaaaa caagcgtatg gcaatcttct
ttatcttctc aatgctgctg 660 ggcgcggaac tgcagattac caacatgttc
ggtaatacct tcctgcacag cttcgacaaa 720 gatccgatgt ttgccagcag
ctttattgtg cagcatgcgt caatcatcat gtcgatttcg 780 cagatctctg
aaaccctgtt cattctgacc atcccgttct tcttaagccg ctacggtatt 840
aagaacgtaa tgatgatcag tattgtggcg tggatcctgc gttttgcgct gtttgcttac
900 ggcgacccga ctccgttcgg tactgtactg ctggtactgt cgatgatcgt
ttacggttgc 960 gcattcgact tcttcaacat ctctggttcg gtgtttgtcg
aaaaagaagt tagcccggca 1020 attcgcgcca gtgcacaagg gatgttcctg
atgatgacta acggcttcgg ctgtatcctc 1080 ggcggcatcg tgagcggtaa
agttgttgag atgtacaccc aaaacggcat taccgactgg 1140 cagaccgtat
ggttgatttt cgctggttac tccgtggttc tggccttcgc gttcatggcg 1200
atgttcaaat ataaacacgt tcgtgtcccg acaggcacac agacggttag ccactaa 1257
<210> SEQ ID NO 126 <211> LENGTH: 1257 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1257) <223> OTHER INFORMATION: xapB - Escherichia coli
K-12 W3110 (P45562; JW2397) <400> SEQUENCE: 126 atgagcatcg
cgatgcgctt aaaggtaatg tcctttttgc aatattttat ctgggggagc 60
tggctggtta ccctcggctc ttacatgatt aatactcttc atttcaccgg cgctaatgtt
120 ggcatggttt acagttccaa agggatcgcc gcgattatta tgcctggtat
aatggggatc 180 atcgcagaca aatggctgcg cgcagaacgt gcatacatgc
tgtgtcacct ggtgtgtgcg 240 ggcgtacttt tttatgcggc atccgtaact
gatccggata tgatgttttg ggtgatgtta 300 gtcaatgcga tggcgtttat
gccgactatt gcgttatcga acagcgtctc ttattcctgt 360 cttgcccagg
cagggcttga cccggtgacc gctttcccgc ccattcgcgt ttttggtacg 420
gtggggttca ttgtcgcgat gtgggcagta agcctgctgc atctggaatt gagtagtctg
480 cagctgtata tcgcgtccgg tgcgtcattg ctgctgtcgg cttatgcgct
gactttgccg 540 aagattccgg ttgcggagaa aaaagcgacc acatcgcttg
ccagcaagct gggtctggat 600 gccttcgtgc tgtttaaaaa tccacgcatg
gccatctttt tcctctttgc catgatgctg 660 ggtgcggtac tgcaaattac
caacgttttt ggtaatccgt tcctacatga tttcgcccgt 720 aacccggagt
ttgctgacag ttttgtggtg aaatatccct ccattttact gtcagtttca 780
cagatggcag aagtgggctt tatactgact atcccattct ttttaaagcg atttggcatt
840 aaaaccgtca tgctgatgag tatggtggcc tggacgctgc gctttggctt
cttcgcctat 900 ggcgatccgt caacaaccgg atttattttg ctgctgctgt
cgatgattgt ttatggctgt 960 gcattcgatt tcttcaatat ttctggttcg
gtatttgtcg aacaggaagt tgattccagc 1020 attcgtgcca gcgcgcaggg
gctctttatg accatggtaa atggtgtcgg cgcatgggtt 1080 ggctcgattc
tgagtggcat ggcagtagat tacttttcgg tggatggcgt aaaagactgg 1140
caaactatct ggctggtgtt tgcaggatat gctctttttc tcgcagtgat atttttcttt
1200 gggtttaaat ataatcatga ccctgaaaag ataaagcatc gagcggtgac tcattaa
1257 <210> SEQ ID NO 127 <211> LENGTH: 1242 <212>
TYPE: DNA <213> ORGANISM: Caulobacter crescentus CB15
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1242) <223> OTHER INFORMATION: CC1628 -
Caulobacter crescentus CB15 (AAK23606) <400> SEQUENCE: 127
atggggacga gtttccgtct gttcgtgatg atggtgctgc agctggcgat ctggggcgcc
60 tgggcgccca agatcttccc ctacatgggc atgctgggct tcgcgccctg
gcagcagtcg 120 ctggtcggca gcgcctgggg cgtggcggcg ctggtgggca
tcttcttctc gaatcagttc 180 gccgaccgga acttctcggc cgagcggttc
ctggcggtca gccacctgat cggcggcgtg 240 gcgctgctgg gcacggcctt
ctcgacggag ttctggccgt tctttgcctg ttacctcgtt 300 ttcagcctgg
tctatgtgcc gacgctgtcg gtcaccaact cgatcgcctt cgccaatctg 360
cgcgatccgg cggccggctt cggcggggtg cggatgggcg gaaccgtcgg ctgggtgctg
420 gtcagctggc ccttcgtgtt cctgctgggc gcccaagcga cggtggagca
ggtccgctgg 480 atcttcctgg tggcggcgat cgtctccttc gttttcgccg
gttacgctct gaccctgccg 540 cacacgccgc cgcgcaaggc cgatgacgct
gtcgacaagc tggcctggcg acgggcgttc 600 aagctactgg gcgcgccctt
cgtgtttgtc ctctttgtcg tgaccttcat cgattccgtg 660 atccacaacg
gctacttcgt gatggccgac gccttcctga ccaaccgggt cgggatcgcg 720
ggcaatctca gcatggtcgt gctgagcctg ggccaggtgg ccgaaatcat caccatgctg
780 ctgttgggcc gcgtgctggc caagctgggc tggaaggtca ccatgatcgt
cggcgtgctg 840 ggccacgccg cgcgctttgc ggtcttcgcc tacttcgccg
acagcgtgcc ggtcatcgtg 900 gcggtgcagc tgctgcacgg cgtctgctac
gccttcttct tcgccacggt ttacatcttc 960 gtcgacgccg tcttcccgaa
agatgtccgc tccagcgcgc agggtctgtt caacttgctg 1020 atcctgggcg
tcggcaatgt ggccgccagc ttcatcttcc ccgcgctgat cggtcgcctg 1080
accaccgatg ggtccgtcga ctacacgacg ctgttcctcg tgccgaccgc catggctttg
1140 gcggcggtct gcctgctggc gctgttcttc cggccgccca cgcggggacc
tgtttcggag 1200 gcggattccg cttcatccgc cgccagttcg gcccaagcct ag 1242
<210> SEQ ID NO 128 <211> LENGTH: 1260 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1260) <223> OTHER INFORMATION: codB - Escherichia coli
K-12 W3110 (P25525; JW0327) <400> SEQUENCE: 128 gtgtcgcaag
ataacaactt tagccagggg ccagtcccgc agtcggcgcg gaaaggggta 60
ttggcattga cgttcgtcat gctgggatta accttctttt ccgccagtat gtggaccggc
120 ggcactctcg gaaccggtct tagctatcat gatttcttcc tcgcagttct
catcggtaat 180 cttctcctcg gtatttacac ttcatttctc ggttacattg
gcgcaaaaac cggcctgacc 240 actcatcttc ttgctcgctt ctcgtttggt
gttaaaggct catggctgcc ttcactgcta 300 ctgggcggaa ctcaggttgg
ctggtttggc gtcggtgtgg cgatgtttgc cattccggtg 360 ggtaaggcaa
ccgggctgga tattaatttg ctgattgccg tttccggttt actgatgacc 420
gtcaccgtct tttttggcat ttcggcgctg acggttcttt cggtgattgc ggttccggct
480 atcgcctgcc tgggcggtta ttccgtgtgg ctggctgtta acggcatggg
cggcctggac 540 gcattaaaag cggtcgttcc cgcacaaccg ttagatttca
atgtcgcgct ggcgctggtt 600 gtggggtcat ttatcagtgc gggtacgctc
accgctgact ttgtccggtt tggtcgcaat 660 gccaaactgg cggtgctggt
ggcgatggtg gcctttttcc tcggcaactc gttgatgttt 720 attttcggtg
cagcgggcgc tgcggcactg ggcatggcgg atatctctga tgtgatgatt 780
gctcagggcc tgctgctgcc tgcgattgtg gtgctggggc tgaatatctg gaccaccaac
840 gataacgcac tctatgcgtc gggtttaggt ttcgccaaca ttaccgggat
gtcgagcaaa 900 accctttcgg taatcaacgg tattatcggt acggtctgcg
cattatggct gtataacaat 960 tttgtcggct ggttgacctt cctttcggca
gctattcctc cagtgggtgg cgtgatcatc 1020 gccgactatc tgatgaaccg
tcgccgctat gagcactttg cgaccacgcg tatgatgagt 1080 gtcaattggg
tggcgattct ggcggtcgcc ttggggattg ctgcaggcca ctggttaccg 1140
ggaattgttc cggtcaacgc ggtattaggt ggcgcgctga gctatctgat ccttaacccg
1200 attttgaatc gtaaaacgac agcagcaatg acgcatgtgg aggctaacag
tgtcgaataa 1260 <210> SEQ ID NO 129 <211> LENGTH: 1656
<212> TYPE: DNA <213> ORGANISM: Corynebacterium sp.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1656) <223> OTHER INFORMATION: mctC
Corynebacterium <400> SEQUENCE: 129 atgaattcca ctattctcct
tgcacaagac gctgtttctg agggcgtcgg taatccgatt 60 cttaacatca
gtgtcttcgt cgtcttcatt attgtgacga tgaccgtggt gcttcgcgtg 120
ggcaagagca ccagcgaatc caccgacttc tacaccggtg gtgcttcctt ctccggaacc
180 cagaacggtc tggctatcgc aggtgactac ctgtctgcag cgtccttcct
cggaatcgtt 240 ggtgcaattt cactcaacgg ttacgacgga ttcctttact
ccatcggctt cttcgtcgca 300 tggcttgttg cactgctgct cgtggcagag
ccacttcgta acgtgggccg cttcaccatg 360 gctgacgtgc tgtccttccg
actgcgtcag aaaccagtcc gcgtcgctgc ggcctgcggt 420 accctcgcgg
ttaccctctt ttacttgatc gctcagatgg ctggtgcagg ttcgcttgtg 480
tccgttctgc tggacatcca cgagttcaag tggcaggcag ttgttgtcgg tatcgttggc
540 attgtcatga tcgcctacgt tcttcttggc ggtatgaagg gcaccacata
cgttcagatg 600 attaaggcag ttctgctggt cggtggcgtt gccattatga
ccgttctgac cttcgtcaag 660 gtgtctggtg gcctgaccac ccttttaaat
gacgctgttg agaagcacgc cgcttcagat 720 tacgctgcca ccaaggggta
cgatccaacc cagatcctgg agcctggtct gcagtacggt 780 gcaactctga
ccactcagct ggacttcatt tccttggctc tcgctctgtg tcttggaacc 840
gctggtctgc cacacgttct gatgcgcttc tacaccgttc ctaccgccaa ggaagcacgt
900 aagtctgtga cctgggctat cgtcctcatt ggtgcgttct acctgatgac
cctggtcctt 960 ggttacggcg ctgcggcact ggtcggtcca gaccgcgtca
ttgccgcacc aggtgctgct 1020 aatgctgctg ctcctctgct ggccttcgag
cttggtggtt ccatcttcat ggcgctgatt 1080 tccgcagttg cgttcgctac
cgttctcgcc gtggtcgcag gtcttgcaat taccgcatcc 1140 gctgctgttg
gtcacgacat ctacaacgct gttatccgca acggtcagtc caccgaagcg 1200
gagcaggtcc gagtatcccg catcaccgtt gtcgtcattg gcctgatttc cattgtcctg
1260 ggaattcttg caatgaccca gaacgttgcg ttcctcgtgg ccctggcctt
cgcagttgca 1320 gcatccgcta acctgccaac catcctgtac tccctgtact
ggaagaagtt caacaccacc 1380 ggcgctgtgg ccgctatcta caccggtctc
atctccgcgc tgctgctgat cttcctgtcc 1440 ccagcagtct ccggtaatga
cagcgcaatg gttccaggtg cagactgggc aatcttccca 1500 ctgaagaacc
caggcctcgt ctccatccca ctggcattca tcgctggttg gatcggcact 1560
ttggttggca agccagacaa catggatgat cttgctgccg aaatggaagt tcgttccctc
1620 accggtgtcg gtgttgaaaa ggctgttgat cactaa 1656 <210> SEQ
ID NO 130 <211> LENGTH: 1521 <212> TYPE: DNA
<213> ORGANISM: Virgibacillus sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1521) <223> OTHER INFORMATION: putP_6 - Virgibacillus
sp. <400> SEQUENCE: 130 atggatctta cgacattaat aacttttata
gtatatctac tagggatgtt ggcgattggc 60 ctcatcatgt attatcgaac
caataattta tcagattatg ttcttggtgg acgtgatctt 120 ggtccaggcg
tagctgcatt gagtgctggt gcatcggata tgagtggttg gctgttatta 180
ggtttgcctg gagcgattta tgcatctggt atgtctgaag cttggatggg gatcgggtta
240 gctgtaggtg cttatttaaa ttggcaattt gtagctaagc gattacgcgt
ttataccgag 300 gtatcaaata attccattac gatcccagat tattttgaaa
atcggtttaa agataactca 360 catattcttc gtgttatatc tgctatcgta
attttgttat tcttcacttt ttatacatct 420 tcaggaatgg ttgcaggagc
aaaattattt gaggcttcat tcggtctcca atacgaaact 480 gctctgtgga
ttggtgcggt tgtagttgta tcttatacgt tacttggagg atttctagcg 540
gttgcatgga cagactttat tcaaggtatt cttatgttcc ttgcactaat tgttgttcca
600 atcgtcgcat tagatcaaat gggtggctgg aatcaagcgg tacaagctgt
tggtgaaatt 660 aatccttccc acctcaatat ggttgaaggt gttggaataa
tggcaattat ttcatcactt 720 gcttggggct taggttattt tggacagcca
catattattg ttcgttttat ggcattacgt 780 tcggcgaaag atgttccgaa
agcgaaattt attggaacag cttggatgat tttaggactt 840 tatggagcaa
tctttactgg ttttgtagga ctagcattta tcagtacaca agaagtaccg 900
attctgtctg aattcgggat tcaagtagtt aatgagaatg gtttacaaat gttagccgat
960 cctgaaaaga tatttattgc tttctcccaa atactattcc atccagtagt
tgccggtatc 1020 ttactagcgg caatcttgtc tgcaattatg agtaccgttg
attcacagtt acttgtatca 1080 tcttcagcgg ttgcagaaga tttctataaa
gctattttcc gtaaaaaagc tactggtaaa 1140 gagcttgttt gggttggacg
tattgctaca gtgataattg cgattgttgc tttaattatt 1200 gcaatgaacc
cagatagctc tgtattggat ctagttagtt atgcatgggc tggatttggt 1260
gcagcatttg gaccaattat catcttgtca ttattctgga agagaatcac aagaaatggt
1320 gcactagcgg gtatcattgt aggtgccatt acggtaattg tatggggaga
ctttctatct 1380 ggaggtatct ttgacctcta cgaaattgtt ccaggcttta
tcttaaatat gattgtcacc 1440 gttattgtga gtcttatcga taaaccgaat
ccagatttag aagctgactt tgatgaaacc 1500 gtagaaaaaa tgaaagaata a 1521
<210> SEQ ID NO 131 <211> LENGTH: 1323 <212>
TYPE: DNA <213> ORGANISM: Lactobacillus johnsonii <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1323) <223> OTHER INFORMATION: cbsT1 - Lactobacillus
johnsonii <400> SEQUENCE: 131 atgtcgacca caccgacaca
gccatcatca cgaaaacagg ctgtttaccc gtacttgatc 60 gtgctgtcgg
gcatcgtctt cacggccatc ccggtatcgc tggtctgcag ttgcgcaggt 120
atcttcttca cgcctgtcag cagctacttc catgttccca aggccgcatt caccggatat
180 ttcagcatat tcagcatcac catggtcgcc ttcctgccgg tggccggatg
gctgatgcac 240 cgctacgatc tgcgcatcgt actgaccgca agcaccgtcc
tggctggact gggctgcctg 300 ggtatgtccc gatcatccgc catgtggcag
ttctatctat gcggagtggt tctgggaatc 360 ggcatgccgg ccgtcctcta
tctgtcagtg ccaacactca tcaacgcctg gttccgcaag 420 cgggtcgggt
tcttcatcgg cctgtgcatg gccttcaccg gcataggcgg cgtgatcttc 480
aaccagatag gcaccatgat catcagatcc gcccctgatg gatggaggcg gggatatctg
540 gttttcgcta ttctcatcct ggtgatcacc ctgcccttca ccattttcgt
cattcgcagc 600 acacccgaac agatgggtct gcatccctac ggcgccgacc
aggagcctga tgcagctgag 660 acggccacca atagtgcagg caccgggagc
aaagaccaaa agagtcctga gcctgcagcg 720 tcaaccgtag gcatgactgc
ctcccaggcc ttgcgctccc ctgccttctg ggcgctggcg 780 ctcttctgcg
gtctgatcac catgaatcag accatttacc agttcctgcc ctcctacgcg 840
gcatccctgc catccatggc agcctacacg ggactgatcg cctcctcctg catggccggc
900 caggccatcg gcaagatcat cctgggcatg gtcaacgacg gcagcatcgt
aggcggtctc 960 tgtctgggca tcggcggcgg cattctcggc gtctgcctca
tggtcgcctt ccccggattg 1020 cccgtgctcc tcctgctggg agcctttgcc
ttcggccttg tctacgcctg cactactgtg 1080 cagacaccaa tcctggttac
agcggtcttc ggctcgcgcg actacaccaa catctatgca 1140 cgtatccaga
tggttgggtc cctagcctcg gccttcgcag ctctcttctg gggcgccatc 1200
gctgaccagc cccacggcta catcatcatg ttcggtctga gcatcctgat catggttgtg
1260 gccttgttcc taggcattat ccctctgaaa ggtacgcgca agttgaccga
tcagatcgcc 1320 tga 1323 <210> SEQ ID NO 132 <211>
LENGTH: 1356 <212> TYPE: DNA <213> ORGANISM:
Lactobacillus johnsonii <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1356) <223> OTHER
INFORMATION: cbsT2 - Lactobacillus johnsonii <400> SEQUENCE:
132 atgtctactg atgccgctac taaagataaa gtagtaagca agggctataa
atacttcatg 60 gttttccttt gtatgttaac ccaagctatt ccttatggaa
ttgctcaaaa cattcagcct 120 ttgtttatcc accctttagt taatactttc
cactttacct tagcatcgta cacattaatt 180 tttacgtttg gtgcggtttt
tgcttcagtt gcttctccat ttattggtaa ggcattagaa 240 aaagttaact
tccgactaat gtatttaatt ggtattggtc tttctgctat tgcctacgta 300
atttttggaa ttagtacaaa actacccggt ttctatattg ccgctatcat ttgtatggtt
360 ggttcaacct tttactccgg ccaaggtgtt ccctgggtta ttaaccactg
gttcccagca 420 aagggacgtg gggctgcctt aggaattgcc ttctgcggtg
gttctattgg taatatcttt 480 ttacaaccag caacccaagc tattttaaaa
cactacatga caggtaatac taagaccggt 540 catttaacct ctatggcacc
attctttatc tttgccgtag ctttattagt aatcggtgta 600 attatcgcct
gcttcattag aacccctaag aaagacgaaa ttgttgtttc tgatgcagaa 660
ctagctgaaa gcaagaaagc tgaagccgca gccaaagcta aagagtttaa aggctggact
720 agtaaacaag tgttacaaat gaaatggttc tggattttca gccttggttt
cttaatcatt 780 ggtttaggct tagcttcttt aaatgaagac tatgccgcct
tccttgatac taagctttct 840 ttaaccgatg ttggtttagt tgggtcaatg
tacggtgttg gttgtttaat cggaaatatt 900 tctggtggtt tcttatttga
taaatttggt acagcaaaat caatgaccta tgctggttgt 960 atgtatattt
tatctattct gatgatgatc tttattagct tccagccata tggttcatct 1020
attagtaagg ctgctggcat tggctatgct atcttttgcg gcttagctgt atttagttac
1080 atgtcaggcc cagccttcat ggcaaaagac ctctttggtt caagagatca
aggtgtcatg 1140 cttggatacg ttggtttagc ttatgcaatt ggctatgcca
ttggtgctcc actatttggg 1200 attattaagg gagcggcaag ctttacagtt
gcttggtact ttatgattgc ctttgttgca 1260 attggtttta tcattttagt
atttgccgtt atccaaatta agagatacca aaagaaatac 1320 attgcagagc
aagcagcaaa agctaatgct aaataa 1356 <210> SEQ ID NO 133
<211> LENGTH: 1287 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1287)
<223> OTHER INFORMATION: amtB - Escherichia coli K-12 MG1655
(B0451; 945084) <400> SEQUENCE: 133 atgaagatag cgacgataaa
aactgggctt gcttcactgg cgatgcttcc gggactggta 60 atggctgcac
ctgcggtggc cgataaagcc gacaatgcgt ttatgatgat ttgtactgcg 120
ctggtgctgt ttatgactat tccggggatt gccctgtttt acggtgggtt gattcgcggc
180 aaaaacgtgc tgtcgatgct gacgcaggtg acggtgacat ttgcactggt
ctgtattctc 240 tgggtggttt acggttactc gctggcgttt ggtgagggca
acaacttctt cggcaacatt 300 aactggttga tgctgaaaaa catcgaactg
acggcggtga tgggcagcat ttatcagtat 360 atccacgtgg cgtttcaggg
atcgtttgcc tgcattaccg tcggcttgat agttggggcg 420 ctggcggaac
gaatccgctt ctcagctgtg ttgattttcg tggtggtatg gctgacgctc 480
tcttacattc cgattgcgca tatggtgtgg ggcggtggtt tgctggcttc tcacggtgcg
540 ctggatttcg cgggtggcac cgtggtgcac attaacgccg caatcgccgg
tctggtgggc 600 gcgtatctga taggaaaacg cgtgggcttc ggtaaagagg
cgtttaaacc gcacaacctg 660 ccgatggtct tcaccgggac tgccattctc
tatatcggtt ggtttggctt taacgccggg 720 tcagcgggca cggcgaatga
aatcgcggca ctggcatttg tgaatactgt ggtcgcaacg 780 gcggcggcaa
ttcttggctg gatcttcggt gaatgggcgc tgcgtggtaa gccttcactg 840
ctgggggcgt gttctggcgc gattgccggt ctggtcggcg tgacgccagc ctgcggctac
900 attggggttg gcggcgcgtt gattatcggc gtggtagctg gtctggcggg
cttgtggggc 960 gttaccatgc tcaaacgctt gctgcgggtg gatgatccct
gcgatgtctt cggtgtgcac 1020 ggcgtttgtg gcattgtcgg ctgtatcatg
accgggattt ttgccgccag ctcgctgggc 1080 ggcgtgggct tcgctgaagg
tgtgacgatg ggccatcagt tgctggtaca gctggaaagc 1140 atcgccatta
cgatcgtctg gtccggtgtt gtggcattta tcggctacaa attggcggat 1200
ctgacggttg gtctgcgtgt accggaagag caggagcgag aagggctgga tgtcaacagc
1260 cacggcgaga atgcctataa cgcgtaa 1287 <210> SEQ ID NO 134
<211> LENGTH: 1401 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1401)
<223> OTHER INFORMATION: GABA permease GabP Escherichia coli
<400> SEQUENCE: 134 atggggcaat catcgcaacc acatgagtta
ggcggcgggc tgaagtcacg ccacgtcacc 60 atgttgtcta ttgccggtgt
tatcggcgca agtctgtttg tcggttccag cgtcgccatc 120 gccgaagcgg
gcccggcggt attactggcc tatctgttcg ccggattact ggtggttatg 180
attatgcgga tgttggcgga aatggcagtt gccacgcccg ataccggttc gttttccacc
240 tatgccgata aagccattgg ccgctgggcg ggttatacca tcggctggct
gtactggtgg 300 ttttgggtac tggttatccc gctggaagcc aacatcgccg
ctatgatcct gcactcgtgg 360 gttccaggca ttcccatctg gttattttcc
ctcgtcatta ccctcgcctt aactggcagt 420 aatttattaa gcgttaaaaa
ctacggcgaa tttgagttct ggctggcgct gtgcaaagtc 480 atcgctatcc
tggcctttat tttccttggt gcagtcgcaa ttagcggttt ttacccttat 540
gccgaagtga gcgggatctc aagattgtgg gatagcggcg gctttatgcc caacggtttc
600 ggtgcggtat taagcgcgat gttgatcacc atgttctcgt ttatgggcgc
agaaattgtc 660 accattgccg ccgcggaatc cgacacgccg gaaaaacata
ttgtccgcgc cactaactcg 720 gttatctggc gtatttctat cttctatttg
tgctctattt ttgtcgtagt ggcgttaata 780 ccgtggaata tgccggggct
gaaagccgtt ggttcttatc gctcggttct ggaattgctc 840 aatattcccc
atgcgaaatt aatcatggac tgcgtgatat tactttccgt aaccagctgt 900
ctgaactcgg cgctgtatac cgcgtcaagg atgctctact ccttaagccg tcgcggtgat
960 gcgcccgcgg taatgggcaa aatcaaccgc agtaaaaccc cgtatgtggc
ggtgttactc 1020 tccaccggag cggcattttt aacggtggtg gtgaactatt
acgcacctgc gaaagtgttt 1080 aaattcctga tagacagctc cggtgctatc
gccctgctgg tttatttagt catcgccgtt 1140 tcacagttgc ggatgcgtaa
aattctgcga gcagaaggaa gcgaaattcg cttgcgcatg 1200 tggctttacc
cgtggctcac ctggctggta ataggcttta ttacctttgt gttggtagtg 1260
atgctattcc gcccggcgca acagttagaa gtgatctcta ccggcttatt agcgataggg
1320 attatctgta ccgtgccgat tatggcgcgc tggaaaaagc tggtattgtg
gcaaaaaaca 1380 cccgttcata atacgcgctg a 1401 <210> SEQ ID NO
135 <211> LENGTH: 1239 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1239)
<223> OTHER INFORMATION: mtnH Escherichia coli <400>
SEQUENCE: 135 atgacgaact atcgcgttga gagtagcagc ggacgggcgg
cgcgcaagat gaggctcgca 60 ttaatgggac ctgcgttcat tgcggcgatt
ggttatatcg atcccggtaa ctttgcgacc 120 aatattcagg cgggtgccag
cttcggctat cagctactgt gggttgtcgt ttgggccaac 180 ctgatggcga
tgctgattca gatcctctct gccaaactag ggattgccac cggtaaaaat 240
ctggcggagc agattcgcga tcactatccg cgtcccgtag tgtggttcta ttgggttcag
300 gcagaaatta ttgcgatggc aaccgacctg gcggaattta ttggtgcggc
gatcggtttt 360 aaactcattc ttggtgtctc gttgttgcag ggcgcggtgc
tgacggggat cgcgactttc 420 ctgattttaa tgctgcaacg tcgcgggcaa
aaaccgctgg agaaagtgat tggcgggtta 480 ctgttgtttg ttgccgcggc
ttacattgtc gagttgattt tctcccagcc taacctggcg 540 cagctgggta
aaggaatggt gatcccgagt ttacctactt cggaggcggt cttcctggca 600
gcaggcgtgt taggggcgac gattatgccg catgtgattt atttgcactc ctcgctcact
660 cagcatttac atggcggttc gcgtcaacaa cgttattccg ccaccaaatg
ggatgtggct 720 atcgccatga cgattgccgg ttttgtcaat ctggcgatga
tggctacagc tgcggcggcg 780 ttccactttt ctggtcatac tggtgttgcc
gatcttgatg aggcttatct gacgctgcaa 840 ccgctgttaa gccatgctgc
ggcaacggtc tttgggttaa gtctggttgc tgccggactg 900 tcctcaacgg
tggtggggac actggcgggg caggtggtga tgcagggatt cattcgcttc 960
catatcccgc tgtgggtgcg tcgtacagtc accatgttgc cgtcatttat tgtcattctg
1020 atgggattag atccgacacg gattctggtt atgagtcagg tgctgttaag
ttttggtatc 1080 gccctggcgc tggttccact gctgattttc accagtgaca
gcaagttgat gggcgatctg 1140 gtgaacagca aacgcgtaaa acagacaggc
tgggtgattg tagtgctggt cgtggcgctg 1200 aatatctggt tgttggtggg
gacggcgctg ggattgtag 1239 <210> SEQ ID NO 136 <211>
LENGTH: 287 <212> TYPE: PRT <213> ORGANISM: Salmonella
enterica <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(287) <223> OTHER INFORMATION: Tsx
- Salmonella enterica subsp. enterica serovar Typhimurium LT2
(STM0413) <400> SEQUENCE: 136 Met Lys Lys Thr Leu Leu Ala Val
Ser Ala Ala Leu Ala Leu Thr Ser 1 5 10 15 Ser Phe Thr Ala Asn Ala
Ala Glu Asn Asp Gln Pro Gln Tyr Leu Ser 20 25 30 Asp Trp Trp His
Gln Ser Val Asn Val Val Gly Ser Tyr His Thr Arg 35 40 45 Phe Ser
Pro Lys Leu Asn Asn Asp Val Tyr Leu Glu Tyr Glu Ala Phe 50 55 60
Ala Lys Lys Asp Trp Phe Asp Phe Tyr Gly Tyr Ile Asp Ile Pro Lys 65
70 75 80 Thr Phe Asp Trp Gly Asn Gly Asn Asp Lys Gly Ile Trp Ser
Asp Gly 85 90 95 Ser Pro Leu Phe Met Glu Ile Glu Pro Arg Phe Ser
Ile Asp Lys Leu 100 105 110 Thr Gly Ala Asp Leu Ser Phe Gly Pro Phe
Lys Glu Trp Tyr Phe Ala 115 120 125 Asn Asn Tyr Ile Tyr Asp Met Gly
Asp Asn Lys Ala Ser Arg Gln Ser 130 135 140 Thr Trp Tyr Met Gly Leu
Gly Thr Asp Ile Asp Thr Gly Leu Pro Met 145 150 155 160 Gly Leu Ser
Leu Asn Val Tyr Ala Lys Tyr Gln Trp Gln Asn Tyr Gly 165 170 175 Ala
Ser Asn Glu Asn Glu Trp Asp Gly Tyr Arg Phe Lys Val Lys Tyr 180 185
190 Phe Val Pro Ile Thr Asp Leu Trp Gly Gly Lys Leu Ser Tyr Ile Gly
195 200 205 Phe Thr Asn Phe Asp Trp Gly Ser Asp Leu Gly Asp Asp Pro
Asn Arg 210 215 220 Thr Ser Asn Ser Ile Ala Ser Ser His Ile Leu Ala
Leu Asn Tyr Asp 225 230 235 240 His Trp His Tyr Ser Val Val Ala Arg
Tyr Phe His Asn Gly Gly Gln 245 250 255 Trp Gln Asn Gly Ala Lys Leu
Asn Trp Gly Asp Gly Asp Phe Ser Ala 260 265 270 Lys Ser Thr Gly Trp
Gly Gly Tyr Leu Val Val Gly Tyr Asn Phe 275 280 285 <210> SEQ
ID NO 137 <211> LENGTH: 406 <212> TYPE: PRT <213>
ORGANISM: Bacillus halodurans <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(406) <223>
OTHER INFORMATION: BH1446 - Bacillus halodurans (BAB05165)
<400> SEQUENCE: 137 Met Asn Ile Leu Trp Gly Leu Leu Gly Ile
Val Val Val Phe Leu Ile 1 5 10 15 Ala Phe Ala Phe Ser Thr Asn Arg
Arg Ala Ile Lys Pro Arg Thr Ile 20 25 30 Leu Gly Gly Leu Ala Ile
Gln Leu Leu Phe Ala Ile Ile Val Leu Lys 35 40 45 Ile Pro Ala Gly
Gln Ala Leu Leu Glu Ser Leu Thr Asn Val Val Leu 50 55 60 Asn Ile
Ile Ser Tyr Ala Asn Glu Gly Ile Asp Phe Val Phe Gly Gly 65 70 75 80
Phe Phe Glu Glu Gly Ser Gly Val Gly Phe Val Phe Ala Ile Asn Val 85
90 95 Leu Ser Val Val Ile Phe Phe Ser Ala Leu Ile Ser Ile Leu Tyr
Tyr 100 105 110 Leu Gly Ile Met Gln Phe Val Ile Lys Ile Ile Gly Gly
Ala Leu Ser 115 120 125 Trp Leu Leu Gly Thr Ser Lys Ala Glu Ser Met
Ser Ala Ala Ala Asn 130 135 140 Ile Phe Val Gly Gln Thr Glu Ala Pro
Leu Val Val Lys Pro Tyr Leu 145 150 155 160 Pro Lys Met Thr Gln Ser
Glu Leu Phe Ala Val Met Thr Gly Gly Leu 165 170 175 Ala Ser Val Ala
Gly Ser Val Leu Ile Gly Tyr Ser Leu Leu Gly Val 180 185 190 Pro Leu
Gln Tyr Leu Leu Ala Ala Ser Phe Met Ala Ala Pro Ala Gly 195 200 205
Leu Ile Met Ala Lys Met Ile Met Pro Glu Thr Glu Lys Thr Thr Asp 210
215 220 Ala Glu Asp Asp Phe Lys Leu Ala Lys Asp Glu Glu Ser Thr Asn
Leu 225 230 235 240 Ile Asp Ala Ala Ala Asn Gly Ala Ser Thr Gly Leu
Met Leu Val Leu 245 250 255 Asn Ile Ala Ala Met Leu Leu Ala Phe Val
Ala Leu Ile Ala Leu Ile 260 265 270 Asn Gly Ile Leu Gly Trp Ile Gly
Gly Leu Phe Gly Ala Ser Gln Leu 275 280 285 Ser Leu Glu Leu Ile Leu
Gly Tyr Val Phe Ala Pro Leu Ala Phe Val 290 295 300 Ile Gly Ile Pro
Trp Ala Glu Ala Leu Gln Ala Gly Ser Tyr Ile Gly 305 310 315 320 Gln
Lys Leu Val Val Asn Glu Phe Val Ala Tyr Leu Ser Phe Ala Pro 325 330
335 Glu Ile Glu Asn Leu Ser Asp Lys Ala Val Met Val Ile Ser Phe Ala
340 345 350 Leu Cys Gly Phe Ala Asn Phe Ser Ser Leu Gly Ile Leu Leu
Gly Gly 355 360 365 Leu Gly Lys Leu Ala Pro Ser Arg Arg Pro Asp Ile
Ala Arg Leu Gly 370 375 380 Leu Arg Ala Ile Leu Ala Gly Thr Leu Ala
Ser Leu Leu Ser Ala Ser 385 390 395 400 Ile Ala Gly Met Leu Phe 405
<210> SEQ ID NO 138 <211> LENGTH: 393 <212> TYPE:
PRT <213> ORGANISM: Bacillus subtilis <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(393)
<223> OTHER INFORMATION: nupC Bacillus subtilis subsp.
subtilis 168 (BSU39410; CAA57663) <400> SEQUENCE: 138 Met Lys
Tyr Leu Ile Gly Ile Ile Gly Leu Ile Val Phe Leu Gly Leu 1 5 10 15
Ala Trp Ile Ala Ser Ser Gly Lys Lys Arg Ile Lys Ile Arg Pro Ile 20
25 30 Val Val Met Leu Ile Leu Gln Phe Ile Leu Gly Tyr Ile Leu Leu
Asn 35 40 45 Thr Gly Ile Gly Asn Phe Leu Val Gly Gly Phe Ala Lys
Gly Phe Gly 50 55 60 Tyr Leu Leu Glu Tyr Ala Ala Glu Gly Ile Asn
Phe Val Phe Gly Gly 65 70 75 80 Leu Val Asn Ala Asp Gln Thr Thr Phe
Phe Met Asn Val Leu Leu Pro 85 90 95 Ile Val Phe Ile Ser Ala Leu
Ile Gly Ile Leu Gln Lys Trp Lys Val 100 105 110 Leu Pro Phe Ile Ile
Arg Tyr Ile Gly Leu Ala Leu Ser Lys Val Asn 115 120 125 Gly Met Gly
Arg Leu Glu Ser Tyr Asn Ala Val Ala Ser Ala Ile Leu 130 135 140 Gly
Gln Ser Glu Val Phe Ile Ser Leu Lys Lys Glu Leu Gly Leu Leu 145 150
155 160 Asn Gln Gln Arg Leu Tyr Thr Leu Cys Ala Ser Ala Met Ser Thr
Val 165 170 175 Ser Met Ser Ile Val Gly Ala Tyr Met Thr Met Leu Lys
Pro Glu Tyr 180 185 190 Val Val Thr Ala Leu Val Leu Asn Leu Phe Gly
Gly Phe Ile Ile Ala 195 200 205 Ser Ile Ile Asn Pro Tyr Glu Val Ala
Lys Glu Glu Asp Met Leu Arg 210 215 220 Val Glu Glu Glu Glu Lys Gln
Ser Phe Phe Glu Val Leu Gly Glu Tyr 225 230 235 240 Ile Leu Asp Gly
Phe Lys Val Ala Val Val Val Ala Ala Met Leu Ile 245 250 255 Gly Phe
Val Ala Ile Ile Ala Leu Ile Asn Gly Ile Phe Asn Ala Val 260 265 270
Phe Gly Ile Ser Phe Gln Gly Ile Leu Gly Tyr Val Phe Ala Pro Phe 275
280 285 Ala Phe Leu Val Gly Ile Pro Trp Asn Glu Ala Val Asn Ala Gly
Ser 290 295 300 Ile Met Ala Thr Lys Met Val Ser Asn Glu Phe Val Ala
Met Thr Ser 305 310 315 320 Leu Thr Gln Asn Gly Phe His Phe Ser Gly
Arg Thr Thr Ala Ile Val 325 330 335 Ser Val Phe Leu Val Ser Phe Ala
Asn Phe Ser Ser Ile Gly Ile Ile 340 345 350 Ala Gly Ala Val Lys Gly
Leu Asn Glu Lys Gln Gly Asn Val Val Ala 355 360 365 Arg Phe Gly Leu
Lys Leu Leu Tyr Gly Ala Thr Leu Val Ser Phe Leu 370 375 380 Ser Ala
Ala Ile Val Gly Leu Ile Tyr 385 390 <210> SEQ ID NO 139
<211> LENGTH: 404 <212> TYPE: PRT <213> ORGANISM:
Bacillus subtilis <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(404) <223> OTHER
INFORMATION: yutK - Bacillus subtilis subsp. subtilis 168: BSU32180
<400> SEQUENCE: 139 Met Asn Val Leu Trp Gly Leu Leu Gly Ala
Val Ala Ile Ile Ala Ile 1 5 10 15 Ala Phe Leu Phe Ser Glu Lys Lys
Ser Asn Ile Lys Ile Arg Thr Val 20 25 30 Ile Val Gly Leu Cys Thr
Gln Val Ala Phe Gly Tyr Ile Val Leu Lys 35 40 45 Trp Glu Ala Gly
Arg Ala Val Phe Leu Trp Phe Ser Ser Arg Val Gln 50 55 60 Leu Leu
Ile Asp Tyr Ala Asn Glu Gly Ile Ser Phe Ile Phe Gly Pro 65 70 75 80
Leu Leu Lys Val Gly Asp Ser Pro Ala Phe Ala Leu Ser Val Leu Pro 85
90 95 Val Ile Ile Phe Phe Ser Ala Leu Ile Ala Val Leu Tyr His Leu
Lys 100 105 110 Ile Met Gln Leu Val Phe Arg Val Ile Gly Gly Gly Leu
Ser Lys Leu 115 120 125 Leu Gly Thr Ser Lys Thr Glu Ser Leu Ala Ala
Ala Ala Asn Ile Phe 130 135 140 Val Gly Gln Ser Glu Ser Pro Leu Val
Ile Lys Pro Leu Ile Ala Gly 145 150 155 160 Leu Thr Arg Ser Glu Leu
Phe Thr Ile Met Thr Ser Gly Leu Ser Ala 165 170 175 Val Ala Gly Ser
Thr Leu Phe Gly Tyr Ala Leu Leu Gly Ile Pro Ile 180 185 190 Glu Tyr
Leu Leu Ala Ala Ser Phe Met Ala Ala Pro Ala Gly Leu Val 195 200 205
Phe Gly Lys Leu Ile Ile Pro Glu Thr Glu Lys Thr Gln Thr Val Lys 210
215 220 Ser Asp Phe Lys Met Asp Glu Gly Glu Gly Ala Ala Asn Val Ile
Asp 225 230 235 240 Ala Ala Ala Lys Gly Ala Ser Thr Gly Leu Gln Ile
Ala Leu Asn Val 245 250 255 Gly Ala Met Leu Leu Ala Phe Val Ala Leu
Ile Ala Val Val Asn Gly 260 265 270 Ile Leu Gly Gly Ala Phe Gly Leu
Phe Gly Leu Lys Gly Val Thr Leu 275 280 285 Glu Ser Ile Leu Gly Tyr
Val Phe Ser Pro Ile Ala Phe Leu Ile Gly 290 295 300 Val Pro Trp His
Glu Ala Leu Gln Ala Gly Ser Tyr Ile Gly Gln Lys 305 310 315 320 Leu
Val Leu Asn Glu Phe Val Ala Tyr Ser Asn Phe Gly Ser His Ile 325 330
335 Gly Glu Phe Ser Lys Lys Thr Ala Thr Ile Ile Ser Phe Ala Leu Cys
340 345 350 Gly Phe Ala Asn Phe Ser Ser Ile Ala Ile Met Leu Gly Thr
Leu Gly 355 360 365 Gly Leu Ala Pro Ser Arg Arg Ser Asp Ile Ala Arg
Leu Gly Leu Lys 370 375 380 Ala Val Leu Ala Gly Thr Leu Ala Asn Leu
Leu Ser Ala Ala Ile Ala 385 390 395 400 Gly Met Phe Ile <210>
SEQ ID NO 140 <211> LENGTH: 397 <212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(397)
<223> OTHER INFORMATION: yxjA - Bacillus subtilis subsp.
spizizenii W23 (BSUW23_19355) <400> SEQUENCE: 140 Met Tyr Phe
Leu Leu Asn Leu Val Gly Leu Ile Val Ile Met Ala Val 1 5 10 15 Val
Phe Leu Cys Ser Pro Gln Lys Lys Lys Ile Gln Trp Arg Pro Ile 20 25
30 Ile Thr Leu Ile Val Leu Glu Leu Leu Ile Thr Trp Phe Met Leu Gly
35 40 45 Thr Lys Val Gly Ser Trp Ala Ile Gly Lys Ile Gly Asp Phe
Phe Thr 50 55 60 Trp Leu Ile Ala Cys Ala Ser Asp Gly Ile Ala Phe
Ala Phe Pro Ser 65 70 75 80 Val Met Ala Asn Glu Thr Val Asp Phe Phe
Phe Ser Ala Leu Leu Pro 85 90 95 Ile Ile Phe Ile Val Thr Phe Phe
Asp Ile Leu Thr Tyr Phe Gly Ile 100 105 110 Leu Pro Trp Leu Ile Asp
Lys Ile Gly Trp Val Ile Ser Lys Ala Ser 115 120 125 Arg Leu Pro Lys
Leu Glu Ser Phe Phe Ser Ile Gln Met Met Phe Leu 130 135 140 Gly Asn
Thr Glu Ala Leu Ala Val Ile Arg Gln Gln Leu Thr Val Leu 145 150 155
160 Asn Asn Asn Arg Leu Leu Thr Phe Gly Leu Met Ser Met Ser Ser Ile
165 170 175 Ser Gly Ser Ile Ile Gly Ser Tyr Leu Ser Met Val Pro Ala
Thr Tyr 180 185 190 Val Phe Thr Ala Ile Pro Leu Asn Cys Leu Asn Ala
Leu Ile Ile Ala 195 200 205 Asn Leu Leu Asn Pro Val His Val Pro Glu
Asp Glu Asp Ile Ile Tyr 210 215 220 Thr Pro Pro Lys Glu Glu Lys Lys
Asp Phe Phe Ser Thr Ile Ser Asn 225 230 235 240 Ser Met Leu Val Gly
Met Asn Met Val Ile Val Ile Leu Ala Met Val 245 250 255 Ile Gly Tyr
Val Ala Leu Thr Ser Ala Val Asn Gly Ile Leu Gly Val 260 265 270 Phe
Val His Gly Leu Thr Ile Gln Thr Ile Phe Ala Tyr Leu Phe Ser 275 280
285 Pro Phe Ala Phe Leu Leu Gly Leu Pro Val His Asp Ala Met Tyr Val
290 295 300 Ala Gln Leu Met Gly Met Lys Leu Ala Thr Asn Glu Phe Val
Ala Met 305 310 315 320 Leu Asp Leu Lys Asn Asn Leu Thr Thr Leu Pro
Pro His Thr Val Ala 325 330 335 Val Ala Thr Thr Phe Leu Thr Ser Phe
Ala Asn Phe Ser Thr Val Gly 340 345 350 Met Ile Tyr Gly Thr Tyr Asn
Ser Ile Leu Asp Gly Glu Lys Ser Thr 355 360 365 Val Ile Gly Lys Asn
Val Trp Lys Leu Leu Val Ser Gly Ile Ala Val 370 375 380 Ser Leu Leu
Ser Ala Ala Ile Val Gly Leu Phe Val Trp 385 390 395 <210> SEQ
ID NO 141 <211> LENGTH: 426 <212> TYPE: PRT <213>
ORGANISM: Caulobacter crescentus CB15 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(426)
<223> OTHER INFORMATION: ccCNT (CC2089) - Caulobacter
crescentus CB15 (AAK24060) <400> SEQUENCE: 141 Met Phe Arg
Pro Glu Asn Val Gln Ala Leu Ala Gly Leu Ala Leu Thr 1 5 10 15 Leu
Gly Leu Cys Trp Leu Val Ser Glu Asn Arg Lys Arg Phe Pro Trp 20 25
30 Gly Leu Ala Ile Gly Ala Val Val Ile Gln Val Leu Leu Val Leu Val
35 40 45 Leu Phe Gly Leu Pro Gln Ala Gln Gln Met Leu Arg Gly Val
Asn Gly 50 55 60 Ala Val Glu Gly Leu Ala Ala Ser Thr Gln Ala Gly
Thr Ala Phe Val 65 70 75 80 Phe Gly Phe Leu Ala Gly Gly Asp Gln Pro
Tyr Pro Val Ser Asn Pro 85 90 95 Gly Ala Gly Phe Ile Phe Ala Phe
Arg Val Leu Pro Val Ile Leu Val 100 105 110 Val Cys Ala Leu Ser Ala
Leu Leu Trp His Trp Lys Ile Leu Lys Trp 115 120 125 Leu Ala Gln Gly
Phe Gly Phe Val Phe Gln Lys Thr Leu Gly Leu Arg 130 135 140 Gly Pro
Pro Ala Leu Ala Thr Ala Ala Thr Ile Phe Met Gly Gln Val 145 150 155
160 Glu Gly Pro Ile Phe Ile Arg Ala Tyr Leu Asp Lys Leu Ser Arg Ser
165 170 175 Glu Leu Phe Met Leu Ile Ala Val Gly Met Ala Cys Val Ser
Gly Ser 180 185 190 Thr Met Val Ala Tyr Ala Thr Ile Leu Ala Asp Val
Leu Pro Asn Ala 195 200 205 Ala Ala His Val Leu Thr Ala Ser Ile Ile
Ser Ala Pro Ala Gly Val 210 215 220 Leu Leu Ala Arg Ile Ile Val Pro
Ser Asp Pro Met Glu Lys Ser Ala 225 230 235 240 Asp Leu Asp Leu Ser
Thr Glu Asp Lys Thr Tyr Gly Ser Ser Ile Asp 245 250 255 Ala Val Met
Lys Gly Thr Thr Asp Gly Leu Gln Ile Ala Leu Asn Val 260 265 270 Gly
Ala Thr Leu Ile Val Phe Val Ala Leu Ala Thr Met Val Asp Lys 275 280
285 Val Leu Gly Ala Phe Pro Pro Val Gly Gly Glu Pro Leu Ser Ile Ala
290 295 300 Arg Gly Leu Gly Val Val Phe Ala Pro Leu Ala Trp Ser Met
Gly Ile 305 310 315 320 Pro Trp Lys Glu Ala Gly Thr Ala Gly Gly Leu
Leu Gly Val Lys Leu 325 330 335 Ile Leu Thr Glu Phe Thr Ala Phe Ile
Gln Leu Ser Lys Val Gly Glu 340 345 350 Ala Leu Leu Asp Glu Arg Thr
Arg Met Ile Met Thr Tyr Ala Leu Cys 355 360 365 Gly Phe Ala Asn Ile
Gly Ser Val Gly Met Asn Val Ala Gly Phe Ser 370 375 380 Val Leu Val
Pro Gln Arg Arg Gln Glu Val Leu Gly Leu Val Trp Lys 385 390 395 400
Ala Met Met Ala Gly Phe Leu Ala Thr Cys Leu Thr Ala Ser Leu Val 405
410 415 Gly Leu Met Pro Arg Ser Leu Phe Gly Leu 420 425 <210>
SEQ ID NO 142 <211> LENGTH: 416 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: yeiJ - Escherichia coli K-12 W3110
(AAC75222; JW2148) <400> SEQUENCE: 142 Met Asp Val Met Arg
Ser Val Leu Gly Met Val Val Leu Leu Thr Ile 1 5 10 15 Ala Phe Leu
Leu Ser Val Asn Lys Lys Lys Ile Ser Leu Arg Thr Val 20 25 30 Gly
Ala Ala Leu Val Leu Gln Val Val Ile Gly Gly Ile Met Leu Trp 35 40
45 Leu Pro Pro Gly Arg Trp Val Ala Glu Lys Val Ala Phe Gly Val His
50 55 60 Lys Val Met Ala Tyr Ser Asp Ala Gly Ser Ala Phe Ile Phe
Gly Ser 65 70 75 80 Leu Val Gly Pro Lys Met Asp Thr Leu Phe Asp Gly
Ala Gly Phe Ile 85 90 95 Phe Gly Phe Arg Val Leu Pro Ala Ile Ile
Phe Val Thr Ala Leu Val 100 105 110 Ser Ile Leu Tyr Tyr Ile Gly Val
Met Gly Ile Leu Ile Arg Ile Leu 115 120 125 Gly Gly Ile Phe Gln Lys
Ala Leu Asn Ile Ser Lys Ile Glu Ser Phe 130 135 140 Val Ala Val Thr
Thr Ile Phe Leu Gly Gln Asn Glu Ile Pro Ala Ile 145 150 155 160 Val
Lys Pro Phe Ile Asp Arg Leu Asn Arg Asn Glu Leu Phe Thr Ala 165 170
175 Ile Cys Ser Gly Met Ala Ser Ile Ala Gly Ser Thr Met Ile Gly Tyr
180 185 190 Ala Ala Leu Gly Val Pro Val Glu Tyr Leu Leu Ala Ala Ser
Leu Met 195 200 205 Ala Ile Pro Gly Gly Ile Leu Phe Ala Arg Leu Leu
Ser Pro Ala Thr 210 215 220 Glu Ser Ser Gln Val Ser Phe Asn Asn Leu
Ser Phe Thr Glu Thr Pro 225 230 235 240 Pro Lys Ser Ile Ile Glu Ala
Ala Ala Thr Gly Ala Met Thr Gly Leu 245 250 255 Lys Ile Ala Ala Gly
Val Ala Thr Val Val Met Ala Phe Val Ala Ile 260 265 270 Ile Ala Leu
Ile Asn Gly Ile Ile Gly Gly Val Gly Gly Trp Phe Gly 275 280 285 Phe
Glu His Ala Ser Leu Glu Ser Ile Leu Gly Tyr Leu Leu Ala Pro 290 295
300 Leu Ala Trp Val Met Gly Val Asp Trp Ser Asp Ala Asn Leu Ala Gly
305 310 315 320 Ser Leu Ile Gly Gln Lys Leu Ala Ile Asn Glu Phe Val
Ala Tyr Leu 325 330 335 Asn Phe Ser Pro Tyr Leu Gln Thr Ala Gly Thr
Leu Asp Ala Lys Thr 340 345 350 Val Ala Ile Ile Ser Phe Ala Leu Cys
Gly Phe Ala Asn Phe Gly Ser 355 360 365 Ile Gly Val Val Val Gly Ala
Phe Ser Ala Val Ala Pro His Arg Ala 370 375 380 Pro Glu Ile Ala Gln
Leu Gly Leu Arg Ala Leu Ala Ala Ala Thr Leu 385 390 395 400 Ser Asn
Leu Met Ser Ala Thr Ile Ala Gly Phe Phe Ile Gly Leu Ala 405 410 415
<210> SEQ ID NO 143 <211> LENGTH: 416 <212> TYPE:
PRT <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: yeiM - Escherichia coli K-12 W3110
(AAC75225; JW2151) <400> SEQUENCE: 143 Met Asp Ile Met Arg
Ser Val Val Gly Met Val Val Leu Leu Ala Ile 1 5 10 15 Ala Phe Leu
Leu Ser Val Asn Lys Lys Ser Ile Ser Leu Arg Thr Val 20 25 30 Gly
Ala Ala Leu Leu Leu Gln Ile Ala Ile Gly Gly Ile Met Leu Tyr 35 40
45 Phe Pro Pro Gly Lys Trp Ala Val Glu Gln Ala Ala Leu Gly Val His
50 55 60 Lys Val Met Ser Tyr Ser Asp Ala Gly Ser Ala Phe Ile Phe
Gly Ser 65 70 75 80 Leu Val Gly Pro Lys Met Asp Val Leu Phe Asp Gly
Ala Gly Phe Ile 85 90 95 Phe Ala Phe Arg Val Leu Pro Ala Ile Ile
Phe Val Thr Ala Leu Ile 100 105 110 Ser Leu Leu Tyr Tyr Ile Gly Val
Met Gly Leu Leu Ile Arg Ile Leu 115 120 125 Gly Ser Ile Phe Gln Lys
Ala Leu Asn Ile Ser Lys Ile Glu Ser Phe 130 135 140 Val Ala Val Thr
Thr Ile Phe Leu Gly Gln Asn Glu Ile Pro Ala Ile 145 150 155 160 Val
Lys Pro Phe Ile Asp Arg Met Asn Arg Asn Glu Leu Phe Thr Ala 165 170
175 Ile Cys Ser Gly Met Ala Ser Ile Ala Gly Ser Met Met Ile Gly Tyr
180 185 190 Ala Gly Met Gly Val Pro Ile Asp Tyr Leu Leu Ala Ala Ser
Leu Met 195 200 205 Ala Ile Pro Gly Gly Ile Leu Phe Ala Arg Ile Leu
Ser Pro Ala Thr 210 215 220 Glu Pro Ser Gln Val Thr Phe Glu Asn Leu
Ser Phe Ser Glu Thr Pro 225 230 235 240 Pro Lys Ser Phe Ile Glu Ala
Ala Ala Ser Gly Ala Met Thr Gly Leu 245 250 255 Lys Ile Ala Ala Gly
Val Ala Thr Val Val Met Ala Phe Val Ala Ile 260 265 270 Ile Ala Leu
Ile Asn Gly Ile Ile Gly Gly Ile Gly Gly Trp Phe Gly 275 280 285 Phe
Ala Asn Ala Ser Leu Glu Ser Ile Phe Gly Tyr Val Leu Ala Pro 290 295
300 Leu Ala Trp Ile Met Gly Val Asp Trp Ser Asp Ala Asn Leu Ala Gly
305 310 315 320 Ser Leu Ile Gly Gln Lys Leu Ala Ile Asn Glu Phe Val
Ala Tyr Leu 325 330 335 Ser Phe Ser Pro Tyr Leu Gln Thr Gly Gly Thr
Leu Glu Val Lys Thr 340 345 350 Ile Ala Ile Ile Ser Phe Ala Leu Cys
Gly Phe Ala Asn Phe Gly Ser 355 360 365 Ile Gly Val Val Val Gly Ala
Phe Ser Ala Ile Ser Pro Lys Arg Ala 370 375 380 Pro Glu Ile Ala Gln
Leu Gly Leu Arg Ala Leu Ala Ala Ala Thr Leu 385 390 395 400 Ser Asn
Leu Met Ser Ala Thr Ile Ala Gly Phe Phe Ile Gly Leu Ala 405 410 415
<210> SEQ ID NO 144 <211> LENGTH: 417 <212> TYPE:
PRT <213> ORGANISM: Haemophilus influenzae <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(417) <223> OTHER INFORMATION: HI0519 - Haemophilus
influenzae Rd KW20 serotype d(AAC22177) <400> SEQUENCE: 144
Met Ser Val Leu Ser Ser Ile Leu Gly Met Val Val Leu Ile Ala Ile 1 5
10 15 Ala Val Leu Leu Ser Asn Asn Arg Lys Ala Ile Ser Ile Arg Thr
Val 20 25 30 Val Gly Ala Leu Ala Ile Gln Val Gly Phe Ala Ala Leu
Ile Leu Tyr 35 40 45 Val Pro Ala Gly Lys Gln Ala Leu Gly Ala Ala
Ala Asp Met Val Ser 50 55 60 Asn Val Ile Ala Tyr Gly Asn Asp Gly
Ile Asn Phe Val Phe Gly Gly 65 70 75 80 Leu Ala Asp Pro Ser Lys Pro
Ser Gly Phe Ile Phe Ala Val Lys Val 85 90 95 Leu Pro Ile Ile Val
Phe Phe Ser Gly Leu Ile Ser Val Leu Tyr Tyr 100 105 110 Leu Gly Ile
Met Gln Val Val Ile Lys Val Leu Gly Gly Ala Leu Gln 115 120 125 Lys
Ala Leu Gly Thr Ser Lys Ala Glu Ser Met Ser Ala Ala Ala Asn 130 135
140 Ile Phe Val Gly Gln Thr Glu Ala Pro Leu Val Val Arg Pro Tyr Ile
145 150 155 160 Lys Asn Met Thr Gln Ser Glu Leu Phe Ala Ile Met Val
Gly Gly Thr 165 170 175 Ala Ser Ile Ala Gly Ser Val Met Ala Gly Tyr
Ala Gly Met Gly Val 180 185 190 Pro Leu Thr Tyr Leu Ile Ala Ala Ser
Phe Met Ala Ala Pro Ala Gly 195 200 205 Leu Leu Phe Ala Lys Leu Met
Phe Pro Gln Thr Glu Gln Phe Thr Asp 210 215 220 Lys Gln Pro Glu Asp
Asn Asp Ser Glu Lys Pro Thr Asn Val Leu Glu 225 230 235 240 Ala Met
Ala Gly Gly Ala Ser Ala Gly Met Gln Leu Ala Leu Asn Val 245 250 255
Gly Ala Met Leu Ile Ala Phe Val Gly Leu Ile Ala Leu Ile Asn Gly 260
265 270 Ile Leu Ser Gly Val Gly Gly Trp Phe Gly Tyr Gly Asp Leu Thr
Leu 275 280 285 Gln Ser Ile Phe Gly Leu Ile Phe Lys Pro Leu Ala Tyr
Leu Ile Gly 290 295 300 Val Thr Asp Gly Ala Glu Ala Gly Ile Ala Gly
Gln Met Ile Gly Met 305 310 315 320 Lys Leu Ala Val Asn Glu Phe Val
Gly Tyr Leu Glu Phe Ala Lys Tyr 325 330 335 Leu Gln Pro Asp Ser Ala
Ile Val Leu Thr Glu Lys Thr Lys Ala Ile 340 345 350 Ile Thr Phe Ala
Leu Cys Gly Phe Ala Asn Phe Ser Ser Ile Ala Ile 355 360 365 Leu Ile
Gly Gly Leu Gly Gly Met Ala Pro Ser Arg Arg Ser Asp Val 370 375 380
Ala Arg Leu Gly Ile Lys Ala Val Ile Ala Gly Thr Leu Ala Asn Leu 385
390 395 400 Met Ser Ala Thr Ile Ala Gly Leu Phe Ile Gly Leu Gly Ala
Ala Ala 405 410 415 Leu <210> SEQ ID NO 145 <211>
LENGTH: 418 <212> TYPE: PRT <213> ORGANISM:
Helicobacter pylori 26695 <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(418) <223>
OTHER INFORMATION: nupC (HP1180) - Helicobacter pylori 26695
(AAD08224) <400> SEQUENCE: 145 Met Ile Phe Ser Ser Leu Phe
Ser Val Val Gly Met Ala Val Leu Phe 1 5 10 15 Leu Ile Ala Trp Val
Phe Ser Ser Asn Lys Arg Ala Ile Asn Tyr Arg 20 25 30 Thr Ile Val
Ser Ala Phe Val Ile Gln Val Ala Leu Gly Ala Leu Ala 35 40 45 Leu
Tyr Val Pro Leu Gly Arg Glu Met Leu Gln Gly Leu Ala Ser Gly 50 55
60 Ile Gln Ser Val Ile Ser Tyr Gly Tyr Glu Gly Val Arg Phe Leu Phe
65 70 75 80 Gly Asn Leu Ala Pro Asn Ala Lys Gly Asp Gln Gly Ile Gly
Gly Phe 85 90 95 Val Phe Ala Ile Asn Val Leu Ala Ile Ile Ile Phe
Phe Ala Ser Leu 100 105 110 Ile Ser Leu Leu Tyr Tyr Leu Lys Ile Met
Pro Leu Phe Ile Asn Leu 115 120 125 Ile Gly Gly Ala Leu Gln Lys Cys
Leu Gly Thr Ser Arg Ala Glu Ser 130 135 140 Met Ser Ala Ala Ala Asn
Ile Phe Val Ala His Thr Glu Ala Pro Leu 145 150 155 160 Val Ile Lys
Pro Tyr Leu Lys Ser Met Ser Asp Ser Glu Ile Phe Ala 165 170 175 Val
Met Cys Val Gly Met Ala Ser Val Ala Gly Pro Val Leu Ala Gly 180 185
190 Tyr Ala Ser Met Gly Ile Pro Leu Pro Tyr Leu Ile Ala Ala Ser Phe
195 200 205 Met Ser Ala Pro Gly Gly Leu Leu Phe Ala Lys Ile Ile Tyr
Pro Gln 210 215 220 Asn Glu Thr Ile Ser Ser His Ala Asp Val Ser Ile
Glu Lys His Val 225 230 235 240 Asn Ala Ile Glu Ala Ile Ala Asn Gly
Ala Ser Thr Gly Leu Asn Leu 245 250 255 Ala Leu His Val Gly Ala Met
Leu Leu Ala Phe Val Gly Met Leu Ala 260 265 270 Leu Ile Asn Gly Leu
Leu Gly Val Val Gly Gly Phe Leu Gly Met Glu 275 280 285 His Leu Ser
Leu Gly Leu Ile Leu Gly Thr Leu Leu Lys Pro Leu Ala 290 295 300 Phe
Met Leu Gly Ile Pro Trp Ser Gln Ala Gly Ile Ala Gly Glu Ile 305 310
315 320 Ile Gly Ile Lys Ile Ala Leu Asn Glu Phe Val Gly Tyr Met Gln
Leu 325 330 335 Leu Pro Tyr Leu Gly Asp Asn Pro Pro Leu Ile Leu Ser
Glu Lys Thr 340 345 350 Lys Ala Ile Ile Thr Phe Ala Leu Cys Gly Phe
Ala Asn Leu Ser Ser 355 360 365 Val Ala Met Leu Ile Gly Gly Leu Gly
Ser Leu Val Pro Lys Lys Lys 370 375 380 Asp Leu Ile Val Arg Leu Ala
Leu Lys Ala Val Leu Val Gly Thr Leu 385 390 395 400 Ser Asn Phe Met
Ser Ala Thr Ile Ala Gly Leu Phe Ile Gly Leu Asn 405 410 415 Ala His
<210> SEQ ID NO 146 <211> LENGTH: 409 <212> TYPE:
PRT <213> ORGANISM: Staphylococcus aureus <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(409) <223> OTHER INFORMATION: nupC (SA0600) -
Staphylococcus aureus subsp. aureus N315 (BAB41833) <400>
SEQUENCE: 146 Met Phe Leu Leu Ile Asn Ile Ile Gly Leu Ile Val Phe
Leu Gly Ile 1 5 10 15 Ala Val Leu Phe Ser Arg Asp Arg Lys Asn Ile
Gln Trp Gln Ser Ile 20 25 30 Gly Ile Leu Val Val Leu Asn Leu Phe
Leu Ala Trp Phe Phe Ile Tyr 35 40 45 Phe Asp Trp Gly Gln Lys Ala
Val Arg Gly Ala Ala Asn Gly Ile Ala 50 55 60 Trp Val Val Gln Ser
Ala His Ala Gly Thr Gly Phe Ala Phe Ala Ser 65 70 75 80 Leu Thr Asn
Val Lys Met Met Asp Met Ala Val Ala Ala Leu Phe Pro 85 90 95 Ile
Leu Leu Ile Val Pro Leu Phe Asp Ile Leu Met Tyr Phe Asn Ile 100 105
110 Leu Pro Lys Ile Ile Gly Gly Ile Gly Trp Leu Leu Ala Lys Val Thr
115 120 125 Arg Gln Pro Lys Phe Glu Ser Phe Phe Gly Ile Glu Met Met
Phe Leu 130 135 140 Gly Asn Thr Glu Ala Leu Ala Val Ser Ser Glu Gln
Leu Lys Arg Met 145 150 155 160 Asn Glu Met Arg Val Leu Thr Ile Ala
Met Met Ser Met Ser Ser Val 165 170 175 Ser Gly Ala Ile Val Gly Ala
Tyr Val Gln Met Val Pro Gly Glu Leu 180 185 190 Val Leu Thr Ala Ile
Pro Leu Asn Ile Val Asn Ala Ile Ile Val Ser 195 200 205 Cys Leu Leu
Asn Pro Val Ser Val Glu Glu Lys Glu Asp Ile Ile Tyr 210 215 220 Ser
Leu Lys Asn Asn Glu Val Glu Arg Gln Pro Phe Phe Ser Phe Leu 225 230
235 240 Gly Asp Ser Val Leu Ala Ala Gly Lys Leu Val Leu Ile Ile Ile
Ala 245 250 255 Phe Val Ile Ser Phe Val Ala Leu Ala Asp Leu Phe Asp
Arg Phe Ile 260 265 270 Asn Leu Ile Thr Gly Leu Ile Ala Gly Trp Ile
Gly Ile Lys Gly Ser 275 280 285 Phe Gly Leu Asn Gln Ile Leu Gly Val
Phe Met Tyr Pro Phe Ala Leu 290 295 300 Leu Leu Gly Leu Pro Tyr Asp
Glu Ala Trp Leu Val Ala Gln Gln Met 305 310 315 320 Ala Lys Lys Ile
Val Thr Asn Glu Phe Val Val Met Gly Glu Ile Ser 325 330 335 Lys Asp
Ile Ala Ser Tyr Thr Pro His His Arg Ala Val Ile Thr Thr 340 345 350
Phe Leu Ile Ser Phe Ala Asn Phe Ser Thr Ile Gly Met Ile Ile Gly 355
360 365 Thr Leu Lys Gly Ile Val Asp Lys Lys Thr Ser Asp Phe Val Ser
Lys 370 375 380 Tyr Val Pro Met Met Leu Leu Ser Gly Ile Leu Val Ser
Leu Leu Thr 385 390 395 400 Ala Ala Phe Val Gly Leu Phe Ala Trp 405
<210> SEQ ID NO 147 <211> LENGTH: 409 <212> TYPE:
PRT <213> ORGANISM: Staphylococcus aureus <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(409) <223> OTHER INFORMATION: nupC (SAV0645)
-Staphylococcus aureus subsp. aureus Mu50 (BAB56807) <400>
SEQUENCE: 147 Met Phe Leu Leu Ile Asn Ile Ile Gly Leu Ile Val Phe
Leu Gly Ile 1 5 10 15 Ala Val Leu Phe Ser Arg Asp Arg Lys Asn Ile
Gln Trp Gln Ser Ile 20 25 30 Gly Ile Leu Val Val Leu Asn Leu Phe
Leu Ala Trp Phe Phe Ile Tyr 35 40 45 Phe Asp Trp Gly Gln Lys Ala
Val Arg Gly Ala Ala Asn Gly Ile Ala 50 55 60 Trp Val Val Gln Ser
Ala His Ala Gly Thr Gly Phe Ala Phe Ala Ser 65 70 75 80 Leu Thr Asn
Val Lys Met Met Asp Met Ala Val Ala Ala Leu Phe Pro 85 90 95 Ile
Leu Leu Ile Val Pro Leu Phe Asp Ile Leu Met Tyr Phe Asn Ile 100 105
110 Leu Pro Lys Ile Ile Gly Gly Ile Gly Trp Leu Leu Ala Lys Val Thr
115 120 125 Arg Gln Pro Lys Phe Glu Ser Phe Phe Gly Ile Glu Met Met
Phe Leu 130 135 140 Gly Asn Thr Glu Ala Leu Ala Val Ser Ser Glu Gln
Leu Lys Arg Met 145 150 155 160 Asn Glu Met Arg Val Leu Thr Ile Ala
Met Met Ser Met Ser Ser Val 165 170 175 Ser Gly Ala Ile Val Gly Ala
Tyr Val Gln Met Val Pro Gly Glu Leu 180 185 190 Val Leu Thr Ala Ile
Pro Leu Asn Ile Val Asn Ala Ile Ile Val Ser 195 200 205 Cys Leu Leu
Asn Pro Val Ser Val Glu Glu Lys Glu Asp Ile Ile Tyr 210 215 220 Ser
Leu Lys Asn Asn Glu Val Glu Arg Gln Pro Phe Phe Ser Phe Leu 225 230
235 240 Gly Asp Ser Val Leu Ala Ala Gly Lys Leu Val Leu Ile Ile Ile
Ala 245 250 255 Phe Val Ile Ser Phe Val Ala Leu Ala Asp Leu Phe Asp
Arg Phe Ile 260 265 270 Asn Leu Ile Thr Gly Leu Ile Ala Gly Trp Ile
Gly Ile Lys Gly Ser 275 280 285 Phe Gly Leu Asn Gln Ile Leu Gly Val
Phe Met Tyr Pro Phe Ala Leu 290 295 300 Leu Leu Gly Leu Pro Tyr Asp
Glu Ala Trp Leu Val Ala Gln Gln Met 305 310 315 320 Ala Lys Lys Ile
Val Thr Asn Glu Phe Val Val Met Gly Glu Ile Ser 325 330 335 Lys Asp
Ile Ala Ser Tyr Thr Pro His His Arg Ala Val Ile Thr Thr 340 345 350
Phe Leu Ile Ser Phe Ala Asn Phe Ser Thr Ile Gly Met Ile Ile Gly 355
360 365 Thr Leu Lys Gly Ile Val Asp Lys Lys Thr Ser Asp Phe Val Ser
Lys 370 375 380 Tyr Val Pro Met Met Leu Leu Ser Gly Ile Leu Val Ser
Leu Leu Thr 385 390 395 400 Ala Ala Phe Val Gly Leu Phe Ala Trp 405
<210> SEQ ID NO 148 <211> LENGTH: 400 <212> TYPE:
PRT <213> ORGANISM: Streptococcus pyogenes SF370 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(400) <223> OTHER INFORMATION: nupC (SpNupC) -
Streptococcus pyogenes SF370 serotype M1 (AAK34582) <400>
SEQUENCE: 148 Met Gln Phe Ile Tyr Ser Ile Ile Gly Ile Leu Leu Val
Leu Gly Ile 1 5 10 15 Val Tyr Ala Ile Ser Phe Asn Arg Lys Ser Val
Ser Leu Ser Leu Ile 20 25 30 Gly Lys Ala Leu Ile Val Gln Phe Ile
Ile Ala Leu Ile Leu Val Arg 35 40 45 Ile Pro Leu Gly Gln Gln Ile
Val Ser Val Val Ser Thr Gly Val Thr 50 55 60 Ser Val Ile Asn Cys
Gly Gln Ala Gly Leu Asn Phe Val Phe Gly Ser 65 70 75 80 Leu Ala Asp
Ser Gly Ala Lys Thr Gly Phe Ile Phe Ala Ile Gln Thr 85 90 95 Leu
Gly Asn Ile Val Phe Leu Ser Ala Leu Val Ser Leu Leu Tyr Tyr 100 105
110 Val Gly Ile Leu Gly Phe Val Val Lys Trp Ile Gly Lys Gly Val Gly
115 120 125 Lys Ile Met Lys Ser Ser Glu Val Glu Ser Phe Val Ala Val
Ala Asn 130 135 140 Met Phe Leu Gly Gln Thr Asp Ser Pro Ile Leu Val
Ser Lys Tyr Leu 145 150 155 160 Gly Arg Met Thr Asp Ser Glu Ile Met
Val Val Leu Val Ser Gly Met 165 170 175 Gly Ser Met Ser Val Ser Ile
Leu Gly Gly Tyr Ile Ala Leu Gly Ile 180 185 190 Pro Met Glu Tyr Leu
Leu Ile Ala Ser Thr Met Val Pro Ile Gly Ser 195 200 205 Ile Leu Ile
Ala Lys Ile Leu Leu Pro Gln Thr Glu Pro Val Gln Lys 210 215 220 Ile
Asp Asp Ile Lys Met Asp Asn Lys Gly Asn Asn Ala Asn Val Ile 225 230
235 240 Asp Ala Ile Ala Glu Gly Ala Ser Thr Gly Ala Gln Met Ala Phe
Ser 245 250 255 Ile Gly Ala Ser Leu Ile Ala Phe Val Gly Leu Val Ser
Leu Ile Asn 260 265 270 Met Met Leu Ser Gly Leu Gly Ile Arg Leu Glu
Gln Ile Phe Ser Tyr 275 280 285 Val Phe Ala Pro Phe Gly Phe Leu Met
Gly Phe Asp His Lys Asn Ile 290 295 300 Leu Leu Glu Gly Asn Leu Leu
Gly Ser Lys Leu Ile Leu Asn Glu Phe 305 310 315 320 Val Ser Phe Gln
Gln Leu Gly His Leu Ile Lys Ser Leu Asp Tyr Arg 325 330 335 Thr Ala
Leu Val Ala Thr Ile Ser Leu Cys Gly Phe Ala Asn Leu Ser 340 345 350
Ser Leu Gly Ile Cys Val Ser Gly Ile Ala Val Leu Cys Pro Glu Lys 355
360 365 Arg Ser Thr Leu Ala Arg Leu Val Phe Arg Ala Met Ile Gly Gly
Ile 370 375 380 Ala Val Ser Met Leu Ser Ala Phe Ile Val Gly Ile Val
Thr Leu Phe 385 390 395 400 <210> SEQ ID NO 149 <211>
LENGTH: 418 <212> TYPE: PRT <213> ORGANISM: Vibrio
cholerae O1 biovar El Tor N16961 <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(418) <223>
OTHER INFORMATION: nupC (VC2352) - Vibrio cholerae O1 biovar El Tor
N16961 (AAF95495) <400> SEQUENCE: 149 Met Ser Leu Phe Met Ser
Leu Ile Gly Met Ala Val Leu Leu Gly Ile 1 5 10 15 Ala Val Leu Leu
Ser Ser Asn Arg Lys Ala Ile Asn Leu Arg Thr Val 20 25 30 Gly Gly
Ala Phe Ala Ile Gln Phe Ser Leu Gly Ala Phe Ile Leu Tyr 35 40 45
Val Pro Trp Gly Gln Glu Leu Leu Arg Gly Phe Ser Asp Ala Val Ser 50
55 60 Asn Val Ile Asn Tyr Gly Asn Asp Gly Thr Ser Phe Leu Phe Gly
Gly 65 70 75 80 Leu Val Ser Gly Lys Met Phe Glu Val Phe Gly Gly Gly
Gly Phe Ile 85 90 95 Phe Ala Phe Arg Val Leu Pro Thr Leu Ile Phe
Phe Ser Ala Leu Ile 100 105 110 Ser Val Leu Tyr Tyr Leu Gly Val Met
Gln Trp Val Ile Arg Ile Leu 115 120 125 Gly Gly Gly Leu Gln Lys Ala
Leu Gly Thr Ser Arg Ala Glu Ser Met 130 135 140 Ser Ala Ala Ala Asn
Ile Phe Val Gly Gln Thr Glu Ala Pro Leu Val 145 150 155 160 Val Arg
Pro Phe Val Pro Lys Met Thr Gln Ser Glu Leu Phe Ala Val 165 170 175
Met Cys Gly Gly Leu Ala Ser Ile Ala Gly Gly Val Leu Ala Gly Tyr 180
185 190 Ala Ser Met Gly Val Lys Ile Glu Tyr Leu Val Ala Ala Ser Phe
Met 195 200 205 Ala Ala Pro Gly Gly Leu Leu Phe Ala Lys Leu Met Met
Pro Glu Thr 210 215 220 Glu Lys Pro Gln Asp Asn Glu Asp Ile Thr Leu
Asp Gly Gly Asp Asp 225 230 235 240 Lys Pro Ala Asn Val Ile Asp Ala
Ala Ala Gly Gly Ala Ser Ala Gly 245 250 255 Leu Gln Leu Ala Leu Asn
Val Gly Ala Met Leu Ile Ala Phe Ile Gly 260 265 270 Leu Ile Ala Leu
Ile Asn Gly Met Leu Gly Gly Ile Gly Gly Trp Phe 275 280 285 Gly Met
Pro Glu Leu Lys Leu Glu Met Leu Leu Gly Trp Leu Phe Ala 290 295 300
Pro Leu Ala Phe Leu Ile Gly Val Pro Trp Asn Glu Ala Thr Val Ala 305
310 315 320 Gly Glu Phe Ile Gly Leu Lys Thr Val Ala Asn Glu Phe Val
Ala Tyr 325 330 335 Ser Gln Phe Ala Pro Tyr Leu Thr Glu Ala Ala Pro
Val Val Leu Ser 340 345 350 Glu Lys Thr Lys Ala Ile Ile Ser Phe Ala
Leu Cys Gly Phe Ala Asn 355 360 365 Leu Ser Ser Ile Ala Ile Leu Leu
Gly Gly Leu Gly Ser Leu Ala Pro 370 375 380 Lys Arg Arg Gly Asp Ile
Ala Arg Met Gly Val Lys Ala Val Ile Ala 385 390 395 400 Gly Thr Leu
Ser Asn Leu Met Ala Ala Thr Ile Ala Gly Phe Phe Leu 405 410 415 Ser
Phe <210> SEQ ID NO 150 <211> LENGTH: 405 <212>
TYPE: PRT <213> ORGANISM: Vibrio cholerae O1 biovar El Tor
N16961 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(405) <223> OTHER INFORMATION:
nupC (VC1953) - Vibrio cholerae O1 biovar El Tor N16961 (AAF95101)
<400> SEQUENCE: 150 Met Gly Gly Val Met Ser Ser Leu Leu Gly
Met Gly Ala Ile Leu Leu 1 5 10 15 Val Ala Trp Leu Phe Ser Thr Asn
Arg Lys Asn Ile Asn Leu Arg Thr 20 25 30 Val Ser Leu Ala Leu Leu
Leu Gln Ile Phe Phe Ala Leu Leu Val Leu 35 40 45 Tyr Val Pro Ala
Gly Lys Glu Ala Leu Asn Arg Val Thr Gly Ala Val 50 55 60 Ser Gln
Leu Ile Asn Tyr Gly Gln Asp Gly Ile Gly Phe Val Phe Gly 65 70 75 80
Gly Leu Ala Asn Gly Ser Val Gly Phe Val Phe Ala Ile Asn Val Leu 85
90 95 Gly Ile Ile Ile Phe Phe Ser Ala Leu Ile Ser Gly Leu Tyr His
Leu 100 105 110 Gly Ile Met Pro Lys Val Ile Asn Leu Ile Gly Gly Gly
Leu Gln Lys 115 120 125 Leu Leu Gly Thr Gly Arg Ala Glu Ser Leu Ser
Ala Thr Ala Asn Ile 130 135 140 Phe Val Gly Met Ile Glu Ala Pro Leu
Val Val Lys Pro Tyr Leu His 145 150 155 160 Lys Met Thr Asp Ser Gln
Phe Phe Ala Val Met Thr Gly Gly Leu Ala 165 170 175 Ser Val Ala Gly
Gly Thr Leu Val Gly Tyr Ala Ser Leu Gly Val Glu 180 185 190 Leu Asn
Tyr Leu Ile Ala Ala Ala Phe Met Ser Ala Pro Ala Gly Leu 195 200 205
Leu Met Ala Lys Ile Met Leu Pro Glu Thr Glu His Val Asp Ala Ala 210
215 220 Ile Ala Gln Asp Glu Leu Asp Leu Pro Lys Ser Thr Asn Val Val
Glu 225 230 235 240 Ala Ile Ala Asp Gly Ala Met Ser Gly Val Lys Ile
Ala Val Ala Val 245 250 255 Gly Ala Thr Leu Leu Ala Phe Val Ser Val
Ile Ala Leu Leu Asn Gly 260 265 270 Leu Leu Gly Trp Phe Gly Gly Trp
Phe Gly Ile Glu Leu Ser Phe Glu 275 280 285 Leu Ile Met Gly Tyr Val
Phe Ala Pro Val Ala Trp Leu Ile Gly Ile 290 295 300 Pro Trp His Glu
Ala Ile Thr Ala Gly Ser Leu Ile Gly Asn Lys Val 305 310 315 320 Val
Val Asn Glu Phe Val Ala Phe Ile Gln Leu Ile Glu Val Lys Glu 325 330
335 Gln Leu Ser Ala His Ser Gln Ala Ile Val Thr Phe Ala Leu Cys Gly
340 345 350 Phe Ala Asn Ile Ser Thr Met Ala Ile Leu Ile Gly Gly Leu
Gly Ser 355 360 365 Leu Val Pro Glu Arg Arg Ser Phe Ile Ser Gln Tyr
Gly Phe Arg Ala 370 375 380 Ile Gly Ala Gly Val Leu Ala Asn Leu Met
Ser Ala Ser Ile Ala Gly 385 390 395 400 Val Ile Leu Ser Leu 405
<210> SEQ ID NO 151 <211> LENGTH: 425 <212> TYPE:
PRT <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(425)
<223> OTHER INFORMATION: yegT - Escherichia coli K-12 W3110
(P76417; JW2085) <400> SEQUENCE: 151 Met Lys Thr Thr Ala Lys
Leu Ser Phe Met Met Phe Val Glu Trp Phe 1 5 10 15 Ile Trp Gly Ala
Trp Phe Val Pro Leu Trp Leu Trp Leu Ser Lys Ser 20 25 30 Gly Phe
Ser Ala Gly Glu Ile Gly Trp Ser Tyr Ala Cys Thr Ala Ile 35 40 45
Ala Ala Ile Leu Ser Pro Ile Leu Val Gly Ser Ile Thr Asp Arg Phe 50
55 60 Phe Ser Ala Gln Lys Val Leu Ala Val Leu Met Phe Ala Gly Ala
Leu 65 70 75 80 Leu Met Tyr Phe Ala Ala Gln Gln Thr Thr Phe Ala Gly
Phe Phe Pro 85 90 95 Leu Leu Leu Ala Tyr Ser Leu Thr Tyr Met Pro
Thr Ile Ala Leu Thr 100 105 110 Asn Ser Ile Ala Phe Ala Asn Val Pro
Asp Val Glu Arg Asp Phe Pro 115 120 125 Arg Ile Arg Val Met Gly Thr
Ile Gly Trp Ile Ala Ser Gly Leu Ala 130 135 140 Cys Gly Phe Leu Pro
Gln Ile Leu Gly Tyr Ala Asp Ile Ser Pro Thr 145 150 155 160 Asn Ile
Pro Leu Leu Ile Thr Ala Gly Ser Ser Ala Leu Leu Gly Val 165 170 175
Phe Ala Phe Phe Leu Pro Asp Thr Pro Pro Lys Ser Thr Gly Lys Met 180
185 190 Asp Ile Lys Val Met Leu Gly Leu Asp Ala Leu Ile Leu Leu Arg
Asp 195 200 205 Lys Asn Phe Leu Val Phe Phe Phe Cys Ser Phe Leu Phe
Ala Met Pro 210 215 220 Leu Ala Phe Tyr Tyr Ile Phe Ala Asn Gly Tyr
Leu Thr Glu Val Gly 225 230 235 240 Met Lys Asn Ala Thr Gly Trp Met
Thr Leu Gly Gln Phe Ser Glu Ile 245 250 255 Phe Phe Met Leu Ala Leu
Pro Phe Phe Thr Lys Arg Phe Gly Ile Lys 260 265 270 Lys Val Leu Leu
Leu Gly Leu Val Thr Ala Ala Ile Arg Tyr Gly Phe 275 280 285 Phe Ile
Tyr Gly Ser Ala Asp Glu Tyr Phe Thr Tyr Ala Leu Leu Phe 290 295 300
Leu Gly Ile Leu Leu His Gly Val Ser Tyr Asp Phe Tyr Tyr Val Thr 305
310 315 320 Ala Tyr Ile Tyr Val Asp Lys Lys Ala Pro Val His Met Arg
Thr Ala 325 330 335 Ala Gln Gly Leu Ile Thr Leu Cys Cys Gln Gly Phe
Gly Ser Leu Leu 340 345 350 Gly Tyr Arg Leu Gly Gly Val Met Met Glu
Lys Met Phe Ala Tyr Gln 355 360 365 Glu Pro Val Asn Gly Leu Thr Phe
Asn Trp Ser Gly Met Trp Thr Phe 370 375 380 Gly Ala Val Met Ile Ala
Ile Ile Ala Val Leu Phe Met Ile Phe Phe 385 390 395 400 Arg Glu Ser
Asp Asn Glu Ile Thr Ala Ile Lys Val Asp Asp Arg Asp 405 410 415 Ile
Ala Leu Thr Gln Gly Glu Val Lys 420 425 <210> SEQ ID NO 152
<211> LENGTH: 418 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(418) <223> OTHER
INFORMATION: nupG - Escherichia coli K-12 W3110 (P09452; JW2932
<400> SEQUENCE: 152 Met Asn Leu Lys Leu Gln Leu Lys Ile Leu
Ser Phe Leu Gln Phe Cys 1 5 10 15 Leu Trp Gly Ser Trp Leu Thr Thr
Leu Gly Ser Tyr Met Phe Val Thr 20 25 30 Leu Lys Phe Asp Gly Ala
Ser Ile Gly Ala Val Tyr Ser Ser Leu Gly 35 40 45 Ile Ala Ala Val
Phe Met Pro Ala Leu Leu Gly Ile Val Ala Asp Lys 50 55 60 Trp Leu
Ser Ala Lys Trp Val Tyr Ala Ile Cys His Thr Ile Gly Ala 65 70 75 80
Ile Thr Leu Phe Met Ala Ala Gln Val Thr Thr Pro Glu Ala Met Phe 85
90 95 Leu Val Ile Leu Ile Asn Ser Phe Ala Tyr Met Pro Thr Leu Gly
Leu 100 105 110 Ile Asn Thr Ile Ser Tyr Tyr Arg Leu Gln Asn Ala Gly
Met Asp Ile 115 120 125 Val Thr Asp Phe Pro Pro Ile Arg Ile Trp Gly
Thr Ile Gly Phe Ile 130 135 140 Met Ala Met Trp Val Val Ser Leu Ser
Gly Phe Glu Leu Ser His Met 145 150 155 160 Gln Leu Tyr Ile Gly Ala
Ala Leu Ser Ala Ile Leu Val Leu Phe Thr 165 170 175 Leu Thr Leu Pro
His Ile Pro Val Ala Lys Gln Gln Ala Asn Gln Ser 180 185 190 Trp Thr
Thr Leu Leu Gly Leu Asp Ala Phe Ala Leu Phe Lys Asn Lys 195 200 205
Arg Met Ala Ile Phe Phe Ile Phe Ser Met Leu Leu Gly Ala Glu Leu 210
215 220 Gln Ile Thr Asn Met Phe Gly Asn Thr Phe Leu His Ser Phe Asp
Lys 225 230 235 240 Asp Pro Met Phe Ala Ser Ser Phe Ile Val Gln His
Ala Ser Ile Ile 245 250 255 Met Ser Ile Ser Gln Ile Ser Glu Thr Leu
Phe Ile Leu Thr Ile Pro 260 265 270 Phe Phe Leu Ser Arg Tyr Gly Ile
Lys Asn Val Met Met Ile Ser Ile 275 280 285 Val Ala Trp Ile Leu Arg
Phe Ala Leu Phe Ala Tyr Gly Asp Pro Thr 290 295 300 Pro Phe Gly Thr
Val Leu Leu Val Leu Ser Met Ile Val Tyr Gly Cys 305 310 315 320 Ala
Phe Asp Phe Phe Asn Ile Ser Gly Ser Val Phe Val Glu Lys Glu 325 330
335 Val Ser Pro Ala Ile Arg Ala Ser Ala Gln Gly Met Phe Leu Met Met
340 345 350 Thr Asn Gly Phe Gly Cys Ile Leu Gly Gly Ile Val Ser Gly
Lys Val 355 360 365 Val Glu Met Tyr Thr Gln Asn Gly Ile Thr Asp Trp
Gln Thr Val Trp 370 375 380 Leu Ile Phe Ala Gly Tyr Ser Val Val Leu
Ala Phe Ala Phe Met Ala 385 390 395 400 Met Phe Lys Tyr Lys His Val
Arg Val Pro Thr Gly Thr Gln Thr Val 405 410 415 Ser His <210>
SEQ ID NO 153 <211> LENGTH: 418 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(418)
<223> OTHER INFORMATION: xapB - Escherichia coli K-12 W3110
(P45562; JW2397) <400> SEQUENCE: 153 Met Ser Ile Ala Met Arg
Leu Lys Val Met Ser Phe Leu Gln Tyr Phe 1 5 10 15 Ile Trp Gly Ser
Trp Leu Val Thr Leu Gly Ser Tyr Met Ile Asn Thr 20 25 30 Leu His
Phe Thr Gly Ala Asn Val Gly Met Val Tyr Ser Ser Lys Gly 35 40 45
Ile Ala Ala Ile Ile Met Pro Gly Ile Met Gly Ile Ile Ala Asp Lys 50
55 60 Trp Leu Arg Ala Glu Arg Ala Tyr Met Leu Cys His Leu Val Cys
Ala 65 70 75 80 Gly Val Leu Phe Tyr Ala Ala Ser Val Thr Asp Pro Asp
Met Met Phe 85 90 95 Trp Val Met Leu Val Asn Ala Met Ala Phe Met
Pro Thr Ile Ala Leu 100 105 110 Ser Asn Ser Val Ser Tyr Ser Cys Leu
Ala Gln Ala Gly Leu Asp Pro 115 120 125 Val Thr Ala Phe Pro Pro Ile
Arg Val Phe Gly Thr Val Gly Phe Ile 130 135 140 Val Ala Met Trp Ala
Val Ser Leu Leu His Leu Glu Leu Ser Ser Leu 145 150 155 160 Gln Leu
Tyr Ile Ala Ser Gly Ala Ser Leu Leu Leu Ser Ala Tyr Ala 165 170 175
Leu Thr Leu Pro Lys Ile Pro Val Ala Glu Lys Lys Ala Thr Thr Ser 180
185 190 Leu Ala Ser Lys Leu Gly Leu Asp Ala Phe Val Leu Phe Lys Asn
Pro 195 200 205 Arg Met Ala Ile Phe Phe Leu Phe Ala Met Met Leu Gly
Ala Val Leu 210 215 220 Gln Ile Thr Asn Val Phe Gly Asn Pro Phe Leu
His Asp Phe Ala Arg 225 230 235 240 Asn Pro Glu Phe Ala Asp Ser Phe
Val Val Lys Tyr Pro Ser Ile Leu 245 250 255 Leu Ser Val Ser Gln Met
Ala Glu Val Gly Phe Ile Leu Thr Ile Pro 260 265 270 Phe Phe Leu Lys
Arg Phe Gly Ile Lys Thr Val Met Leu Met Ser Met 275 280 285 Val Ala
Trp Thr Leu Arg Phe Gly Phe Phe Ala Tyr Gly Asp Pro Ser 290 295 300
Thr Thr Gly Phe Ile Leu Leu Leu Leu Ser Met Ile Val Tyr Gly Cys 305
310 315 320 Ala Phe Asp Phe Phe Asn Ile Ser Gly Ser Val Phe Val Glu
Gln Glu 325 330 335 Val Asp Ser Ser Ile Arg Ala Ser Ala Gln Gly Leu
Phe Met Thr Met 340 345 350 Val Asn Gly Val Gly Ala Trp Val Gly Ser
Ile Leu Ser Gly Met Ala 355 360 365 Val Asp Tyr Phe Ser Val Asp Gly
Val Lys Asp Trp Gln Thr Ile Trp 370 375 380 Leu Val Phe Ala Gly Tyr
Ala Leu Phe Leu Ala Val Ile Phe Phe Phe 385 390 395 400 Gly Phe Lys
Tyr Asn His Asp Pro Glu Lys Ile Lys His Arg Ala Val 405 410 415 Thr
His <210> SEQ ID NO 154 <211> LENGTH: 413 <212>
TYPE: PRT <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(413) <223> OTHER INFORMATION: CC1628 - Caulobacter
crescentus CB15 (AAK23606) <400> SEQUENCE: 154 Met Gly Thr
Ser Phe Arg Leu Phe Val Met Met Val Leu Gln Leu Ala 1 5 10 15 Ile
Trp Gly Ala Trp Ala Pro Lys Ile Phe Pro Tyr Met Gly Met Leu 20 25
30 Gly Phe Ala Pro Trp Gln Gln Ser Leu Val Gly Ser Ala Trp Gly Val
35 40 45 Ala Ala Leu Val Gly Ile Phe Phe Ser Asn Gln Phe Ala Asp
Arg Asn 50 55 60 Phe Ser Ala Glu Arg Phe Leu Ala Val Ser His Leu
Ile Gly Gly Val 65 70 75 80 Ala Leu Leu Gly Thr Ala Phe Ser Thr Glu
Phe Trp Pro Phe Phe Ala 85 90 95 Cys Tyr Leu Val Phe Ser Leu Val
Tyr Val Pro Thr Leu Ser Val Thr 100 105 110 Asn Ser Ile Ala Phe Ala
Asn Leu Arg Asp Pro Ala Ala Gly Phe Gly 115 120 125 Gly Val Arg Met
Gly Gly Thr Val Gly Trp Val Leu Val Ser Trp Pro 130 135 140 Phe Val
Phe Leu Leu Gly Ala Gln Ala Thr Val Glu Gln Val Arg Trp 145 150 155
160 Ile Phe Leu Val Ala Ala Ile Val Ser Phe Val Phe Ala Gly Tyr Ala
165 170 175 Leu Thr Leu Pro His Thr Pro Pro Arg Lys Ala Asp Asp Ala
Val Asp 180 185 190 Lys Leu Ala Trp Arg Arg Ala Phe Lys Leu Leu Gly
Ala Pro Phe Val 195 200 205 Phe Val Leu Phe Val Val Thr Phe Ile Asp
Ser Val Ile His Asn Gly 210 215 220 Tyr Phe Val Met Ala Asp Ala Phe
Leu Thr Asn Arg Val Gly Ile Ala 225 230 235 240 Gly Asn Leu Ser Met
Val Val Leu Ser Leu Gly Gln Val Ala Glu Ile 245 250 255 Ile Thr Met
Leu Leu Leu Gly Arg Val Leu Ala Lys Leu Gly Trp Lys 260 265 270 Val
Thr Met Ile Val Gly Val Leu Gly His Ala Ala Arg Phe Ala Val 275 280
285 Phe Ala Tyr Phe Ala Asp Ser Val Pro Val Ile Val Ala Val Gln Leu
290 295 300 Leu His Gly Val Cys Tyr Ala Phe Phe Phe Ala Thr Val Tyr
Ile Phe 305 310 315 320 Val Asp Ala Val Phe Pro Lys Asp Val Arg Ser
Ser Ala Gln Gly Leu 325 330 335 Phe Asn Leu Leu Ile Leu Gly Val Gly
Asn Val Ala Ala Ser Phe Ile 340 345 350 Phe Pro Ala Leu Ile Gly Arg
Leu Thr Thr Asp Gly Ser Val Asp Tyr 355 360 365 Thr Thr Leu Phe Leu
Val Pro Thr Ala Met Ala Leu Ala Ala Val Cys 370 375 380 Leu Leu Ala
Leu Phe Phe Arg Pro Pro Thr Arg Gly Pro Val Ser Glu 385 390 395 400
Ala Asp Ser Ala Ser Ser Ala Ala Ser Ser Ala Gln Ala 405 410
<210> SEQ ID NO 155 <211> LENGTH: 419 <212> TYPE:
PRT <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(419)
<223> OTHER INFORMATION: codB - Escherichia coli K-12 W3110
(P25525; JW0327) <400> SEQUENCE: 155 Met Ser Gln Asp Asn Asn
Phe Ser Gln Gly Pro Val Pro Gln Ser Ala 1 5 10 15 Arg Lys Gly Val
Leu Ala Leu Thr Phe Val Met Leu Gly Leu Thr Phe 20 25 30 Phe Ser
Ala Ser Met Trp Thr Gly Gly Thr Leu Gly Thr Gly Leu Ser 35 40 45
Tyr His Asp Phe Phe Leu Ala Val Leu Ile Gly Asn Leu Leu Leu Gly 50
55 60 Ile Tyr Thr Ser Phe Leu Gly Tyr Ile Gly Ala Lys Thr Gly Leu
Thr 65 70 75 80 Thr His Leu Leu Ala Arg Phe Ser Phe Gly Val Lys Gly
Ser Trp Leu 85 90 95 Pro Ser Leu Leu Leu Gly Gly Thr Gln Val Gly
Trp Phe Gly Val Gly 100 105 110 Val Ala Met Phe Ala Ile Pro Val Gly
Lys Ala Thr Gly Leu Asp Ile 115 120 125 Asn Leu Leu Ile Ala Val Ser
Gly Leu Leu Met Thr Val Thr Val Phe 130 135 140 Phe Gly Ile Ser Ala
Leu Thr Val Leu Ser Val Ile Ala Val Pro Ala 145 150 155 160 Ile Ala
Cys Leu Gly Gly Tyr Ser Val Trp Leu Ala Val Asn Gly Met 165 170 175
Gly Gly Leu Asp Ala Leu Lys Ala Val Val Pro Ala Gln Pro Leu Asp 180
185 190 Phe Asn Val Ala Leu Ala Leu Val Val Gly Ser Phe Ile Ser Ala
Gly 195 200 205 Thr Leu Thr Ala Asp Phe Val Arg Phe Gly Arg Asn Ala
Lys Leu Ala 210 215 220 Val Leu Val Ala Met Val Ala Phe Phe Leu Gly
Asn Ser Leu Met Phe 225 230 235 240 Ile Phe Gly Ala Ala Gly Ala Ala
Ala Leu Gly Met Ala Asp Ile Ser 245 250 255 Asp Val Met Ile Ala Gln
Gly Leu Leu Leu Pro Ala Ile Val Val Leu 260 265 270 Gly Leu Asn Ile
Trp Thr Thr Asn Asp Asn Ala Leu Tyr Ala Ser Gly 275 280 285 Leu Gly
Phe Ala Asn Ile Thr Gly Met Ser Ser Lys Thr Leu Ser Val 290 295 300
Ile Asn Gly Ile Ile Gly Thr Val Cys Ala Leu Trp Leu Tyr Asn Asn 305
310 315 320 Phe Val Gly Trp Leu Thr Phe Leu Ser Ala Ala Ile Pro Pro
Val Gly 325 330 335 Gly Val Ile Ile Ala Asp Tyr Leu Met Asn Arg Arg
Arg Tyr Glu His 340 345 350 Phe Ala Thr Thr Arg Met Met Ser Val Asn
Trp Val Ala Ile Leu Ala 355 360 365 Val Ala Leu Gly Ile Ala Ala Gly
His Trp Leu Pro Gly Ile Val Pro 370 375 380 Val Asn Ala Val Leu Gly
Gly Ala Leu Ser Tyr Leu Ile Leu Asn Pro 385 390 395 400 Ile Leu Asn
Arg Lys Thr Thr Ala Ala Met Thr His Val Glu Ala Asn 405 410 415 Ser
Val Glu <210> SEQ ID NO 156 <211> LENGTH: 428
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(428) <223> OTHER INFORMATION: amtB -
Escherichia coli K-12 MG1655 (B0451; 945084) <400> SEQUENCE:
156 Met Lys Ile Ala Thr Ile Lys Thr Gly Leu Ala Ser Leu Ala Met Leu
1 5 10 15 Pro Gly Leu Val Met Ala Ala Pro Ala Val Ala Asp Lys Ala
Asp Asn 20 25 30 Ala Phe Met Met Ile Cys Thr Ala Leu Val Leu Phe
Met Thr Ile Pro 35 40 45 Gly Ile Ala Leu Phe Tyr Gly Gly Leu Ile
Arg Gly Lys Asn Val Leu 50 55 60 Ser Met Leu Thr Gln Val Thr Val
Thr Phe Ala Leu Val Cys Ile Leu 65 70 75 80 Trp Val Val Tyr Gly Tyr
Ser Leu Ala Phe Gly Glu Gly Asn Asn Phe 85 90 95 Phe Gly Asn Ile
Asn Trp Leu Met Leu Lys Asn Ile Glu Leu Thr Ala 100 105 110 Val Met
Gly Ser Ile Tyr Gln Tyr Ile His Val Ala Phe Gln Gly Ser 115 120 125
Phe Ala Cys Ile Thr Val Gly Leu Ile Val Gly Ala Leu Ala Glu Arg 130
135 140 Ile Arg Phe Ser Ala Val Leu Ile Phe Val Val Val Trp Leu Thr
Leu 145 150 155 160 Ser Tyr Ile Pro Ile Ala His Met Val Trp Gly Gly
Gly Leu Leu Ala 165 170 175 Ser His Gly Ala Leu Asp Phe Ala Gly Gly
Thr Val Val His Ile Asn 180 185 190 Ala Ala Ile Ala Gly Leu Val Gly
Ala Tyr Leu Ile Gly Lys Arg Val 195 200 205 Gly Phe Gly Lys Glu Ala
Phe Lys Pro His Asn Leu Pro Met Val Phe 210 215 220 Thr Gly Thr Ala
Ile Leu Tyr Ile Gly Trp Phe Gly Phe Asn Ala Gly 225 230 235 240 Ser
Ala Gly Thr Ala Asn Glu Ile Ala Ala Leu Ala Phe Val Asn Thr 245 250
255 Val Val Ala Thr Ala Ala Ala Ile Leu Gly Trp Ile Phe Gly Glu Trp
260 265 270 Ala Leu Arg Gly Lys Pro Ser Leu Leu Gly Ala Cys Ser Gly
Ala Ile 275 280 285 Ala Gly Leu Val Gly Val Thr Pro Ala Cys Gly Tyr
Ile Gly Val Gly 290 295 300 Gly Ala Leu Ile Ile Gly Val Val Ala Gly
Leu Ala Gly Leu Trp Gly 305 310 315 320 Val Thr Met Leu Lys Arg Leu
Leu Arg Val Asp Asp Pro Cys Asp Val 325 330 335 Phe Gly Val His Gly
Val Cys Gly Ile Val Gly Cys Ile Met Thr Gly 340 345 350 Ile Phe Ala
Ala Ser Ser Leu Gly Gly Val Gly Phe Ala Glu Gly Val 355 360 365 Thr
Met Gly His Gln Leu Leu Val Gln Leu Glu Ser Ile Ala Ile Thr 370 375
380 Ile Val Trp Ser Gly Val Val Ala Phe Ile Gly Tyr Lys Leu Ala Asp
385 390 395 400 Leu Thr Val Gly Leu Arg Val Pro Glu Glu Gln Glu Arg
Glu Gly Leu 405 410 415 Asp Val Asn Ser His Gly Glu Asn Ala Tyr Asn
Ala 420 425 <210> SEQ ID NO 157 <211> LENGTH: 466
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(466) <223> OTHER INFORMATION: GABA permease
GabP Escherichia coli <400> SEQUENCE: 157 Met Gly Gln Ser Ser
Gln Pro His Glu Leu Gly Gly Gly Leu Lys Ser 1 5 10 15 Arg His Val
Thr Met Leu Ser Ile Ala Gly Val Ile Gly Ala Ser Leu 20 25 30 Phe
Val Gly Ser Ser Val Ala Ile Ala Glu Ala Gly Pro Ala Val Leu 35 40
45 Leu Ala Tyr Leu Phe Ala Gly Leu Leu Val Val Met Ile Met Arg Met
50 55 60 Leu Ala Glu Met Ala Val Ala Thr Pro Asp Thr Gly Ser Phe
Ser Thr 65 70 75 80 Tyr Ala Asp Lys Ala Ile Gly Arg Trp Ala Gly Tyr
Thr Ile Gly Trp 85 90 95 Leu Tyr Trp Trp Phe Trp Val Leu Val Ile
Pro Leu Glu Ala Asn Ile 100 105 110 Ala Ala Met Ile Leu His Ser Trp
Val Pro Gly Ile Pro Ile Trp Leu 115 120 125 Phe Ser Leu Val Ile Thr
Leu Ala Leu Thr Gly Ser Asn Leu Leu Ser 130 135 140 Val Lys Asn Tyr
Gly Glu Phe Glu Phe Trp Leu Ala Leu Cys Lys Val 145 150 155 160 Ile
Ala Ile Leu Ala Phe Ile Phe Leu Gly Ala Val Ala Ile Ser Gly 165 170
175 Phe Tyr Pro Tyr Ala Glu Val Ser Gly Ile Ser Arg Leu Trp Asp Ser
180 185 190 Gly Gly Phe Met Pro Asn Gly Phe Gly Ala Val Leu Ser Ala
Met Leu 195 200 205 Ile Thr Met Phe Ser Phe Met Gly Ala Glu Ile Val
Thr Ile Ala Ala 210 215 220 Ala Glu Ser Asp Thr Pro Glu Lys His Ile
Val Arg Ala Thr Asn Ser 225 230 235 240 Val Ile Trp Arg Ile Ser Ile
Phe Tyr Leu Cys Ser Ile Phe Val Val 245 250 255 Val Ala Leu Ile Pro
Trp Asn Met Pro Gly Leu Lys Ala Val Gly Ser 260 265 270 Tyr Arg Ser
Val Leu Glu Leu Leu Asn Ile Pro His Ala Lys Leu Ile 275 280 285 Met
Asp Cys Val Ile Leu Leu Ser Val Thr Ser Cys Leu Asn Ser Ala 290 295
300 Leu Tyr Thr Ala Ser Arg Met Leu Tyr Ser Leu Ser Arg Arg Gly Asp
305 310 315 320 Ala Pro Ala Val Met Gly Lys Ile Asn Arg Ser Lys Thr
Pro Tyr Val 325 330 335 Ala Val Leu Leu Ser Thr Gly Ala Ala Phe Leu
Thr Val Val Val Asn 340 345 350 Tyr Tyr Ala Pro Ala Lys Val Phe Lys
Phe Leu Ile Asp Ser Ser Gly 355 360 365 Ala Ile Ala Leu Leu Val Tyr
Leu Val Ile Ala Val Ser Gln Leu Arg 370 375 380 Met Arg Lys Ile Leu
Arg Ala Glu Gly Ser Glu Ile Arg Leu Arg Met 385 390 395 400 Trp Leu
Tyr Pro Trp Leu Thr Trp Leu Val Ile Gly Phe Ile Thr Phe 405 410 415
Val Leu Val Val Met Leu Phe Arg Pro Ala Gln Gln Leu Glu Val Ile 420
425 430 Ser Thr Gly Leu Leu Ala Ile Gly Ile Ile Cys Thr Val Pro Ile
Met 435 440 445 Ala Arg Trp Lys Lys Leu Val Leu Trp Gln Lys Thr Pro
Val His Asn 450 455 460 Thr Arg 465 <210> SEQ ID NO 158
<211> LENGTH: 412 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(412) <223> OTHER
INFORMATION: mtnH Escherichia coli <400> SEQUENCE: 158 Met
Thr Asn Tyr Arg Val Glu Ser Ser Ser Gly Arg Ala Ala Arg Lys 1 5 10
15 Met Arg Leu Ala Leu Met Gly Pro Ala Phe Ile Ala Ala Ile Gly Tyr
20 25 30 Ile Asp Pro Gly Asn Phe Ala Thr Asn Ile Gln Ala Gly Ala
Ser Phe 35 40 45 Gly Tyr Gln Leu Leu Trp Val Val Val Trp Ala Asn
Leu Met Ala Met 50 55 60 Leu Ile Gln Ile Leu Ser Ala Lys Leu Gly
Ile Ala Thr Gly Lys Asn 65 70 75 80 Leu Ala Glu Gln Ile Arg Asp His
Tyr Pro Arg Pro Val Val Trp Phe 85 90 95 Tyr Trp Val Gln Ala Glu
Ile Ile Ala Met Ala Thr Asp Leu Ala Glu 100 105 110 Phe Ile Gly Ala
Ala Ile Gly Phe Lys Leu Ile Leu Gly Val Ser Leu 115 120 125 Leu Gln
Gly Ala Val Leu Thr Gly Ile Ala Thr Phe Leu Ile Leu Met 130 135 140
Leu Gln Arg Arg Gly Gln Lys Pro Leu Glu Lys Val Ile Gly Gly Leu 145
150 155 160 Leu Leu Phe Val Ala Ala Ala Tyr Ile Val Glu Leu Ile Phe
Ser Gln 165 170 175 Pro Asn Leu Ala Gln Leu Gly Lys Gly Met Val Ile
Pro Ser Leu Pro 180 185 190 Thr Ser Glu Ala Val Phe Leu Ala Ala Gly
Val Leu Gly Ala Thr Ile 195 200 205 Met Pro His Val Ile Tyr Leu His
Ser Ser Leu Thr Gln His Leu His 210 215 220 Gly Gly Ser Arg Gln Gln
Arg Tyr Ser Ala Thr Lys Trp Asp Val Ala 225 230 235 240 Ile Ala Met
Thr Ile Ala Gly Phe Val Asn Leu Ala Met Met Ala Thr 245 250 255 Ala
Ala Ala Ala Phe His Phe Ser Gly His Thr Gly Val Ala Asp Leu 260 265
270 Asp Glu Ala Tyr Leu Thr Leu Gln Pro Leu Leu Ser His Ala Ala Ala
275 280 285 Thr Val Phe Gly Leu Ser Leu Val Ala Ala Gly Leu Ser Ser
Thr Val 290 295 300 Val Gly Thr Leu Ala Gly Gln Val Val Met Gln Gly
Phe Ile Arg Phe 305 310 315 320 His Ile Pro Leu Trp Val Arg Arg Thr
Val Thr Met Leu Pro Ser Phe 325 330 335 Ile Val Ile Leu Met Gly Leu
Asp Pro Thr Arg Ile Leu Val Met Ser 340 345 350 Gln Val Leu Leu Ser
Phe Gly Ile Ala Leu Ala Leu Val Pro Leu Leu 355 360 365 Ile Phe Thr
Ser Asp Ser Lys Leu Met Gly Asp Leu Val Asn Ser Lys 370 375 380 Arg
Val Lys Gln Thr Gly Trp Val Ile Val Val Leu Val Val Ala Leu 385 390
395 400 Asn Ile Trp Leu Leu Val Gly Thr Ala Leu Gly Leu 405 410
<210> SEQ ID NO 159 <211> LENGTH: 1320 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1320) <223> OTHER INFORMATION: Nucleotide sequence:
BCAA transporter BrnQ from E. coli <400> SEQUENCE: 159
atgacccatc aattaagatc gcgcgatatc atcgctctgg gctttatgac atttgcgttg
60 ttcgtcggcg caggtaacat tattttccct ccaatggtcg gcttgcaggc
aggcgaacac 120 gtctggactg cggcattcgg cttcctcatt actgccgttg
gcctaccggt attaacggta 180 gtggcgctgg caaaagttgg cggcggtgtt
gacagtctca gcacgccaat tggtaaagtc 240 gctggcgtac tgctggcaac
agtttgttac ctggcggtgg ggccgctttt tgctacgccg 300 cgtacagcta
ccgtttcttt tgaagtgggc attgcgccgc tgacgggtga ttccgcgctg 360
ccgctgttta tttacagcct ggtctatttc gctatcgtta ttctggtttc gctctatccg
420 ggcaagctgc tggataccgt gggcaacttc cttgcgccgc tgaaaattat
cgcgctggtc 480 atcctgtctg ttgccgcaat tatctggccg gcgggttcta
tcagtacggc gactgaggct 540 tatcaaaacg ctgcgttttc taacggcttc
gtcaacggct atctgaccat ggatacgctg 600 ggcgcaatgg tgtttggtat
cgttattgtt aacgcggcgc gttctcgtgg cgttaccgaa 660 gcgcgtctgc
tgacccgtta taccgtctgg gctggcctga tggcgggtgt tggtctgact 720
ctgctgtacc tggcgctgtt ccgtctgggt tcagacagcg cgtcgctggt cgatcagtct
780 gcaaacggtg cggcgatcct gcatgcttac gttcagcata cctttggcgg
cggcggtagc 840 ttcctgctgg cggcgttaat cttcatcgcc tgcctggtca
cggcggttgg cctgacctgt 900 gcttgtgcag aattcttcgc ccagtacgta
ccgctctctt atcgtacgct ggtgtttatc 960 ctcggcggct tctcgatggt
ggtgtctaac ctcggcttga gccagctgat tcagatctct 1020 gtaccggtgc
tgaccgccat ttatccgccg tgtatcgcac tggttgtatt aagttttaca 1080
cgctcatggt ggcataattc gtcccgcgtg attgctccgc cgatgtttat cagcctgctt
1140 tttggtattc tcgacgggat caaggcatct gcattcagcg atatcttacc
gtcctgggcg 1200 cagcgtttac cgctggccga acaaggtctg gcgtggttaa
tgccaacagt ggtgatggtg 1260 gttctggcca ttatctggga tcgtgcggca
ggtcgtcagg tgacctccag cgctcactaa 1320 <210> SEQ ID NO 160
<211> LENGTH: 439 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(439) <223> OTHER
INFORMATION: AA sequence: BCAA transporter BrnQ from E. coli
<400> SEQUENCE: 160 Met Thr His Gln Leu Arg Ser Arg Asp Ile
Ile Ala Leu Gly Phe Met 1 5 10 15 Thr Phe Ala Leu Phe Val Gly Ala
Gly Asn Ile Ile Phe Pro Pro Met 20 25 30 Val Gly Leu Gln Ala Gly
Glu His Val Trp Thr Ala Ala Phe Gly Phe 35 40 45 Leu Ile Thr Ala
Val Gly Leu Pro Val Leu Thr Val Val Ala Leu Ala 50 55 60 Lys Val
Gly Gly Gly Val Asp Ser Leu Ser Thr Pro Ile Gly Lys Val 65 70 75 80
Ala Gly Val Leu Leu Ala Thr Val Cys Tyr Leu Ala Val Gly Pro Leu 85
90 95 Phe Ala Thr Pro Arg Thr Ala Thr Val Ser Phe Glu Val Gly Ile
Ala 100 105 110 Pro Leu Thr Gly Asp Ser Ala Leu Pro Leu Phe Ile Tyr
Ser Leu Val 115 120 125 Tyr Phe Ala Ile Val Ile Leu Val Ser Leu Tyr
Pro Gly Lys Leu Leu 130 135 140 Asp Thr Val Gly Asn Phe Leu Ala Pro
Leu Lys Ile Ile Ala Leu Val 145 150 155 160 Ile Leu Ser Val Ala Ala
Ile Ile Trp Pro Ala Gly Ser Ile Ser Thr 165 170 175 Ala Thr Glu Ala
Tyr Gln Asn Ala Ala Phe Ser Asn Gly Phe Val Asn 180 185 190 Gly Tyr
Leu Thr Met Asp Thr Leu Gly Ala Met Val Phe Gly Ile Val 195 200 205
Ile Val Asn Ala Ala Arg Ser Arg Gly Val Thr Glu Ala Arg Leu Leu 210
215 220 Thr Arg Tyr Thr Val Trp Ala Gly Leu Met Ala Gly Val Gly Leu
Thr 225 230 235 240 Leu Leu Tyr Leu Ala Leu Phe Arg Leu Gly Ser Asp
Ser Ala Ser Leu 245 250 255 Val Asp Gln Ser Ala Asn Gly Ala Ala Ile
Leu His Ala Tyr Val Gln 260 265 270 His Thr Phe Gly Gly Gly Gly Ser
Phe Leu Leu Ala Ala Leu Ile Phe 275 280 285 Ile Ala Cys Leu Val Thr
Ala Val Gly Leu Thr Cys Ala Cys Ala Glu 290 295 300 Phe Phe Ala Gln
Tyr Val Pro Leu Ser Tyr Arg Thr Leu Val Phe Ile 305 310 315 320 Leu
Gly Gly Phe Ser Met Val Val Ser Asn Leu Gly Leu Ser Gln Leu 325 330
335 Ile Gln Ile Ser Val Pro Val Leu Thr Ala Ile Tyr Pro Pro Cys Ile
340 345 350 Ala Leu Val Val Leu Ser Phe Thr Arg Ser Trp Trp His Asn
Ser Ser 355 360 365 Arg Val Ile Ala Pro Pro Met Phe Ile Ser Leu Leu
Phe Gly Ile Leu 370 375 380 Asp Gly Ile Lys Ala Ser Ala Phe Ser Asp
Ile Leu Pro Ser Trp Ala 385 390 395 400 Gln Arg Leu Pro Leu Ala Glu
Gln Gly Leu Ala Trp Leu Met Pro Thr 405 410 415 Val Val Met Val Val
Leu Ala Ile Ile Trp Asp Arg Ala Ala Gly Arg 420 425 430 Gln Val Thr
Ser Ser Ala His 435 <210> SEQ ID NO 161 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic:
consensus sequence <400> SEQUENCE: 161 ttgttgayry rtcaacwa 18
<210> SEQ ID NO 162 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: consensus sequence
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (8)..(8) <223> OTHER INFORMATION: n is a, c, g, or
t <400> SEQUENCE: 162 ttataatnat tataa 15 <210> SEQ ID
NO 163 <211> LENGTH: 355 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic: katG Regulatory sequence <400>
SEQUENCE: 163 tgtggctttt atgaaaatca cacagtgatc acaaatttta
aacagagcac aaaatgctgc 60 ctcgaaatga gggcgggaaa ataaggttat
cagccttgtt ttctccctca ttacttgaag 120 gatatgaagc taaaaccctt
ttttataaag catttgtccg aattcggaca taatcaaaaa 180 agcttaatta
agatcaattt gatctacatc tctttaacca acaatatgta agatctcaac 240
tatcgcatcc gtggattaat tcaattataa cttctctcta acgctgtgta tcgtaacggt
300 aacactgtag aggggagcac attgatgcga attcattaaa gaggagaaag gtacc
355 <210> SEQ ID NO 164 <211> LENGTH: 228 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic: dps Regulatory
sequence <400> SEQUENCE: 164 ttccgaaaat tcctggcgag cagataaata
agaattgttc ttatcaatat atctaactca 60 ttgaatcttt attagttttg
tttttcacgc ttgttaccac tattagtgtg ataggaacag 120 ccagaatagc
ggaacacata gccggtgcta tacttaatct cgttaattac tgggacataa 180
catcaagagg atatgaaatt cgaattcatt aaagaggaga aaggtacc 228
<210> SEQ ID NO 165 <211> LENGTH: 334 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: ahpC Regulatory sequence
<400> SEQUENCE: 165 gcttagatca ggtgattgcc ctttgtttat
gagggtgttg taatccatgt cgttgttgca 60 tttgtaaggg caacacctca
gcctgcaggc aggcactgaa gataccaaag ggtagttcag 120 attacacggt
cacctggaaa gggggccatt ttacttttta tcgccgctgg cggtgcaaag 180
ttcacaaagt tgtcttacga aggttgtaag gtaaaactta tcgatttgat aatggaaacg
240 cattagccga atcggcaaaa attggttacc ttacatctca tcgaaaacac
ggaggaagta 300 tagatgcgaa ttcattaaag aggagaaagg tacc 334
<210> SEQ ID NO 166 <211> LENGTH: 134 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: oxyS Regulatory sequence
<400> SEQUENCE: 166 ctcgagttca ttatccatcc tccatcgcca
cgatagttca tggcgatagg tagaatagca 60 atgaacgatt atccctatca
agcattctga ctgataattg ctcacacgaa ttcattaaag 120 aggagaaagg tacc 134
<210> SEQ ID NO 167 <211> LENGTH: 416 <212> TYPE:
PRT <213> ORGANISM: Pseudomonas sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: Pseudomonas kynureninase <400>
SEQUENCE: 167 Met Thr Thr Arg Asn Asp Cys Leu Ala Leu Asp Ala Gln
Asp Ser Leu 1 5 10 15 Ala Pro Leu Arg Gln Gln Phe Ala Leu Pro Glu
Gly Val Ile Tyr Leu 20 25 30 Asp Gly Asn Ser Leu Gly Ala Arg Pro
Val Ala Ala Leu Ala Arg Ala 35 40 45 Gln Ala Val Ile Ala Glu Glu
Trp Gly Asn Gly Leu Ile Arg Ser Trp 50 55 60 Asn Ser Ala Gly Trp
Arg Asp Leu Ser Glu Arg Leu Gly Asn Arg Leu 65 70 75 80 Ala Thr Leu
Ile Gly Ala Arg Asp Gly Glu Val Val Val Thr Asp Thr 85 90 95 Thr
Ser Ile Asn Leu Phe Lys Val Leu Ser Ala Ala Leu Arg Val Gln 100 105
110 Ala Thr Arg Ser Pro Glu Arg Arg Val Ile Val Thr Glu Thr Ser Asn
115 120 125 Phe Pro Thr Asp Leu Tyr Ile Ala Glu Gly Leu Ala Asp Met
Leu Gln 130 135 140 Gln Gly Tyr Thr Leu Arg Leu Val Asp Ser Pro Glu
Glu Leu Pro Gln 145 150 155 160 Ala Ile Asp Gln Asp Thr Ala Val Val
Met Leu Thr His Val Asn Tyr 165 170 175 Lys Thr Gly Tyr Met His Asp
Met Gln Ala Leu Thr Ala Leu Ser His 180 185 190 Glu Cys Gly Ala Leu
Ala Ile Trp Asp Leu Ala His Ser Ala Gly Ala 195 200 205 Val Pro Val
Asp Leu His Gln Ala Gly Ala Asp Tyr Ala Ile Gly Cys 210 215 220 Thr
Tyr Lys Tyr Leu Asn Gly Gly Pro Gly Ser Gln Ala Phe Val Trp 225 230
235 240 Val Ser Pro Gln Leu Cys Asp Leu Val Pro Gln Pro Leu Ser Gly
Trp 245 250 255 Phe Gly His Ser Arg Gln Phe Ala Met Glu Pro Arg Tyr
Glu Pro Ser 260 265 270 Asn Gly Ile Ala Arg Tyr Leu Cys Gly Thr Gln
Pro Ile Thr Ser Leu 275 280 285 Ala Met Val Glu Cys Gly Leu Asp Val
Phe Ala Gln Thr Asp Met Ala 290 295 300 Ser Leu Arg Arg Lys Ser Leu
Ala Leu Thr Asp Leu Phe Ile Glu Leu 305 310 315 320 Val Glu Gln Arg
Cys Ala Ala His Glu Leu Thr Leu Val Thr Pro Arg 325 330 335 Glu His
Ala Lys Arg Gly Ser His Val Ser Phe Glu His Pro Glu Gly 340 345 350
Tyr Ala Val Ile Gln Ala Leu Ile Asp Arg Gly Val Ile Gly Asp Tyr 355
360 365 Arg Glu Pro Arg Ile Met Arg Phe Gly Phe Thr Pro Leu Tyr Thr
Thr 370 375 380 Phe Thr Glu Val Trp Asp Ala Val Gln Ile Leu Gly Glu
Ile Leu Asp 385 390 395 400 Arg Lys Thr Trp Ala Gln Ala Gln Phe Gln
Val Arg His Ser Val Thr 405 410 415 <210> SEQ ID NO 168
<211> LENGTH: 465 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(465) <223> OTHER
INFORMATION: Human kynureninase <400> SEQUENCE: 168 Met Glu
Pro Ser Ser Leu Glu Leu Pro Ala Asp Thr Val Gln Arg Ile 1 5 10 15
Ala Ala Glu Leu Lys Cys His Pro Thr Asp Glu Arg Val Ala Leu His 20
25 30 Leu Asp Glu Glu Asp Lys Leu Arg His Phe Arg Glu Cys Phe Tyr
Ile 35 40 45 Pro Lys Ile Gln Asp Leu Pro Pro Val Asp Leu Ser Leu
Val Asn Lys 50 55 60 Asp Glu Asn Ala Ile Tyr Phe Leu Gly Asn Ser
Leu Gly Leu Gln Pro 65 70 75 80 Lys Met Val Lys Thr Tyr Leu Glu Glu
Glu Leu Asp Lys Trp Ala Lys 85 90 95 Ile Ala Ala Tyr Gly His Glu
Val Gly Lys Arg Pro Trp Ile Thr Gly 100 105 110 Asp Glu Ser Ile Val
Gly Leu Met Lys Asp Ile Val Gly Ala Asn Glu 115 120 125 Lys Glu Ile
Ala Leu Met Asn Ala Leu Thr Val Asn Leu His Leu Leu 130 135 140 Met
Leu Ser Phe Phe Lys Pro Thr Pro Lys Arg Tyr Lys Ile Leu Leu 145 150
155 160 Glu Ala Lys Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser Gln
Leu 165 170 175 Gln Leu His Gly Leu Asn Ile Glu Glu Ser Met Arg Met
Ile Lys Pro 180 185 190 Arg Glu Gly Glu Glu Thr Leu Arg Ile Glu Asp
Ile Leu Glu Val Ile 195 200 205 Glu Lys Glu Gly Asp Ser Ile Ala Val
Ile Leu Phe Ser Gly Val His 210 215 220 Phe Tyr Thr Gly Gln His Phe
Asn Ile Pro Ala Ile Thr Lys Ala Gly 225 230 235 240 Gln Ala Lys Gly
Cys Tyr Val Gly Phe Asp Leu Ala His Ala Val Gly 245 250 255 Asn Val
Glu Leu Tyr Leu His Asp Trp Gly Val Asp Phe Ala Cys Trp 260 265 270
Cys Ser Tyr Lys Tyr Leu Asn Ala Gly Ala Gly Gly Ile Ala Gly Ala 275
280 285 Phe Ile His Glu Lys His Ala His Thr Ile Lys Pro Ala Leu Val
Gly 290 295 300 Trp Phe Gly His Glu Leu Ser Thr Arg Phe Lys Met Asp
Asn Lys Leu 305 310 315 320 Gln Leu Ile Pro Gly Val Cys Gly Phe Arg
Ile Ser Asn Pro Pro Ile 325 330 335 Leu Leu Val Cys Ser Leu His Ala
Ser Leu Glu Ile Phe Lys Gln Ala 340 345 350 Thr Met Lys Ala Leu Arg
Lys Lys Ser Val Leu Leu Thr Gly Tyr Leu 355 360 365 Glu Tyr Leu Ile
Lys His Asn Tyr Gly Lys Asp Lys Ala Ala Thr Lys 370 375 380 Lys Pro
Val Val Asn Ile Ile Thr Pro Ser His Val Glu Glu Arg Gly 385 390 395
400 Cys Gln Leu Thr Ile Thr Phe Ser Val Pro Asn Lys Asp Val Phe Gln
405 410 415 Glu Leu Glu Lys Arg Gly Val Val Cys Asp Lys Arg Asn Pro
Asn Gly 420 425 430 Ile Arg Val Ala Pro Val Pro Leu Tyr Asn Ser Phe
His Asp Val Tyr 435 440 445 Lys Phe Thr Asn Leu Leu Thr Ser Ile Leu
Asp Ser Ala Glu Thr Lys 450 455 460 Asn 465 <210> SEQ ID NO
169 <211> LENGTH: 378 <212> TYPE: PRT <213>
ORGANISM: Shewanella sp. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(378) <223> OTHER
INFORMATION: Shewanella kynureninase <400> SEQUENCE: 169 Met
Leu Leu Asn Val Lys Gln Asp Phe Cys Leu Ala Gly Pro Gly Tyr 1 5 10
15 Leu Leu Asn His Ser Val Gly Arg Pro Leu Lys Ser Thr Glu Gln Ala
20 25 30 Leu Lys Gln Ala Phe Phe Ala Pro Trp Gln Glu Ser Gly Arg
Glu Pro 35 40 45 Trp Gly Gln Trp Leu Gly Val Ile Asp Asn Phe Thr
Ala Ala Leu Ala 50 55 60 Ser Leu Phe Asn Gly Gln Pro Gln Asp Phe
Cys Pro Gln Val Asn Leu 65 70 75 80 Ser Ser Ala Leu Thr Lys Ile Val
Met Ser Leu Asp Arg Leu Thr Arg 85 90 95 Asp Leu Thr Arg Asn Gly
Gly Ala Val Val Leu Met Ser Glu Ile Asp 100 105 110 Phe Pro Ser Met
Gly Phe Ala Leu Lys Lys Ala Leu Pro Ala Ser Cys 115 120 125 Glu Leu
Arg Phe Ile Pro Lys Ser Leu Asp Val Thr Asp Pro Asn Val 130 135 140
Trp Asp Ala His Ile Cys Asp Asp Val Asp Leu Val Phe Val Ser His 145
150 155 160 Ala Tyr Ser Asn Thr Gly Gln Gln Ala Pro Leu Ala Gln Ile
Ile Ser 165 170 175 Leu Ala Arg Glu Arg Gly Cys Leu Ser Leu Val Asp
Val Ala Gln Ser 180 185 190 Ala Gly Ile Leu Pro Leu Asp Leu Ala Lys
Leu Gln Pro Asp Phe Met 195 200 205 Ile Gly Ser Ser Val Lys Trp Leu
Cys Ser Gly Pro Gly Ala Ala Tyr 210 215 220 Leu Trp Val Asn Pro Ala
Ile Leu Pro Glu Cys Gln Pro Gln Asp Val 225 230 235 240 Gly Trp Phe
Ser His Glu Asn Pro Phe Glu Phe Asp Ile His Asp Phe 245 250 255 Arg
Tyr His Pro Thr Ala Leu Arg Phe Trp Gly Gly Thr Pro Ser Ile 260 265
270 Ala Pro Tyr Ala Ile Ala Ala His Ser Ile Glu Tyr Phe Ala Asn Ile
275 280 285 Gly Ser Gln Val Met Arg Glu His Asn Leu Gln Leu Met Glu
Pro Val 290 295 300 Val Gln Ala Leu Asp Asn Glu Leu Val Ser Pro Gln
Glu Val Asp Lys 305 310 315 320 Arg Ser Gly Thr Ile Ile Leu Gln Phe
Gly Glu Arg Gln Pro Gln Ile 325 330 335 Leu Ala Ala Leu Ala Ala Ala
Asn Ile Ser Val Asp Thr Arg Ser Leu 340 345 350 Gly Ile Arg Val Ser
Pro His Ile Tyr Asn Asp Glu Ala Asp Ile Ala 355 360 365 Arg Leu Leu
Gly Val Ile Lys Ala Asn Arg 370 375 <210> SEQ ID NO 170
<211> LENGTH: 1424 <212> TYPE: DNA <213>
ORGANISM: Pseudomonas sp. <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1424)
<223> OTHER INFORMATION: Ptet-kynU(Pseudomonas) <400>
SEQUENCE: 170 atctaatcta gacatcatta attcctaatt tttgttgaca
ctctatcatt gatagagtta 60 ttttaccact ccctatcagt gatagagaaa
agtgaattat ataaaagtgg gaggtgcccg 120 aatgacgacc cgaaatgatt
gcctagcgtt ggatgcacag gacagtctgg ctccgctgcg 180 ccaacaattt
gcgctgccgg agggtgtgat atacctggat ggcaattcgc tgggcgcacg 240
tccggtagct gcgctggctc gcgcgcaggc tgtgatcgca gaagaatggg gcaacgggtt
300 gatccgttca tggaactctg cgggctggcg tgatctgtct gaacgcctgg
gtaatcgcct 360 ggctaccctg attggtgcgc gcgatgggga agtagttgtt
actgatacca cctcgattaa 420 tctgtttaaa gtgctgtcag cggcgctgcg
cgtgcaagct acccgtagcc cggagcgccg 480 tgttatcgtg actgagacct
cgaatttccc gaccgacctg tatattgcgg aagggttggc 540 ggatatgctg
caacaaggtt acactctgcg tttggtggat tcaccggaag agctgccaca 600
ggctatagat caggacaccg cggtggtgat gctgacgcac gtaaattata aaaccggtta
660 tatgcacgac atgcaggctc tgaccgcgtt gagccacgag tgtggggctc
tggcgatttg 720 ggatctggcg cactctgctg gcgctgtgcc ggtggacctg
caccaagcgg gcgcggacta 780 tgcgattggc tgcacgtaca aatacctgaa
tggcggcccg ggttcgcaag cgtttgtttg 840 ggtttcgccg caactgtgcg
acctggtacc gcagccgctg tctggttggt tcggccatag 900 tcgccaattc
gcgatggagc cgcgctacga accttctaac ggcattgctc gctatctgtg 960
cggcactcag cctattacta gcttggctat ggtggagtgc ggcctggatg tgtttgcgca
1020 gacggatatg gcttcgctgc gccgtaaaag tctggcgctg actgatctgt
tcatcgagct 1080 ggttgaacaa cgctgcgctg cacacgaact gaccctggtt
actccacgtg aacacgcgaa 1140 acgcggctct cacgtgtctt ttgaacaccc
cgagggttac gctgttattc aagctctgat 1200 tgatcgtggc gtgatcggcg
attaccgtga gccacgtatt atgcgtttcg gtttcactcc 1260 tctgtatact
acttttacgg aagtttggga tgcagtacaa atcctgggcg aaatcctgga 1320
tcgtaagact tgggcgcagg ctcagtttca ggtgcgccac tctgttactt aaaaataaaa
1380 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttg 1424
<210> SEQ ID NO 171 <211> LENGTH: 1571 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1571) <223> OTHER INFORMATION: Ptet-kynU(Human)
<400> SEQUENCE: 171 atctaatcta gacatcatta attcctaatt
tttgttgaca ctctatcatt gatagagtta 60 ttttaccact ccctatcagt
gatagagaaa agtgaatatc aagacacgag gaggtaagat 120 tatggagcct
tcatctttag aactgccagc ggacacggtg cagcgcatcg cggcggaact 180
gaagtgccat ccgactgatg agcgtgtggc gctgcatctg gacgaagaag ataaactgcg
240 ccactttcgt gaatgttttt atattcctaa aattcaagac ttgccgccgg
tagatttgag 300 tctcgttaac aaagatgaaa acgcgatcta ctttctgggc
aactctctgg gtctgcaacc 360 aaaaatggtt aaaacgtacc tggaggaaga
actggataaa tgggcaaaaa tcgcggctta 420 tggtcacgaa gtgggcaagc
gtccttggat tactggcgac gagtctattg tgggtttgat 480 gaaagatatt
gtgggcgcga atgaaaagga aattgcactg atgaatgctc tgaccgttaa 540
tctgcacctg ctgatgctgt ctttttttaa accgaccccg aaacgctaca aaatactgct
600 ggaagcgaaa gcgtttccgt cggatcacta tgctatagaa agtcaactgc
agttgcatgg 660 tctgaatatc gaggaatcta tgcgcatgat taaaccgcgt
gagggtgaag aaacgctgcg 720 tattgaagac attctggaag ttattgaaaa
agaaggtgat tctatcgcag ttatactgtt 780 ttctggcgtg cacttttata
caggtcagca cttcaatatc ccggcaatca ctaaagcggg 840 gcaggcaaaa
ggctgctatg ttggttttga cctggcgcat gcagtgggga atgttgaact 900
gtatctgcac gattggggcg ttgatttcgc gtgttggtgt agctacaaat atctgaacgc
960 tggcgcgggt ggcattgctg gcgcttttat tcacgaaaaa cacgcgcaca
ccattaaacc 1020 ggctctggtt ggctggttcg gtcatgagct gagtactcgc
tttaaaatgg ataacaaact 1080 gcaattgatt ccgggtgttt gcggcttccg
tatcagcaat ccgccgattc tgctggtttg 1140 cagcctgcac gctagtctgg
aaatctttaa gcaggcgact atgaaagcgc tgcgcaaaaa 1200 atctgtgctg
ctgaccggct atctggagta tctgatcaaa cacaattatg gcaaagataa 1260
agctgcaact aaaaaaccgg tagtgaacat tatcaccccc tcacacgtgg aggagcgcgg
1320 ttgtcagctg actattactt tcagtgtacc taataaagat gtgttccagg
aactggaaaa 1380 acgcggcgtt gtttgtgata aacgtaaccc gaatggtatt
cgcgtggctc ctgtgccgct 1440 gtacaattca ttccacgatg tttataaatt
caccaacctg ctgacttcta ttctcgacag 1500 tgctgagact aaaaattaaa
aataaaacga aaggctcagt cgaaagactg ggcctttcgt 1560 tttatctgtt g 1571
<210> SEQ ID NO 172 <211> LENGTH: 1310 <212>
TYPE: DNA <213> ORGANISM: Shewanella sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1310) <223> OTHER INFORMATION: ptet-kynU(Shewanella)
<400> SEQUENCE: 172 atctaatcta gacatcatta attcctaatt
tttgttgaca ctctatcatt gatagagtta 60 ttttaccact ccctatcagt
gatagagaaa agtgaatggt tcaccaccac aaggagggat 120 tatgctgctg
aatgtaaaac aggacttttg cctggcaggc ccgggctacc tgctgaatca 180
ctcggttggc cgtccgctga aatcaactga gcaagcgctg aaacaagcat tttttgctcc
240 gtggcaagag agcggtcgtg aaccgtgggg ccagtggctg ggtgttattg
ataatttcac 300 tgctgcgctg gcatctctgt ttaatggtca accgcaggat
ttttgtccgc aggttaacct 360 gagcagcgcg ctgactaaaa ttgtgatgtc
actggatcgt ctgactcgcg atctgacccg 420 caatggcggt gctgttgtgc
tgatgtctga aatcgatttc ccatctatgg gcttcgcgtt 480 gaaaaaagcg
ctgccagcga gctgcgaact gcgttttatc ccgaaaagtc tggacgtgac 540
tgatccgaac gtatgggatg cacacatctg tgatgatgta gacctggttt ttgtgtctca
600 cgcctatagt aatacgggcc aacaggctcc gctggcgcaa atcatctctc
tggcgcgtga 660 acgtggctgc ctgtcactgg tggatgtagc gcaatcagcg
gggattttgc cgctggatct 720 ggcgaaactg caaccggact tcatgatcgg
cagttcggtt aaatggctgt gctcgggccc 780 tggtgcggca tatctgtggg
ttaatccggc gattctgccg gaatgtcagc cgcaggatgt 840 gggctggttt
tcacatgaga atccctttga attcgacatc cacgatttcc gctaccaccc 900
gactgcactg cgcttttggg gtggtacgcc gtcgatcgcg ccttatgcga tcgcggcgca
960 ctcgatcgaa tattttgcca atatcggctc gcaagtgatg cgtgaacaca
acctgcaact 1020 gatggaaccg gtggttcagg cgctggacaa tgaactggtg
agcccgcagg aagtggataa 1080 acgctcaggc actattattc tgcaattcgg
tgaacgtcaa ccgcaaattc tggcggctct 1140 ggctgcggcg aacatttcgg
tggacactcg ttctttgggg attcgtgtta gtccgcacat 1200 ttataatgat
gaggcggaca ttgcgcgcct gctgggtgtg atcaaagcaa atcgctaaaa 1260
ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 1310
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 172
<210> SEQ ID NO 1 <211> LENGTH: 290 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(290)
<223> OTHER INFORMATION: FNR Responsive Promoter <400>
SEQUENCE: 1 gtcagcataa caccctgacc tctcattaat tgttcatgcc gggcggcact
atcgtcgtcc 60 ggccttttcc tctcttactc tgctacgtac atctatttct
ataaatccgt tcaatttgtc 120 tgttttttgc acaaacatga aatatcagac
aattccgtga cttaagaaaa tttatacaaa 180 tcagcaatat accccttaag
gagtatataa aggtgaattt gatttacatc aataagcggg 240 gttgctgaat
cgttaaggta ggcggtaata gaaaagaaat cgaggcaaaa 290 <210> SEQ ID
NO 2 <211> LENGTH: 173 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(173) <223>
OTHER INFORMATION: FNR Responsive Promoter <400> SEQUENCE: 2
atttcctctc atcccatccg gggtgagagt cttttccccc gacttatggc tcatgcatgc
60 atcaaaaaag atgtgagctt gatcaaaaac aaaaaatatt tcactcgaca
ggagtattta 120 tattgcgccc gttacgtggg cttcgactgt aaatcagaaa
ggagaaaaca cct 173 <210> SEQ ID NO 3 <211> LENGTH: 305
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(305) <223> OTHER INFORMATION: FNR Responsive
Promoter <400> SEQUENCE: 3 gtcagcataa caccctgacc tctcattaat
tgttcatgcc gggcggcact atcgtcgtcc 60 ggccttttcc tctcttactc
tgctacgtac atctatttct ataaatccgt tcaatttgtc 120 tgttttttgc
acaaacatga aatatcagac aattccgtga cttaagaaaa tttatacaaa 180
tcagcaatat accccttaag gagtatataa aggtgaattt gatttacatc aataagcggg
240 gttgctgaat cgttaaggat ccctctagaa ataattttgt ttaactttaa
gaaggagata 300 tacat 305 <210> SEQ ID NO 4 <211>
LENGTH: 180 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(180) <223> OTHER INFORMATION: FNR
Responsive Promoter <400> SEQUENCE: 4 catttcctct catcccatcc
ggggtgagag tcttttcccc cgacttatgg ctcatgcatg 60 catcaaaaaa
gatgtgagct tgatcaaaaa caaaaaatat ttcactcgac aggagtattt 120
atattgcgcc cggatccctc tagaaataat tttgtttaac tttaagaagg agatatacat
180 <210> SEQ ID NO 5 <211> LENGTH: 199 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(199) <223> OTHER INFORMATION: FNR Responsive Promoter
<400> SEQUENCE: 5 agttgttctt attggtggtg ttgctttatg gttgcatcgt
agtaaatggt tgtaacaaaa 60 gcaatttttc cggctgtctg tatacaaaaa
cgccgtaaag tttgagcgaa gtcaataaac 120 tctctaccca ttcagggcaa
tatctctctt ggatccctct agaaataatt ttgtttaact 180 ttaagaagga
gatatacat 199 <210> SEQ ID NO 6 <211> LENGTH: 1647
<212> TYPE: DNA <213> ORGANISM: Lactococcus lactis
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1647) <223> OTHER INFORMATION: kivD
(Lactococcus lactis IFPL730) <400> SEQUENCE: 6 atgtatacag
taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60
tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccacaaggat
120 atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga
tggctatgct 180 cgtactaaaa aagctgccgc atttcttaca acctttggag
taggtgaatt gagtgcagtt 240 aatggattag caggaagtta cgccgaaaat
ttaccagtag tagaaatagt gggatcacct 300 acatcaaaag ttcaaaatga
aggaaaattt gttcatcata cgctggctga cggtgatttt 360 aaacacttta
tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420
aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc
480 tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc
actccctttg 540 aaaaaggaaa actcaacttc aaatacaagt gaccaagaaa
ttttgaacaa aattcaagaa 600 agcttgaaaa atgccaaaaa accaatcgtg
attacaggac atgaaataat tagttttggc 660 ttagaaaaaa cagtcactca
atttatttca aagacaaaac tacctattac gacattaaac 720 tttggtaaaa
gttcagttga tgaagccctc ccttcatttt taggaatcta taatggtaca 780
ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatctt gatgcttgga
840 gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga
aaataaaatg 900 atttcactga atatagatga aggaaaaata tttaacgaaa
gaatccaaaa ttttgatttt 960 gaatccctca tctcctctct cttagaccta
agcgaaatag aatacaaagg aaaatatatc 1020 gataaaaagc aagaagactt
tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080 caagcagttg
aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140
ttctttggcg cttcatcaat tttcttaaaa tcaaagagtc attttattgg tcaaccctta
1200 tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc
agataaagaa 1260 agcagacacc ttttatttat tggtgatggt tcacttcaac
ttacagtgca agaattagga 1320 ttagcaatca gagaaaaaat taatccaatt
tgctttatta tcaataatga tggttataca 1380 gtcgaaagag aaattcatgg
accaaatcaa agctacaatg atattccaat gtggaattac 1440 tcaaaattac
cagaatcgtt tggagcaaca gaagatcgag tagtctcaaa aatcgttaga 1500
actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac
1560 tggattgagt taattttggc aaaagaaggt gcaccaaaag tactgaaaaa
aatgggcaaa 1620 ctatttgctg aacaaaataa atcataa 1647 <210> SEQ
ID NO 7 <211> LENGTH: 5739 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic: Tet-bkd construct sequence
<400> SEQUENCE: 7 gtaaaacgac ggccagtgaa ttcgttaaga cccactttca
catttaagtt gtttttctaa 60 tccgcatatg atcaattcaa ggccgaataa
gaaggctggc tctgcacctt ggtgatcaaa 120 taattcgata gcttgtcgta
ataatggcgg catactatca gtagtaggtg tttccctttc 180 ttctttagcg
acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac 240
agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa
300 ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat
gtacttttgc 360 tccatcgcga tgacttagta aagcacatct aaaactttta
gcgttattac gtaaaaaatc 420 ttgccagctt tccccttcta aagggcaaaa
gtgagtatgg tgcctatcta acatctcaat 480 ggctaaggcg tcgagcaaag
cccgcttatt ttttacatgc caatacaatg taggctgctc 540 tacacctagc
ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag 600
cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatca ttaattccta
660 atttttgttg acactctatc attgatagag ttattttacc actccctatc
agtgatagag 720 aaaagtgaac tctagaaata attttgttta actttaagaa
ggagatatac atatgagtga 780 ttacgagccg ttgcgtctgc atgtcccgga
gcccaccggg cgtcctggct gcaagaccga 840 cttttcctat ctgcacctgt
cccccgccgg cgaggtacgc aagccgccgg tggatgtcga 900 gcccgccgaa
accagcgacc tggcctacag cctggtacgt gtgctcgacg acgacggcca 960
cgccgtcggt ccctggaatc cgcagctcag caacgaacaa ctgctgcgcg gcatgcgggc
1020 gatgctcaag acccgcctgt tcgacgcgcg catgctcacc gcgcaacggc
agaaaaagct 1080 ttccttctat atgcaatgcc tcggcgagga agccatcgcc
accgcccaca ccctggccct 1140 gcgcgacggc gacatgtgct ttccgaccta
tcgccagcaa ggcatcctga tcacccgcga 1200 atacccgctg gtggacatga
tctgccagct tctctccaac gaggccgacc cgctcaaggg 1260 ccgccagctg
ccgatcatgt actcgagcaa ggaggcaggt ttcttctcca tctccggcaa 1320
cctcgccacc cagttcatcc aggcggtcgg ctggggcatg gcctcggcga tcaagggcga
1380 cacgcgcatc gcctcggcct ggatcggcga cggcgccacc gccgagtcgg
acttccacac 1440 cgccctcacc ttcgcccatg tctaccgcgc gccggtaatc
ctcaacgtgg tcaacaacca 1500 gtgggcgatc tccaccttcc aggccatcgc
cggcggcgaa ggcaccacct tcgccaaccg 1560 tggcgtgggc tgcgggatcg
cctcgctgcg ggtcgacggc aatgacttcc tggcggtcta 1620 cgccgcctcc
gagtgggccg ccgagcgcgc ccggcgcaac ctcgggccga gcctgatcga 1680
atgggtcacc taccgcgccg gcccgcactc gacttcggac gacccgtcca agtaccgccc
1740 cgccgacgac tggaccaact tcccgctggg cgacccgatc gcccgcctga
agcggcacat 1800 gatcggcctc ggcatctggt cggaggaaca gcacgaagcc
acccacaagg ccctcgaagc 1860 cgaagtactg gccgcgcaga aacaggcgga
gagccatggc accctgatcg acggccgggt 1920 gccgagcgcc gccagcatgt
tcgaggacgt ctatgcagaa ctgccggagc atctgcgccg 1980
gcaacgccag gagctcgggg tatgaatgcc atgaacccgc aacacgagaa cgcccagacg
2040 gtcaccagca tgaccatgat ccaggcgctg cgctcggcga tggacatcat
gctcgagcgc 2100 gacgacgacg tggtggtatt cggccaggac gtcggctact
tcggcggcgt gttccgctgc 2160 accgaaggcc tgcagaagaa atacggcacc
tcgcgggtgt tcgatgcgcc gatctccgag 2220 agcggcatca tcggcgccgc
ggtcggcatg ggtgcctacg gcctgcgccc ggtggtggag 2280 atccagttcg
ccgactacgt ctacccggcc tccgaccagt tgatctccga ggcggcgcgc 2340
ctgcgctatc gctcggccgg cgacttcatc gtgccgatga ccgtacgcat gccctgtggc
2400 ggcggcatct acggcgggca aacgcacagc cagagcccgg aggcgatgtt
cacccaggtc 2460 tgcggcctgc gcacggtgat gccgtccaac ccctacgacg
ccaagggcct gctgatcgcc 2520 tgcatcgaga acgacgaccc ggtgatcttc
ctcgagccca agcgcctcta caacggcccg 2580 ttcgatggcc accacgaccg
cccggtgacg ccctggtcca agcatccggc cagccaggtg 2640 ccggacggct
actacaaggt gccgctggac aaggcggcga tcgtccgccc cggcgcggcg 2700
ctgaccgtgc tgacctacgg caccatggtc tacgtggccc aggccgcggc cgacgaaacc
2760 ggcctggacg ccgagatcat cgacctgcgc agcctctggc cgctggacct
ggaaaccatc 2820 gtcgcctcgg tgaagaagac cggccgctgc gtcatcgccc
acgaggcgac ccgcacctgt 2880 gggttcggcg ccgagctgat gtcgctggtg
caggagcact gcttccacca cctggaggcg 2940 ccgatcgagc gcgtcaccgg
ttgggacacc ccctacccgc atgcccagga gtgggcgtat 3000 ttccccggcc
ccgcgcgcgt cggcgcggca ttcaagcgtg tgatggaggt ctgaatgggt 3060
acccatgtga tcaagatgcc ggacatcggg gaaggcatcg ccgaggtcga actggtggag
3120 tggcatgtcc aggtcggcga ctcggtcaat gaagaccagg tcctcgccga
ggtgatgacc 3180 gacaaggcca cggtggagat tccctcgccg gtggccggac
gcatcctcgc cctcggcggc 3240 cagccgggcc aggtgatggc ggtgggcggc
gaactgatcc gcctggaggt ggaaggcgcc 3300 ggcaacctcg ccgagagtcc
ggccgcggcg acgccggccg cgcccgtcgc cgccaccccg 3360 gagaaaccga
aggaagcccc ggtcgcggcg ccgaaagccg ccgccgaagc gccgcgcgcc 3420
ttgcgcgaca gcgaggcgcc acggcagcgg cgccagcccg gcgaacgccc gctggcctcc
3480 cccgcggtgc gccagcgcgc ccgcgacctg ggcatcgagt tgcagttcgt
gcagggcagc 3540 ggtcccgccg gacgcgtcct ccacgaggac ctcgatgcct
acctgaccca ggatggcagc 3600 gtcgcgcgca gcggcggcgc cgcgcagggg
tatgccgagc gacacgacga acaggcggtg 3660 ccggtgatcg gcctgcgtcg
caagatcgcc cagaagatgc aggacgccaa gcgacgcatc 3720 ccgcatttca
gctatgtcga ggaaatcgac gtcaccgatc tggaagccct gcgcgcccat 3780
ctcaaccaga aatggggtgg ccagcgcggc aagctgaccc tgctgccgtt cctggtccgc
3840 gccatggtcg tggcgctgcg cgacttcccg cagttgaacg cgcgctacga
cgacgaggcc 3900 gaggtggtca cccgctacgg cgcggtgcac gtcggcatcg
ccacccagag cgacaacggc 3960 ctgatggtgc cggtgctgcg ccacgccgaa
tcgcgcgacc tctggggcaa cgccagcgaa 4020 gtggcgcgcc tggccgaagc
cgcacgcagc ggcaaggcgc aacgccagga gctgtccggc 4080 tcgaccatca
ccctgagcag cctcggcgtg ctcggcggga tcgtcagcac accggtgatc 4140
aaccatccgg aggtggccat cgtcggcgtc aaccgcatcg tcgagcgacc gatggtggtc
4200 ggcggcaaca tcgtcgtgcg caagatgatg aacctctcct cctccttcga
ccaccgggtg 4260 gtcgacggga tggacgcggc ggccttcatc caggccgtgc
gcggcctgct cgaacatccc 4320 gccaccctgt tcctggagta agcgatgagc
cagatcctga agacttccct gctgatcgtc 4380 ggcggcggtc ccggcggcta
cgtcgcggcg atccgtgccg ggcaactggg cattcccacc 4440 gtactggtgg
agggcgccgc cctcggcggc acctgtctga acgtcggctg catcccgtcg 4500
aaggcgctga tccacgccgc cgaggaatac ctcaaggccc gccactatgc cagccggtcg
4560 gcgctgggca tccaggtaca ggcgccgagc atcgacatcg cccgcaccgt
ggaatggaag 4620 gacgccatcg tcgaccgcct caccagcggc gtcgccgcgc
tgctgaagaa acacggggtc 4680 gatgtcgtcc agggctgggc gaggatcctc
gacggcaaaa gcgtggcggt cgaactcgcc 4740 ggcggcggca gccagcgcat
cgagtgcgag catctgctgc tggccgccgg ctcgcagagc 4800 gtcgagctac
cgatcctgcc gctgggcggc aaggtgatct cctccaccga ggcgctggcg 4860
cccggcagcc tgcccaagcg cctggtggtg gtcggcggcg gctacatcgg cctggagctg
4920 ggtaccgcct accgcaagct cggcgtcgag gtggcggtgg tggaagcgca
accacgcatc 4980 ctgccgggct acgacgaaga actgaccaag ccggtggccc
aggccttgcg caggctgggc 5040 gtcgagctgt acctcgggca cagcctgctg
ggcccgagcg agaacggcgt gcgggtccgc 5100 gacggcgccg gcgaggagcg
cgagatcgcc gccgaccagg tactggtggc ggtcggccgc 5160 aagccgcgca
gcgaaggctg gaacctggaa agcctgggcc tggacatgaa cggccgggcg 5220
gtgaaggtcg acgaccagtg ccgcacctcg atgcgcaatg tctgggccat aggcgatctc
5280 gccggcgagc cgatgctcgc gcaccgggcc atggcccagg gcgagatggt
cgccgagctg 5340 atcgccggca agcgtcgcca gttcgccccg gtggcgatcc
ccgcggtgtg cttcaccgat 5400 ccggaagtgg tggtcgccgg gttgtccccg
gagcaggcga aggatgccgg cctggactgc 5460 ctggtggcga gcttcccgtt
cgccgccaac ggtcgcgcca tgaccctgga ggccaacgaa 5520 ggcttcgtcc
gcgtggtggc gcgtcgcgac aaccacctgg tcgtcggctg gcaggcggtg 5580
ggcaaggcgg tttcggaact gtccacggcc ttcgcccagt cgctggagat gggcgcccgc
5640 ctggaagaca tcgccggcac catccacgcc catccgaccc tcggcgaagc
ggtccaggaa 5700 gccgccctgc gcgcgctggg acacgccctg cacatctga 5739
<210> SEQ ID NO 8 <211> LENGTH: 6780 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: Tet-ldh-bkd construct
<400> SEQUENCE: 8 gtaaaacgac ggccagtgaa ttcgttaaga cccactttca
catttaagtt gtttttctaa 60 tccgcatatg atcaattcaa ggccgaataa
gaaggctggc tctgcacctt ggtgatcaaa 120 taattcgata gcttgtcgta
ataatggcgg catactatca gtagtaggtg tttccctttc 180 ttctttagcg
acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac 240
agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa
300 ttgattttcg agagtttcat actgtttttc tgtaggccgt gtacctaaat
gtacttttgc 360 tccatcgcga tgacttagta aagcacatct aaaactttta
gcgttattac gtaaaaaatc 420 ttgccagctt tccccttcta aagggcaaaa
gtgagtatgg tgcctatcta acatctcaat 480 ggctaaggcg tcgagcaaag
cccgcttatt ttttacatgc caatacaatg taggctgctc 540 tacacctagc
ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag 600
cagctctaat gcgctgttaa tcactttact tttatctaat ctagacatca ttaattccta
660 atttttgttg acactctatc attgatagag ttattttacc actccctatc
agtgatagag 720 aaaagtgaac tctagaaata attttgttta actttaagaa
ggagatatac atatgttcga 780 catgatggac gcggcccggc tcgagggtct
gcacctcgcc caagacccgg ccacgggact 840 caaggccatt atcgccatcc
acagcacgcg actcggcccg gcgctgggtg gttgtcgcta 900 cctgccttac
cccaacgacg aagccgccat cggcgacgcc atccgcctgg cccagggcat 960
gagctacaag gcggccctgg ccgggctgga gcagggcggc ggcaaggcgg tgatcatccg
1020 cccgccgcac ctggacaatc gcggcgcgct gttcgaggcc ttcgggcgct
tcatcgaaag 1080 cctcggcgga cgctacatca ctgcggtgga cagcggtacc
tccagcgccg acatggactg 1140 catcgcccag cagacccgcc acgtcaccag
caccacccag gccggcgacc cctcgccgca 1200 taccgccctc ggcgtgttcg
ccgggattcg cgccagcgcc caggcgcgcc tcggcagcga 1260 cgacctggaa
ggcctgcggg tcgcggtgca ggggctcggc cacgtcggct acgcattggc 1320
cgagcaactg gcggcggtcg gcgccgagct gctggtctgc gacctcgatc ccggccgggt
1380 gcaactggcc gtcgagcagc tcggtgccca tccgctggcg ccggaggcat
tgctctccac 1440 cccttgcgac atcctcgcgc cctgcggcct gggcggcgtg
ctcaccagcc agagcgtcag 1500 ccagttgcgc tgcgcggcgg tggccggggc
ggcgaacaac cagttggagc ggccggaggt 1560 cgccgacgag ctggaggcgc
gcggcatcct ctatgcgccg gactacgtga tcaactccgg 1620 cggcctgatc
tacgtcgccc tcaagcaccg cggcgccgat ccgcacagca tcaccgcgca 1680
cctggcgcgg attcccgcgc ggctcaccga gatctatgcc catgcccagg ccgaccacca
1740 gtcgccggcg cggatcgccg accgtctggc ggaacggatt ctctacggcc
cgcagtgaga 1800 aggagatata catatgagtg attacgagcc gttgcgtctg
catgtcccgg agcccaccgg 1860 gcgtcctggc tgcaagaccg acttttccta
tctgcacctg tcccccgccg gcgaggtacg 1920 caagccgccg gtggatgtcg
agcccgccga aaccagcgac ctggcctaca gcctggtacg 1980 tgtgctcgac
gacgacggcc acgccgtcgg tccctggaat ccgcagctca gcaacgaaca 2040
actgctgcgc ggcatgcggg cgatgctcaa gacccgcctg ttcgacgcgc gcatgctcac
2100 cgcgcaacgg cagaaaaagc tttccttcta tatgcaatgc ctcggcgagg
aagccatcgc 2160 caccgcccac accctggccc tgcgcgacgg cgacatgtgc
tttccgacct atcgccagca 2220 aggcatcctg atcacccgcg aatacccgct
ggtggacatg atctgccagc ttctctccaa 2280 cgaggccgac ccgctcaagg
gccgccagct gccgatcatg tactcgagca aggaggcagg 2340 tttcttctcc
atctccggca acctcgccac ccagttcatc caggcggtcg gctggggcat 2400
ggcctcggcg atcaagggcg acacgcgcat cgcctcggcc tggatcggcg acggcgccac
2460 cgccgagtcg gacttccaca ccgccctcac cttcgcccat gtctaccgcg
cgccggtaat 2520 cctcaacgtg gtcaacaacc agtgggcgat ctccaccttc
caggccatcg ccggcggcga 2580 aggcaccacc ttcgccaacc gtggcgtggg
ctgcgggatc gcctcgctgc gggtcgacgg 2640 caatgacttc ctggcggtct
acgccgcctc cgagtgggcc gccgagcgcg cccggcgcaa 2700 cctcgggccg
agcctgatcg aatgggtcac ctaccgcgcc ggcccgcact cgacttcgga 2760
cgacccgtcc aagtaccgcc ccgccgacga ctggaccaac ttcccgctgg gcgacccgat
2820 cgcccgcctg aagcggcaca tgatcggcct cggcatctgg tcggaggaac
agcacgaagc 2880 cacccacaag gccctcgaag ccgaagtact ggccgcgcag
aaacaggcgg agagccatgg 2940 caccctgatc gacggccggg tgccgagcgc
cgccagcatg ttcgaggacg tctatgcaga 3000 actgccggag catctgcgcc
ggcaacgcca ggagctcggg gtatgaatgc catgaacccg 3060 caacacgaga
acgcccagac ggtcaccagc atgaccatga tccaggcgct gcgctcggcg 3120
atggacatca tgctcgagcg cgacgacgac gtggtggtat tcggccagga cgtcggctac
3180 ttcggcggcg tgttccgctg caccgaaggc ctgcagaaga aatacggcac
ctcgcgggtg 3240 ttcgatgcgc cgatctccga gagcggcatc atcggcgccg
cggtcggcat gggtgcctac 3300 ggcctgcgcc cggtggtgga gatccagttc
gccgactacg tctacccggc ctccgaccag 3360 ttgatctccg aggcggcgcg
cctgcgctat cgctcggccg gcgacttcat cgtgccgatg 3420
accgtacgca tgccctgtgg cggcggcatc tacggcgggc aaacgcacag ccagagcccg
3480 gaggcgatgt tcacccaggt ctgcggcctg cgcacggtga tgccgtccaa
cccctacgac 3540 gccaagggcc tgctgatcgc ctgcatcgag aacgacgacc
cggtgatctt cctcgagccc 3600 aagcgcctct acaacggccc gttcgatggc
caccacgacc gcccggtgac gccctggtcc 3660 aagcatccgg ccagccaggt
gccggacggc tactacaagg tgccgctgga caaggcggcg 3720 atcgtccgcc
ccggcgcggc gctgaccgtg ctgacctacg gcaccatggt ctacgtggcc 3780
caggccgcgg ccgacgaaac cggcctggac gccgagatca tcgacctgcg cagcctctgg
3840 ccgctggacc tggaaaccat cgtcgcctcg gtgaagaaga ccggccgctg
cgtcatcgcc 3900 cacgaggcga cccgcacctg tgggttcggc gccgagctga
tgtcgctggt gcaggagcac 3960 tgcttccacc acctggaggc gccgatcgag
cgcgtcaccg gttgggacac cccctacccg 4020 catgcccagg agtgggcgta
tttccccggc cccgcgcgcg tcggcgcggc attcaagcgt 4080 gtgatggagg
tctgaatggg tacccatgtg atcaagatgc cggacatcgg ggaaggcatc 4140
gccgaggtcg aactggtgga gtggcatgtc caggtcggcg actcggtcaa tgaagaccag
4200 gtcctcgccg aggtgatgac cgacaaggcc acggtggaga ttccctcgcc
ggtggccgga 4260 cgcatcctcg ccctcggcgg ccagccgggc caggtgatgg
cggtgggcgg cgaactgatc 4320 cgcctggagg tggaaggcgc cggcaacctc
gccgagagtc cggccgcggc gacgccggcc 4380 gcgcccgtcg ccgccacccc
ggagaaaccg aaggaagccc cggtcgcggc gccgaaagcc 4440 gccgccgaag
cgccgcgcgc cttgcgcgac agcgaggcgc cacggcagcg gcgccagccc 4500
ggcgaacgcc cgctggcctc ccccgcggtg cgccagcgcg cccgcgacct gggcatcgag
4560 ttgcagttcg tgcagggcag cggtcccgcc ggacgcgtcc tccacgagga
cctcgatgcc 4620 tacctgaccc aggatggcag cgtcgcgcgc agcggcggcg
ccgcgcaggg gtatgccgag 4680 cgacacgacg aacaggcggt gccggtgatc
ggcctgcgtc gcaagatcgc ccagaagatg 4740 caggacgcca agcgacgcat
cccgcatttc agctatgtcg aggaaatcga cgtcaccgat 4800 ctggaagccc
tgcgcgccca tctcaaccag aaatggggtg gccagcgcgg caagctgacc 4860
ctgctgccgt tcctggtccg cgccatggtc gtggcgctgc gcgacttccc gcagttgaac
4920 gcgcgctacg acgacgaggc cgaggtggtc acccgctacg gcgcggtgca
cgtcggcatc 4980 gccacccaga gcgacaacgg cctgatggtg ccggtgctgc
gccacgccga atcgcgcgac 5040 ctctggggca acgccagcga agtggcgcgc
ctggccgaag ccgcacgcag cggcaaggcg 5100 caacgccagg agctgtccgg
ctcgaccatc accctgagca gcctcggcgt gctcggcggg 5160 atcgtcagca
caccggtgat caaccatccg gaggtggcca tcgtcggcgt caaccgcatc 5220
gtcgagcgac cgatggtggt cggcggcaac atcgtcgtgc gcaagatgat gaacctctcc
5280 tcctccttcg accaccgggt ggtcgacggg atggacgcgg cggccttcat
ccaggccgtg 5340 cgcggcctgc tcgaacatcc cgccaccctg ttcctggagt
aagcgatgag ccagatcctg 5400 aagacttccc tgctgatcgt cggcggcggt
cccggcggct acgtcgcggc gatccgtgcc 5460 gggcaactgg gcattcccac
cgtactggtg gagggcgccg ccctcggcgg cacctgtctg 5520 aacgtcggct
gcatcccgtc gaaggcgctg atccacgccg ccgaggaata cctcaaggcc 5580
cgccactatg ccagccggtc ggcgctgggc atccaggtac aggcgccgag catcgacatc
5640 gcccgcaccg tggaatggaa ggacgccatc gtcgaccgcc tcaccagcgg
cgtcgccgcg 5700 ctgctgaaga aacacggggt cgatgtcgtc cagggctggg
cgaggatcct cgacggcaaa 5760 agcgtggcgg tcgaactcgc cggcggcggc
agccagcgca tcgagtgcga gcatctgctg 5820 ctggccgccg gctcgcagag
cgtcgagcta ccgatcctgc cgctgggcgg caaggtgatc 5880 tcctccaccg
aggcgctggc gcccggcagc ctgcccaagc gcctggtggt ggtcggcggc 5940
ggctacatcg gcctggagct gggtaccgcc taccgcaagc tcggcgtcga ggtggcggtg
6000 gtggaagcgc aaccacgcat cctgccgggc tacgacgaag aactgaccaa
gccggtggcc 6060 caggccttgc gcaggctggg cgtcgagctg tacctcgggc
acagcctgct gggcccgagc 6120 gagaacggcg tgcgggtccg cgacggcgcc
ggcgaggagc gcgagatcgc cgccgaccag 6180 gtactggtgg cggtcggccg
caagccgcgc agcgaaggct ggaacctgga aagcctgggc 6240 ctggacatga
acggccgggc ggtgaaggtc gacgaccagt gccgcacctc gatgcgcaat 6300
gtctgggcca taggcgatct cgccggcgag ccgatgctcg cgcaccgggc catggcccag
6360 ggcgagatgg tcgccgagct gatcgccggc aagcgtcgcc agttcgcccc
ggtggcgatc 6420 cccgcggtgt gcttcaccga tccggaagtg gtggtcgccg
ggttgtcccc ggagcaggcg 6480 aaggatgccg gcctggactg cctggtggcg
agcttcccgt tcgccgccaa cggtcgcgcc 6540 atgaccctgg aggccaacga
aggcttcgtc cgcgtggtgg cgcgtcgcga caaccacctg 6600 gtcgtcggct
ggcaggcggt gggcaaggcg gtttcggaac tgtccacggc cttcgcccag 6660
tcgctggaga tgggcgcccg cctggaagac atcgccggca ccatccacgc ccatccgacc
6720 ctcggcgaag cggtccagga agccgccctg cgcgcgctgg gacacgccct
gcacatctga 6780 <210> SEQ ID NO 9 <211> LENGTH: 5597
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic:
Tet-livKHMGF construct <400> SEQUENCE: 9 ccagtgaatt
cgttaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat 60
caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata attcgatagc
120 ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt
ctttagcgac 180 ttgatgctct tgatcttcca atacgcaacc taaagtaaaa
tgccccacag cgctgagtgc 240 atataatgca ttctctagtg aaaaaccttg
ttggcataaa aaggctaatt gattttcgag 300 agtttcatac tgtttttctg
taggccgtgt acctaaatgt acttttgctc catcgcgatg 360 acttagtaaa
gcacatctaa aacttttagc gttattacgt aaaaaatctt gccagctttc 420
cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg ctaaggcgtc
480 gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta
cacctagctt 540 ctgggcgagt ttacgggttg ttaaaccttc gattccgacc
tcattaagca gctctaatgc 600 gctgttaatc actttacttt tatctaatct
agacatcatt aattcctaat ttttgttgac 660 actctatcat tgatagagtt
attttaccac tccctatcag tgatagagaa aagtgaactc 720 tagaaataat
tttgtttaac tttaagaagg agatatacat atgaaacgga atgcgaaaac 780
tatcatcgca gggatgattg cactggcaat ttcacacacc gctatggctg acgatattaa
840 agtcgccgtt gtcggcgcga tgtccggccc gattgcccag tggggcgata
tggaatttaa 900 cggcgcgcgt caggcaatta aagacattaa tgccaaaggg
ggaattaagg gcgataaact 960 ggttggcgtg gaatatgacg acgcatgcga
cccgaaacaa gccgttgcgg tcgccaacaa 1020 aatcgttaat gacggcatta
aatacgttat tggtcatctg tgttcttctt ctacccagcc 1080 tgcgtcagat
atctatgaag acgaaggtat tctgatgatc tcgccgggag cgaccaaccc 1140
ggagctgacc caacgcggtt atcaacacat tatgcgtact gccgggctgg actcttccca
1200 ggggccaacg gcggcaaaat acattcttga gacggtgaag ccccagcgca
tcgccatcat 1260 tcacgacaaa caacagtatg gcgaagggct ggcgcgttcg
gtgcaggacg ggctgaaagc 1320 ggctaacgcc aacgtcgtct tcttcgacgg
tattaccgcc ggggagaaag atttctccgc 1380 gctgatcgcc cgcctgaaaa
aagaaaacat cgacttcgtt tactacggcg gttactaccc 1440 ggaaatgggg
cagatgctgc gccaggcccg ttccgttggc ctgaaaaccc agtttatggg 1500
gccggaaggt gtgggtaatg cgtcgttgtc gaacattgcc ggtgatgccg ccgaaggcat
1560 gttggtcact atgccaaaac gctatgacca ggatccggca aaccagggca
tcgttgatgc 1620 gctgaaagca gacaagaaag atccgtccgg gccttatgtc
tggatcacct acgcggcggt 1680 gcaatctctg gcgactgccc ttgagcgtac
cggcagcgat gagccgctgg cgctggtgaa 1740 agatttaaaa gctaacggtg
caaacaccgt gattgggccg ctgaactggg atgaaaaagg 1800 cgatcttaag
ggatttgatt ttggtgtctt ccagtggcac gccgacggtt catccacggc 1860
agccaagtga tcatcccacc gcccgtaaaa tgcgggcggg tttagaaagg ttaccttatg
1920 tctgagcagt ttttgtattt cttgcagcag atgtttaacg gcgtcacgct
gggcagtacc 1980 tacgcgctga tagccatcgg ctacaccatg gtttacggca
ttatcggcat gatcaacttc 2040 gcccacggcg aggtttatat gattggcagc
tacgtctcat ttatgatcat cgccgcgctg 2100 atgatgatgg gcattgatac
cggctggctg ctggtagctg cgggattcgt cggcgcaatc 2160 gtcattgcca
gcgcctacgg ctggagtatc gaacgggtgg cttaccgccc ggtgcgtaac 2220
tctaagcgcc tgattgcact catctctgca atcggtatgt ccatcttcct gcaaaactac
2280 gtcagcctga ccgaaggttc gcgcgacgtg gcgctgccga gcctgtttaa
cggtcagtgg 2340 gtggtggggc atagcgaaaa cttctctgcc tctattacca
ccatgcaggc ggtgatctgg 2400 attgttacct tcctcgccat gctggcgctg
acgattttca ttcgctattc ccgcatgggt 2460 cgcgcgtgtc gtgcctgcgc
ggaagatctg aaaatggcga gtctgcttgg cattaacacc 2520 gaccgggtga
ttgcgctgac ctttgtgatt ggcgcggcga tggcggcggt ggcgggtgtg 2580
ctgctcggtc agttctacgg cgtcattaac ccctacatcg gctttatggc cgggatgaaa
2640 gcctttaccg cggcggtgct cggtgggatt ggcagcattc cgggagcgat
gattggcggc 2700 ctgattctgg ggattgcgga ggcgctctct tctgcctatc
tgagtacgga atataaagat 2760 gtggtgtcat tcgccctgct gattctggtg
ctgctggtga tgccgaccgg tattctgggt 2820 cgcccggagg tagagaaagt
atgaaaccga tgcatattgc aatggcgctg ctctctgccg 2880 cgatgttctt
tgtgctggcg ggcgtcttta tgggcgtgca actggagctg gatggcacca 2940
aactggtggt cgacacggct tcggatgtcc gttggcagtg ggtgtttatc ggcacggcgg
3000 tggtcttttt cttccagctt ttgcgaccgg ctttccagaa agggttgaaa
agcgtttccg 3060 gaccgaagtt tattctgccc gccattgatg gctccacggt
gaagcagaaa ctgttcctcg 3120 tggcgctgtt ggtgcttgcg gtggcgtggc
cgtttatggt ttcacgcggg acggtggata 3180 ttgccaccct gaccatgatc
tacattatcc tcggtctggg gctgaacgtg gttgttggtc 3240 tttctggtct
gctggtgctg gggtacggcg gtttttacgc catcggcgct tacacttttg 3300
cgctgctcaa tcactattac ggcttgggct tctggacctg cctgccgatt gctggattaa
3360 tggcagcggc ggcgggcttc ctgctcggtt ttccggtgct gcgtttgcgc
ggtgactatc 3420 tggcgatcgt taccctcggt ttcggcgaaa ttgtgcgcat
attgctgctc aataacaccg 3480 aaattaccgg cggcccgaac ggaatcagtc
agatcccgaa accgacactc ttcggactcg 3540 agttcagccg taccgctcgt
gaaggcggct gggacacgtt cagtaatttc tttggcctga 3600 aatacgatcc
ctccgatcgt gtcatcttcc tctacctggt ggcgttgctg ctggtggtgc 3660
taagcctgtt tgtcattaac cgcctgctgc ggatgccgct ggggcgtgcg tgggaagcgt
3720 tgcgtgaaga tgaaatcgcc tgccgttcgc tgggcttaag cccgcgtcgt
atcaagctga 3780 ctgcctttac cataagtgcc gcgtttgccg gttttgccgg
aacgctgttt gcggcgcgtc 3840 agggctttgt cagcccggaa tccttcacct
ttgccgaatc ggcgtttgtg ctggcgatag 3900
tggtgctcgg cggtatgggc tcgcaatttg cggtgattct ggcggcaatt ttgctggtgg
3960 tgtcgcgcga gttgatgcgt gatttcaacg aatacagcat gttaatgctc
ggtggtttga 4020 tggtgctgat gatgatctgg cgtccgcagg gcttgctgcc
catgacgcgc ccgcaactga 4080 agctgaaaaa cggcgcagcg aaaggagagc
aggcatgagt cagccattat tatctgttaa 4140 cggcctgatg atgcgcttcg
gcggcctgct ggcggtgaac aacgtcaatc ttgaactgta 4200 cccgcaggag
atcgtctcgt taatcggccc taacggtgcc ggaaaaacca cggtttttaa 4260
ctgtctgacc ggattctaca aacccaccgg cggcaccatt ttactgcgcg atcagcacct
4320 ggaaggttta ccggggcagc aaattgcccg catgggcgtg gtgcgcacct
tccagcatgt 4380 gcgtctgttc cgtgaaatga cggtaattga aaacctgctg
gtggcgcagc atcagcaact 4440 gaaaaccggg ctgttctctg gcctgttgaa
aacgccatcc ttccgtcgcg cccagagcga 4500 agcgctcgac cgcgccgcga
cctggcttga gcgcattggt ttgctggaac acgccaaccg 4560 tcaggcgagt
aacctggcct atggtgacca gcgccgtctt gagattgccc gctgcatggt 4620
gacgcagccg gagattttaa tgctcgacga acctgcggca ggtcttaacc cgaaagagac
4680 gaaagagctg gatgagctga ttgccgaact gcgcaatcat cacaacacca
ctatcttgtt 4740 gattgaacac gatatgaagc tggtgatggg aatttcggac
cgaatttacg tggtcaatca 4800 ggggacgccg ctggcaaacg gtacgccgga
gcagatccgt aataacccgg acgtgatccg 4860 tgcctattta ggtgaggcat
aagatggaaa aagtcatgtt gtcctttgac aaagtcagcg 4920 cccactacgg
caaaatccag gcgctgcatg aggtgagcct gcatatcaat cagggcgaga 4980
ttgtcacgct gattggcgcg aacggggcgg ggaaaaccac cttgctcggc acgttatgcg
5040 gcgatccgcg tgccaccagc gggcgaattg tgtttgatga taaagacatt
accgactggc 5100 agacagcgaa aatcatgcgc gaagcggtgg cgattgtccc
ggaagggcgt cgcgtcttct 5160 cgcggatgac ggtggaagag aacctggcga
tgggcggttt ttttgctgaa cgcgaccagt 5220 tccaggagcg cataaagtgg
gtgtatgagc tgtttccacg tctgcatgag cgccgtattc 5280 agcgggcggg
caccatgtcc ggcggtgaac agcagatgct ggcgattggt cgtgcgctga 5340
tgagcaaccc gcgtttgcta ctgcttgatg agccatcgct cggtcttgcg ccgattatca
5400 tccagcaaat tttcgacacc atcgagcagc tgcgcgagca ggggatgact
atctttctcg 5460 tcgagcagaa cgccaaccag gcgctaaagc tggcggatcg
cggctacgtg ctggaaaacg 5520 gccatgtagt gctttccgat actggtgatg
cgctgctggc gaatgaagcg gtgagaagtg 5580 cgtatttagg cgggtaa 5597
<210> SEQ ID NO 10 <211> LENGTH: 1104 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1104) <223> OTHER INFORMATION: livJ (Escherichia coli)
<400> SEQUENCE: 10 atgaacataa agggtaaagc gttactggca
ggatgtatcg cgctggcatt cagcaatatg 60 gctctggcag aagatattaa
agtcgcggtc gtgggcgcaa tgtccggtcc ggttgcgcag 120 tacggtgacc
aggagtttac cggcgcagag caggcggttg cggatatcaa cgctaaaggc 180
ggcattaaag gcaacaaact gcaaatcgta aaatatgacg atgcctgtga cccgaaacag
240 gcggttgcgg tggcgaacaa agtcgttaac gacggcatta aatatgtgat
tggtcacctc 300 tgttcttcat caacgcagcc tgcgtctgac atctacgaag
acgaaggcat tttaatgatc 360 accccagcgg caaccgcgcc ggagctgacc
gcccgtggct atcagctgat cctgcgcacc 420 accggcctgg actccgacca
ggggccgacg gcggcgaaat atattcttga gaaagtgaaa 480 ccgcagcgta
ttgctatcgt tcacgacaaa cagcaatacg gcgaaggtct ggcgcgagcg 540
gtgcaggacg gcctgaagaa aggcaatgca aacgtggtgt tctttgatgg catcaccgcc
600 ggggaaaaag atttctcaac gctggtggcg cgtctgaaaa aagagaatat
cgacttcgtt 660 tactacggcg gttatcaccc ggaaatgggg caaatcctgc
gtcaggcacg cgcggcaggg 720 ctgaaaactc agtttatggg gccggaaggt
gtggctaacg tttcgctgtc taacattgcg 780 ggcgaatcag cggaagggct
gctggtgacc aagccgaaga actacgatca ggttccggcg 840 aacaaaccca
ttgttgacgc gatcaaagcg aaaaaacagg acccaagtgg cgcattcgtt 900
tggaccacct acgccgcgct gcaatctttg caggcgggcc tgaatcagtc tgacgatccg
960 gctgaaatcg ccaaatacct gaaagcgaac tccgtggata ccgtaatggg
accgctgacc 1020 tgggatgaga aaggcgatct gaaaggcttt gagttcggcg
tatttgactg gcacgccaac 1080 ggcacggcga ccgatgcgaa gtaa 1104
<210> SEQ ID NO 11 <211> LENGTH: 639 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(639)
<223> OTHER INFORMATION: leucine exporter gene leuE
(Escherichia coli Nissle 1917) <400> SEQUENCE: 11 gtgttcgctg
aatacggggt tctgaattac tggacctatc tggttggggc catttttatt 60
gtgttggtgc cagggccaaa taccctgttt gtactcaaaa atagcgtcag tagcggtatg
120 aaaggcggtt atcttgcggc ctgtggtgta tttattggcg atgcggtatt
gatgtttctg 180 gcatgggctg gagtggcgac attaattaag accaccccga
tattattcaa catcgtacgt 240 tatcttggtg cgttttattt gctctatctg
gggagtaaaa ttctctacgc gaccctgaaa 300 ggtaaaaata gcgagaccaa
atccgatgag ccccaatacg gtgccatttt taaacgcgcg 360 ttaattttga
gcctgactaa tccgaaagcc attttgttct atgtgtcgtt tttcgtacag 420
tttatcgatg ttaatgcccc acatacggga atttcattct ttattctggc gacgacgctg
480 gaactggtga gtttctgcta tttgagcttc ctgattattt ctggggcttt
tgtcacgcag 540 tacatacgta ccaaaaagaa actggctaaa gtgggcaact
cactgattgg tttgatgttc 600 gtgggtttcg ccgcccgact ggcgacgctg
caatcctga 639 <210> SEQ ID NO 12 <211> LENGTH: 2268
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(2268) <223> OTHER INFORMATION: Arginine
decarboxylase (Escherichia coli) <400> SEQUENCE: 12
atgatgaaag tgttaatcgt ggaatctgaa tttctgcacc aggatacgtg ggtcggtaac
60 gctgttgaac gtctggccga tgctttaagc cagcaaaatg tgacagttat
caaatccacc 120 tcttttgacg atggctttgc cattctgtca agcaatgaag
ccatcgattg tctgatgttc 180 tcgtaccaga tggaacaccc cgatgagcac
caaaatgttc gtcagctgat cggcaaactt 240 cacgaacgtc aacagaacgt
accggtcttt ctgttaggcg accgcgaaaa ggccttggcg 300 gctatggatc
gcgatctgct ggagttggtc gacgagtttg cctggattct cgaggatacg 360
gcggatttta ttgccggtcg cgcagtcgcc gccatgacgc gctaccgcca acagctgctc
420 ccgccgctgt tttctgccct gatgaaatac tcggacattc acgaatacag
ctgggcagct 480 cccgggcacc agggcggcgt tggcttcacg aaaaccccag
ctggtcgctt ttatcatgac 540 tactacggcg agaatttatt tcgtaccgac
atgggcattg aacgtaccag cctgggctcg 600 ctgctggacc acacgggcgc
ttttggggaa tcagagaaat atgcagcacg cgtgttcggt 660 gcggaccgca
gttggtccgt cgtggtgggc accagtggta gcaaccgcac cattatgcag 720
gcgtgcatga ccgataatga tgtggtagtg gtggatcgca attgtcataa gagcatcgaa
780 caaggcttga tgctgactgg cgctaaacca gtctatatgg tgccgtcccg
taatcgctat 840 ggtattatcg gcccgattta tcctcaggag atgcagccgg
aaaccctcca gaagaaaatc 900 tcagagtccc cgttaactaa agataaagct
gggcaaaaac cgagttattg tgtagtaact 960 aattgtacgt atgatggtgt
ttgctataac gctaaggagg cccaagatct tctggaaaaa 1020 acaagtgatc
gtcttcattt tgatgaagct tggtacggtt atgcgcgttt caaccctatt 1080
tacgccgacc actatgcgat gcgtggtgaa cctggggatc ataatggccc tactgtgttt
1140 gccacccatt ctacgcataa actcctgaat gcgttgtcac aggcgagtta
catccacgta 1200 cgcgaaggcc gtggcgctat taattttagc cgctttaacc
aggcctatat gatgcacgcg 1260 acgacaagtc cgctgtatgc gatttgcgcg
tccaacgatg ttgcggtcag catgatggac 1320 ggcaacagcg gtctgtcgtt
aacccaggaa gtgattgatg aagcggtcga ctttcgccag 1380 gcgatggccc
gtctgtacaa agaattcacc gccgatggct cgtggttctt caaaccctgg 1440
aataaagaag tcgtgactga cccgcagacg ggcaaaactt atgattttgc agatgccccg
1500 acgaagcttc ttactacggt ccaggattgc tgggtgatgc acccggggga
gtcttggcat 1560 ggcttcaaag atatccctga taactggtct atgctcgacc
caatcaaagt ttcaatttta 1620 gctccaggca tgggcgaaga tggcgaactg
gaagagacgg gggtaccagc tgcgttggtt 1680 accgcctggt taggccgcca
tggtattgtt ccaacacgta ccactgattt tcagattatg 1740 tttctgttca
gtatgggtgt gacgcgcggt aaatggggga cgctggtcaa cactctctgc 1800
tcctttaaac gccattatga tgcgaacacg cccctggcgc aagtcatgcc agagctggtg
1860 gaacaatacc ctgatactta tgcgaacatg ggtatccacg atctgggaga
tactatgttc 1920 gcctggctta aagaaaataa cccgggggcc cgcctgaacg
aagcatatag tggcctgccc 1980 atggcggaaa ttactccgcg tgaagcctat
aatgccatcg ttgataataa cgtcgaatta 2040 gtatccatcg agaacctccc
cggtcgtatt gcggcaaata gcgtaatccc gtacccgccg 2100 ggtattccca
tgctgctcag cggcgaaaac ttcggtgata aaaattcccc gcaagtttct 2160
tatctgcgca gcctgcaatc gtgggaccat cactttcccg ggtttgagca tgaaactgaa
2220 gggacagaga tcatcgatgg catttatcat gtgatgtgcg tcaaggcg 2268
<210> SEQ ID NO 13 <211> LENGTH: 780 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(780)
<223> OTHER INFORMATION: ArgT (Escherichia coli) <400>
SEQUENCE: 13 atgaaaaaaa gcatcctcgc gctgtcactg ttagtgggtc tcagcgccgc
ggccagcagc 60 tatgctgctc ttcctgaaac ggtgcgcatc gggacggata
ccacttatgc accgtttagc 120 agcaaagatg ctaaaggaga cttcgtaggg
tttgatatcg atttaggcaa cgagatgtgc 180
aaacgtatgc aagtgaaatg tacctgggtg gcttcagact ttgatgcatt aatcccgagt
240 ttgaaagcaa aaaaaattga cgcaattatt tcgagcctga gcattacaga
taagcgccaa 300 caagaaattg ccttctcaga taaattatat gccgctgatt
cgcgtcttat cgcggctaaa 360 ggctccccta tccaaccaac gttggacagc
ctgaagggga aacatgtagg ggttctgcaa 420 gggtccacgc aggaagctta
cgccaatgaa acctggcgtt cgaaaggggt cgatgtggtg 480 gcgtacgcca
atcaggactt ggtgtattcc gatctggccg caggtcgtct ggacgcagct 540
ctgcaggacg aagtggcggc gagtgagggt ttcctgaaac agccagcagg caaagatttt
600 gcgttcgccg gctcgagtgt aaaggataaa aaatatttcg gggatggcac
gggtgtcggt 660 ttacgcaaag atgatgcaga actgaccgcg gcgtttaata
aagcccttgg cgaactgcgc 720 caagacggca catatgataa aatggcgaaa
aagtactttg acttcaatgt ttatggtgat 780 <210> SEQ ID NO 14
<211> LENGTH: 726 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(726) <223> OTHER
INFORMATION: artP (Escherichia coli) <400> SEQUENCE: 14
atgtctattc aattaaatgg catcaactgt ttctacggtg cacatcaagc cttatttgac
60 atcacgcttg attgcccgca aggggagaca ctggtgctgc tgggcccgag
tggagccggc 120 aaatcgtcgt tgctgcgggt gttgaacctg ttggagatgc
cgcgctcagg caccctgaat 180 atcgcgggca accatttcga ttttacgaaa
acaccgtccg ataaagctat tcgtgatctt 240 cgtcgcaacg tcggcatggt
gtttcagcag tataatttat gtgctcatct gacggttcag 300 caaaatctga
tcgaagcacc gtgtcgtgtg ttgggcctga gcaaagacca agccctggcc 360
agcgcagaaa aattattaga gcgcctgcgc ttgaaaccat attcggatcg gtacccactt
420 cacttaagcg ggggccagca acagcgcgtt gccatcgctc gtgcgctgat
gatggagccg 480 caagttctcc tttttgatga acctaccgca gcgcttgatc
cggagatcac ggcgcagatc 540 gtcagcatca ttcgtgaact cgctgagacg
aatattacac aagttattgt gacacatgag 600 gtagaagtgg ctcgcaagac
cgcgtctcgc gtagtgtata tggaaaacgg tcatatcgtg 660 gagcaagggg
acgcctcatg ttttacagag ccgcagacag aggcattcaa aaattatctg 720 agccac
726 <210> SEQ ID NO 15 <211> LENGTH: 732 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(732) <223> OTHER INFORMATION: artI (Escherichia coli)
<400> SEQUENCE: 15 atgaaaaaag tgcttattgc cgccctgatt
gcgggcttct ctctgtctgc caccgcggcc 60 gaaaccatcc gttttgccac
tgaagcgtca tatccccctt tcgaaagcat tgacgccaac 120 aaccaaattg
tcggtttcga cgttgacctc gcgcaggccc tgtgcaaaga aattgatgcc 180
acctgcacct tctctaacca agcgtttgac tcattgattc cttcgctgaa atttcgtcgc
240 gtggaagccg tcatgggcgg catggatatc acccccgagc gcgaaaaaca
ggtcttgttt 300 actacaccgt actacgacaa ctcggctttg tttgtcggcc
agcaaggcaa gtatacttct 360 gtcgaccagc tgaaaggtaa aaaagtccgt
tcagtccaga acggcaccac tcaccagaaa 420 ttcatcatgg acaaacatcc
tgagatcact accgtgccgt atgattctta ccagaacgcg 480 aagttagatc
tggaaaatgg tcggattgat ggcgtctttg gcgacaccgc tgtggtacat 540
gaatggctga aagacaatcc taaattagtg gttgtgggag ataaggttac ggataaggat
600 tattttggca ccggtctcgg cattgcagtc cgccaaggta ataccgaatt
gcaacagaaa 660 ttgaataccg cgctggaaaa agtgaaaaaa gacggtacat
acgaaaccat ttacaacaaa 720 tggtttcaaa aa 732 <210> SEQ ID NO
16 <211> LENGTH: 714 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(714) <223>
OTHER INFORMATION: artQ (Escherichia coli) <400> SEQUENCE: 16
atgaacgaat tcttccctct cgcgtctgcg gcaggtatga ccgtgggttt ggcggtttgt
60 gcgctgattg tcggtctcgc tctggcaatg ttctttgccg tatgggagtc
agcgaaatgg 120 cgtccggtcg cctgggcagg ttccgccctg gtaaccattc
tgcgtggtct gccagagatc 180 ctggtagttc tgtttatcta ctttggctct
tctcagttac tgttaacact gtctgacggg 240 tttacgatta acctgggttt
tgtccagatt ccggtccaga tggatattga aaatttcgac 300 gtctcccctt
ttctctgtgg cgtcatcgcg ctgagcttgc tctacgctgc atatgcatca 360
cagacccttc gtggtgcatt aaaagcggtg ccagtaggac agtgggaaag cggccaggcc
420 cttggcctga gcaagagcgc aatttttttc cgccttgtta tgccggccga
tgtccgccat 480 gcgttaccag gtctgggtaa tcaatggctg gtgttgttga
aagacaccgc ccttgtctcg 540 ctgattagcg tgaacgattt aatgctgcaa
accaaatcga ttgcaacccg cactcaggaa 600 ccgtttacct ggtacatcgt
ggcggcagca atctatctgg tgatcacact tctgagccag 660 tatattttaa
aacgtattga cctgcgtgcc acccgctttg agcgccgccc tagc 714 <210>
SEQ ID NO 17 <211> LENGTH: 666 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(666)
<223> OTHER INFORMATION: artM (Escherichia coli) <400>
SEQUENCE: 17 atgtttgaat atctgccgga actgatgaaa ggtttgcata ctagtctgac
gctgaccgtc 60 gcgagtctga tcgttgcgct tatcctggca ctgatcttca
ccattattct gactctcaag 120 accccggtcc tggtgtggct ggtccgcggt
tacattacct tattcaccgg gaccccgctc 180 ttggttcgca tttttcttat
ttactatggt ccgggtcagt ttccgacctt gcaagaatat 240 cctgcgttat
ggcacctgct gtctgaaccg tggctgtgcg ctctgattgc tctgagtgtt 300
aactcggcgg cctatacgac acagctgttc tacggtgcta ttcgtgcgat cccagaaggt
360 caatggcagt cttgtagcgc actgggcatg tcaaagaaag atactcttgc
tattctgctg 420 ccgtacgctt ttaaacgctc tctgagctcg tacagcaatg
aagttgtcct ggttttcaaa 480 agcactagct tagcgtatac gatcacgctg
atggaagtca tgggttatag ccagttatta 540 tatggtcgca cgtacgacgt
catggtgttt ggtgcagcgg gcattatcta tcttgtagtt 600 aatggattac
tgacgttaat gatgcgcttg atcgaacgca aagccgtggc attcgagcgg 660 cgtaat
666 <210> SEQ ID NO 18 <211> LENGTH: 729 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(729) <223> OTHER INFORMATION: artJ (Escherichia coli)
<400> SEQUENCE: 18 atgaaaaaat tggtgcttgc agcactgctg
gccagtttca ctttcggcgc ttcggcggcc 60 gaaaagatta atttcggtgt
cagcgcaact tacccaccgt tcgaaagcat cggtgcgaac 120 aatgagattg
taggatttga tatcgatctg gccaaagcgt tatgcaaaca aatgcaagcg 180
gagtgcactt ttaccaatca tgcgtttgat agcctgatcc cgtcgctgaa gttccgtaaa
240 tacgacgccg tgatttcggg gatggacatc acccctgagc gctcgaaaca
ggtgagcttc 300 accactccat attatgaaaa ctcagcggtg gtgattgcga
aaaaagacac ctataaaaca 360 tttgccgacc tgaaagggaa atgtattggt
atggagaacg gcaccaccca tcagaagtat 420 attcaagacc agcacccgga
ggttaagacc gtaagctacg actcctacca gaatgctttc 480 attgatttaa
aaaatggtcg tattgatggt gtattcggag atacagccgt ggtgaatgag 540
tggctgaaaa ccaatccgca gttgggtgtt gcgaccgaaa aagtgacaga tccacaatac
600 tttgggactg gcctgggcat cgcggtgcgc ccggataaca aagccctgtt
ggagaaactg 660 aacaacgcgt tagctgcgat taaagcggat gggacctatc
agaagatttc agaccaatgg 720 ttcccgcaa 729 <210> SEQ ID NO 19
<211> LENGTH: 633 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(633) <223> OTHER
INFORMATION: ArgO (Escherichia coli) <400> SEQUENCE: 19
atgttctcgt actatttcca aggcttagca ctgggtgcgg ccatgatctt accgctgggc
60 ccacaaaacg cttttgttat gaaccaggga atccgccggc agtaccatat
catgattgcg 120 ctgctgtgtg ccatctcgga tctggtcctg atttgcgccg
gtatttttgg cgggtcggcg 180 ttacttatgc aaagcccttg gctgctggcg
ctggtaacgt ggggcggcgt agcatttctg 240 ctttggtatg gattcggcgc
cttcaaaact gcgatgagtt cgaatatcga gcttgcgagt 300 gctgaggtaa
tgaaacaggg ccgttggaaa attattgcga ccatgttagc cgtgacttgg 360
ttgaacccgc acgtgtacct ggatactttt gtggtgttgg gttcactcgg tgggcaatta
420 gatgtggaac cgaaacgctg gtttgccttg ggcacaatct cggccagttt
tttgtggttc 480 ttcgggctgg cgctgctggc cgcgtggctg gcaccacgtt
tacgcaccgc caaggcccag 540 cgcatcatca acttagtcgt gggctgtgtg
atgtggttca ttgctctgca actggcgcgc 600 gatggcattg cgcacgccca
ggccctgttc tca 633 <210> SEQ ID NO 20 <211> LENGTH:
1203 <212> TYPE: DNA <213> ORGANISM: Flavobacterium
limnosediminis JC2902 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1203) <223> OTHER
INFORMATION: monofunctional lysine-ketoglutarate reductase
(Flavobacterium limnosediminis JC2902)
<400> SEQUENCE: 20 atgatgcgct ttggcatcat taaagaacgt
aagaacccgc cagatcgtcg tgtagtgttt 60 acaccgtccg aactgatcaa
actgaaagaa cagtttccgc tggccgaaat taaggtggaa 120 tcctcagata
ttcgcatttt ttctgatgat gagtatcgta aacttggatt tgaagtaacc 180
gatgacctga gtgattgtga tgtcttgatt ggcgtgaaag aagtaccgat cgatgccctg
240 ctgcccggga aaaagtattt ttttttctct cacacaatta aaaaacagcc
ttacaataaa 300 aaactgctga tcgcctgctt ggaaaaaaac atccgtctga
ttgatcatga gacgatcgtg 360 aatgaagata atcatcgttt gattgggttc
ggccgttacg caggtatcgt gggggcctat 420 aacggtttcc gtgcttttgg
tattaagtac gagctcttta acctgcccaa agcggaaacc 480 ttagcggaca
aaacggcact tgtggaacgc ctgcgtcggc cgatgctgcc gccaatcaaa 540
attgtgttga ccggtcacgg caaagtaggt atgggtgcaa aagagattct ggatgccatg
600 aaaatcaaac aagtttccgt ggaggactac ttaacaaaaa cctatgacaa
gccggtgtat 660 acgcagatcg acgttctgga ctataacaag cggaaagatg
gcaaaccggc ggaacgtgaa 720 cacttttatg ccaatccgca ggagtatgtc
tcggacttcg aacgctttac caaggtgtcg 780 gatctgttca tcgcaggcca
tttctatggc aacggtgcac cggtaattct gactcgcacc 840 atgcttaacg
cttctgataa taaaattaaa gtagttgcgg atattagctg tgatgtcggt 900
ggccctatcg aatgtacgct gcgcagcagc accatcgcag agccgtttta tggttattat
960 ccttccgaag gtaaagaagt cgacgtcaac catccgggcg cggtggttgt
gatggcggtg 1020 gacaatctgc cctgcgagct gcctaaagat gccagcgagg
gtttcggaga aatgtttctc 1080 aaacatgtga ttccagcctt ctacaacaac
gataaggacg gcattcttga gcgggccaaa 1140 atcaccgaaa acggcaaatt
aacaaaacgc ttctcctact tacaggacta tgtcgatggt 1200 gaa 1203
<210> SEQ ID NO 21 <211> LENGTH: 1356 <212> TYPE:
DNA <213> ORGANISM: Flavobacteriumsp. EM1321 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1356) <223> OTHER INFORMATION: saccharopine
dehydrogenase (Flavobacteriumsp. EM1321) <400> SEQUENCE: 21
atgcgtaata ttttgattat cggcgccggt cggtccgctt cctcgctgat tcagtactta
60 ttgaataagt cccaagaaga acagctgcat ttaaccattg ccgatttatc
actcgaactg 120 gctcagaaga aaaccaataa ccatccgaac gctaccgcgc
tggcgctgga tatttataat 180 aaggatgaac gtcgtgcggc catcgagaaa
gcggccattg tgatcagcat gttgccagcg 240 catctgcata tcgaaatcgc
ccgggattgc ctgtatttta aaaagaacct tgttacggcg 300 agctatatta
gtgacgcgat gcaggagctt gatgcggaag ttaaagagaa caaactgatc 360
tttatgaatg aggtcggttt agacccgggt attgatcata tgagcgccat gaaagtcatc
420 gatgaaattc gggaacaagg cggcaaaatg cttctcttcg aaagtttttg
cggcggcctg 480 gtggcaccag aatcagataa caatttatgg aactataaat
ttacctgggc cccacgtaac 540 gtagttctgg ctggccaggg tggtgtggca
aaattcattc aagaaggcac ctataaatat 600 atcccgtatg acagcttatt
tcgccggacc gagtttctgg aagtagaagg atacgggcgt 660 ttcgaagctt
attcgaatcg cgattctctc aaatatcgga gtatttatgg gctcgatgac 720
gttctcaccc tgtttcgtgg tacaatccgt cgcgttggct tctccaaagc ttggaacatg
780 tttgtgcaac tgggcatgac ggacgacagc tatgttatgg aagattctga
gaatatgtcc 840 tatcgtcaat ttattaactc attcctgcct tatcacccaa
ccgatagcgt tgaaattaag 900 acccgttttt tgttaaaaat cgatcaggat
gatatcatgt gggacaaact gctggaactg 960 gatcttttca acgataaaaa
aatggttggg ttgaaaaatg cgacgccggc acagatcctg 1020 gagaaaatcc
tgaacgattc gtggaccctg caaccggaag ataaagatat gatcgtgatg 1080
tatcataaat ttggttacca gatcaacggc gaaaaagtgc agatggattc acagatggtg
1140 tgtatcggcc aggaccaaac gtataccgcg atggcaaaaa ccgtcggcct
gcctgtggca 1200 atggcaactc tgctgattct gaacggtaaa atcaaaacaa
cgggagttca gttgccaatc 1260 aataaagaag tttacctgcc ggtcctggag
gaactggaga aatatggcgt tgtgttcaaa 1320 gaacagatgc tcccatatct
tggatacaaa tatagt 1356 <210> SEQ ID NO 22 <211> LENGTH:
1350 <212> TYPE: DNA <213> ORGANISM: Bacillus
methanolicus PB1 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1350) <223> OTHER
INFORMATION: Lysine aminotransferase (Bacillus methanolicus PB1)
<400> SEQUENCE: 22 atgaagaaaa atcattcctt gcagtcgctt
aaaaaccaag atgagcgttt catttggcac 60 tcgatgaagc cgtataaccc
cgacaagacg atcgttgtca ccaaggccga aggatcatgg 120 attacaacga
gtgatggaaa gaagtatctt gacgcaatgg ccggtctttg gtgcgttaac 180
gtggggtatg gacgcaaaga gcttgccgat gccgcgtacg aacagatgat ggaaatggca
240 tactatccac tgactcagtc acatgtaccc gccattcagt tagcggagaa
gttgaacgat 300 ctgctggaag acgaatacgt aatctttttt agcaattcgg
ggagtgaggc gaacgaggct 360 gcttttaaaa ttgctcgtca gtatcatcaa
caaaaaggag accacaatcg ctataagatt 420 gttgcacgct accgtgcata
tcatgggaac tcaattggag ccttggcagc gacagggcag 480 gcccagcgta
aatataagta tgagcctctg gcctttggat tcgtccatgt tgcccctcct 540
gactcctacc gtgatgaaac taacgtatcc gatccttcgc agttgtccgc agtcaaagaa
600 attgaccgtg taatgacgtg ggagctttcg gaaactatcg ccgcaatgat
catggaaccg 660 attattactg gtggaggcat cttagtgccc ccagaggggt
atatgaaagc ggctaaggag 720 gtttgtgaaa agcacggggc tcttttgatt
gtggacgagg tgatttgcgg gtttggtcgt 780 acgggtaagc cgttcggatt
catgaactat ggagtcaagc cggacattat caccatggct 840 aaaggcatca
ccagtgcgta tcttccgttg tcagcaactg cagtcaaaaa ggaaatctat 900
gatgccttta aaggtgagga cgaatatgag ttcttccgtc atgtcaacac tttcggaggg
960 tcacccgccg catgtgcgct ggctatcaag aacattcaga ttttggagga
ggaaaagctg 1020 tttgaccgct cgggcgacat gggcgaaaaa gttttaacag
aacttcagaa cttgttacgc 1080 gatcacccct acgttggcga cgttcgtgga
aagggtctgt taatcggaat tgaattggtt 1140 aaagacaagc agacgaaaga
gcccttaaat acaagcaaag ttgacgaagt aatcgctctt 1200 tgtaaacagg
aaggacttct gattggaaaa aatggcatga ccgtggcagg ctataacaac 1260
gtccttacac tgtcccctcc gcttaatatc ccagagaccg acttagactt tttgatcaaa
1320 gtactgacgg cgtccttgga gaagattaag 1350 <210> SEQ ID NO 23
<211> LENGTH: 1098 <212> TYPE: DNA <213>
ORGANISM: Agrobacterium tumefaciens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1098) <223> OTHER INFORMATION: lysine dehydrogenase
(Agrobacterium tumefaciens) <400> SEQUENCE: 23 atgaaaaata
tcgtagtgat cggggcaggg aatattggca gcgccattgc gtggatgttg 60
gcagctagcg gggattatcg cattactgta gcagaccgca gcgcggatca gttagctaat
120 gtaccggctc atgaacgtgt cgacattgtt gacattaccg accgccccgc
gctggaagca 180 ctgttaaaag ggaaatttgc ggtacttagc gccgctccca
ccgagtttca tttgactgcc 240 ggaattgcgg aagcggccgt cgcggtaggc
acgcactact tagacttaac agaagatgtg 300 gagtctaccc gcaaggtaaa
agcgctggct gagacggccg agacagcttt aatcccccaa 360 tgtgggctgg
caccaggttt tatttcgatt gttgctgccg atttggctgt gaagtttgat 420
aaattagatt ctgttcgtat gcgcgtcggg gcgctgcctc agtatcccag taacgcattg
480 aattacaatt tgacttggag cacagatggc cttatcaacg agtacattga
gccttgcgag 540 gggtttgtag aaggtcgctt gaccgcggtc ccggctttag
aggaacgcga ggaatttagt 600 cttgatggga tcacctacga ggcattcaac
acctcgggcg gacttgggac cttgtgcgcc 660 acccttgagg gtaaggtgcg
cacaatgaac taccgtacca tccgctatcc gggtcatgta 720 gcaatcatga
aggcacttct taatgacttg aacctgcgta atcgccgtga cgttttgaaa 780
gatctttttg aaaatgcact gcctggaacg atgcaagatg tcgtaattgt ttttgtaaca
840 gtgtgtggca ctcgcaacgg acgctttctg caagagactt atgccaataa
agtgtacgcg 900 gggcctgtgt caggccgcat gatgtccgcg atccagatca
caacagctgc tggaatttgc 960 acagtcctgg atttgttggc cgaaggcgcg
cttccgcaga agggcttcgt tcgtcaagag 1020 gaggtcgcac tgcctaagtt
tttggaaaat cgtttcggac gttattatgg ttctcacgaa 1080 ccgcttgctc
gtgttggt 1098 <210> SEQ ID NO 24 <211> LENGTH: 1179
<212> TYPE: DNA <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: lysine racemase (uncultured
bacterium) <400> SEQUENCE: 24 atggcacata caggccgtat
gtttaagatc gaagccgcgg agatcgtagt ggctcgcctg 60 ccgctgaaat
ttcgttttga gacatctttc ggtgtccaga cacataaagt ggtgccttta 120
ctgatcttac atggcgaagg tgttcaaggg gtcgcggagg ggacaatgga agctcgcccc
180 atgtaccgcg aagaaacgat tgccggagcc cttgatttgt tgcgtggaac
ttttttacct 240 gcgattctgg gccaaacctt tgccaatcca gaagcggtaa
gtgatgccct gggctcttac 300 cgcggcaatc gcatggcacg cgctatggtg
gagatggcag cttgggactt gtgggcccgc 360 acccttggtg tgcctttggg
cacactgttg ggtggtcaca aggaacaagt cgaggtgggt 420 gtatcgttgg
gaatccaggc agatgagcaa gctacagtag acttagtgcg tcgtcatgtt 480
gaacaaggat atcgtcgcat taagttgaag attaagcctg ggtgggacgt tcaacctgta
540 cgtgcgaccc gtgaggcatt catgttaaac acgcttaatg tcggcgcctc
tggttacgcg 600 ggcgcagaac tggttacata cgtgaaccgc cacccccata
tgaacattac ggcgttgacc 660 gtatcagcac agtcaaacga tgcagggaag
ttaatctccg atttgcatcc ccaattaaag 720 ggcatcgttg acttaccatt
gcagccgatg tccgacatct ctgaattcag ccccggggta 780
gatgtagtgt tcctggctac agctcacgaa gtttcacacg acctggcccc gcaatttttg
840 gaggcgggtt gtgtggtctt tgatctgtcc ggcgcttttc gcgttaacga
tgctacattt 900 tacgagaagt attacggttt cacccaccaa tacccagagc
tgctggaaca ggcggcctac 960 gggcttgctg agtggtgtgg caacaaactt
aaggaagcta atcttattgc agttcctgga 1020 tgttacccta ccgccgcaca
gctggcgctg aagccgttaa ttgatgctga cctgctggac 1080 ctgaaccaat
ggccggtgat caatgcgacc agtggcgtat ctggggcggg tcgtaaagcc 1140
gcaatttcaa actccttctg cgaggttagc ttacaaccg 1179 <210> SEQ ID
NO 25 <211> LENGTH: 1407 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1407)
<223> OTHER INFORMATION: Lysine transporter yvsh (Bacillus
subtilis) <400> SEQUENCE: 25 atggagcaga cgaagaaatg gggattttgg
ttactgacgg ccttcgtcgt gggcaacatg 60 gtgggtagtg gaatcttttc
tcttccatcc tccctggcga gcatcgcgtc gcctttcgga 120 gctacgtccg
cttggcttct gacaggtgcg ggggtgttaa tgatcgcctt agtattcgga 180
catttgtcca ttcgtaaacc cgaattgact gccgggcctc aatcatacgc ccgtgcattg
240 ttcagcgatc caaaaaaggg gaatgcggcc gggtttacta tggtttgggg
ttactgggtc 300 gcgagctgga tcagtaacgt agcaatcatt acatctctgg
cggggtatct gaccagcttc 360 ttccccatcc tggtagacaa acgcgaaatg
ttttctattg ggggtcaaga ggtcaccctg 420 gggcagctgc tgacttttgc
cgtttgcacc attctgttgt ggggcaccca tgcgattttg 480 gtcgcatcga
tcaatggcgc aagcaagctg aattttgtga ccacattatc caaggtcttg 540
ggattcgtgt ttttcattgt ggcagggtta ttcgtcttcc agacgacgct ttttggtcat
600 ttctatttcc cggtccaagg cgagaatgga acgagcatcg gtattggggg
acaggtgcat 660 aacgctgcga tttctacact ttgggctttc gtcggaatcg
aaagcgccgt tatcttgtct 720 ggccgcgcgc gcagccagcg cgatgttaaa
cgtgctacca ttaccggact tctgattgca 780 ctgtcgatct atattatcgt
cacgttaatc acgatgggtg ttttacccca cgacaaatta 840 gtaggaagtg
aaaagccatt tgtcgatgtt ttatatgcaa tcgtcgggaa cgctggttca 900
gtaatcatgg cactgctggc catcttgtgc ctttttggaa ccatgttggg gtggatttta
960 ctgggctcgg aggtgcccta ccaagcagcc aaagctggtg atttccccgc
cttctttgcc 1020 aaaactaata agaaaggttc tccagtgatt gcgcttatca
ttaccaatgt catgtcacag 1080 gttttcattt ttagcgtgat cagtcgtaca
atttccgatg cttttacttt tttgactaca 1140 gcggccacgt tggcctatct
gattccctac ttagtttcag cgatttatag tttgaaagtg 1200 gttattaaag
gcgaaaccta tgaccagttg aaaggcagtc gtgtacgtga tggtcttatc 1260
gctatcttgg catgtgcata ctcagtcttc gtaatcgtga cgggtaccgc cgatttgacg
1320 acctttattt taggtattgg gctttttttt gtgggcctta tcgtgtaccc
atttgtctcg 1380 aagaagtttc aaaaggagaa gcaggaa 1407 <210> SEQ
ID NO 26 <211> LENGTH: 1404 <212> TYPE: DNA <213>
ORGANISM: Klebsiella sp. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1404) <223> OTHER
INFORMATION: Lysine Transporter LysP (Klebsiella) <400>
SEQUENCE: 26 atgaccatga ttgctattgg cgggtcgatc ggcacagggc ttttcgttgc
atccggagca 60 acgattagtc aagcaggtcc aggcggggct ctgctgtctt
atattcttat cggcttaatg 120 gtgtattttc tgatgacctc tcttggagag
ctggccgctt ttatgccagt ctccggatcg 180 ttcgctacat atgggcaaaa
ctacgtagag gagggtttcg ggtttgcgct gggttggaat 240 tactggtata
attgggctgt gacgatcgca gttgacttgg tggcttcgca gcttgtgatg 300
agctattggt tccctgacac tccgggctgg atttggtctg ctttgttttt gggcatcatg
360 ttcttgctta actggatctc cgttcgcggg ttcggtgaag ctgagtactg
gttcagtctg 420 attaaagttg cgaccgttat tatcttcatc atcgttggcg
tgatgatgat tgtcggcatt 480 ttcaaagggg cgcaaccggc tggatggtcc
aactggggta tcgctgacgc cccatttgcg 540 gggggcttct cggcgatgat
tggcgttgcc atgattgtcg gtttttcctt tcagggtaca 600 gagttaattg
gaattgctgc tggtgaatcc gagaatcctg agaaaaatat tccacgtgcg 660
gtacgtcagg tattctggcg cattttactg ttttatgttt ttgcaatctt gattatctcg
720 ttgatcatcc cttatactga cccatcctta ttgcgtaacg atgtgaagga
tatttccgtg 780 tctcccttca cgttggtatt tcagtatgct gggctgctta
gtgccgctgc gatcatgaac 840 gcagtcattc ttacggctgt actgagcgct
ggaaactcgg gaatgtacgc ttcaacacgc 900 atgttatata ccttggcatg
tgacgggaaa gcaccgcgta tctttagcaa gctttcccgt 960 ggcggtgtgc
cacgcaatgc tctgtatgca acaactgtaa ttgctgcctt atgctttctt 1020
accagcatgt tcggcaacca aacggtttat ctgtggttgc tgaacacttc gggaatgaca
1080 gggttcatcg cctggctggg tattgctatt tctcactatc gtttccgtcg
cggctacgtg 1140 ctgcagggga atgatatcaa taatcttccg tatcgttcag
gattttttcc tcttggaccc 1200 atttttgcat ttgtattgtg tttgattatt
actcttggcc aaaattatga ggcgttctta 1260 aaagatacta tcgattgggg
tggggtagcc gcaacctaca tcgggattcc cttgttcctt 1320 gttatttggt
ttggatataa gttggctaag ggtacccgct ttgtccgtta ttccgaaatg 1380
accttcccag atcgttttaa acgc 1404 <210> SEQ ID NO 27
<211> LENGTH: 630 <212> TYPE: DNA <213> ORGANISM:
Pseudomonas sp. <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(630) <223> OTHER
INFORMATION: Lysine Exporter (Pseudomonas) <400> SEQUENCE: 27
atgagtatgg aagtctggct ggggtttttt gcagcgtgtt gggtgattag tttgtcaccg
60 ggagccggag ccatcgcctc tatgtcatcg ggtttacaat atggcttctg
gcgtggctac 120 tggaatgcac ttggattgca gcttggttta attatgcaaa
ttgcaattat cgctgcgggc 180 gtcggagccg tcttggcggc ctcggctacg
gccttccagg taattaaatg gttcggagtt 240 gggtatcttg tgtatttagc
atacaaacaa tggcgtgcac tgcccatgga tatgtcggat 300 gaaagcgggg
tgcgtccaat cggcaaacca ttatcgctgg tatttcgtgg atttttggtg 360
aatatctcca acccaaaagc tttagtattc atgttggccg ttttacccca gttcctgaat
420 ccccacgccc ccttgttacc ccaatacgtg gctatcactg tgacaatggt
tacagttgac 480 ttgttagtga tggccggata cacaggttta gcatctcatg
tattacgtat gcttcgtacc 540 ccaaaacagc aaaaacgcct gaaccgcacc
ttcgccggtt tattcatcgg agcggccaca 600 ttccttgcca ctttgcgccg
cgcaccagta 630 <210> SEQ ID NO 28 <211> LENGTH: 1014
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1014) <223> OTHER INFORMATION: Asparaginase
(Escherichia coli) <400> SEQUENCE: 28 atgcagaaga aatcgatcta
cgtcgcgtac acgggcggca ccattgggat gcagcgttcg 60 gagcagggtt
acatccccgt ttccggtcac ttgcagcgcc agctggcctt gatgcccgag 120
ttccatcgcc ccgagatgcc agattttacc attcatgagt acactccact tatggattca
180 tcggacatga cgccggaaga ctggcaacac attgcagaag atatcaaggc
tcactatgat 240 gattatgacg gctttgttat tttacacggt actgacacaa
tggcatacac agcttctgca 300 ctttccttta tgcttgagaa ccttggtaag
cccgtgatcg tgaccgggtc gcagatcccc 360 cttgccgaat tgcgcagtga
cgggcagatc aatcttctta atgcgttata tgtggccgct 420 aactatccga
tcaatgaagt gactttattc ttcaataacc gcttgtaccg tggaaaccgc 480
actacgaaag cccatgctga tggctttgac gcctttgcat ccccaaatct gcctcccctt
540 ttggaagccg ggattcacat ccgtcgttta aatacacccc ccgccccaca
tggagagggg 600 gagcttatcg tacatccaat tacccctcaa cctatcggag
ttgtaacgat ttaccctggt 660 attagtgccg acgtagtccg caatttcctt
cgccagcccg tgaaagcatt gatcttacgt 720 tcctacggtg tagggaacgc
gccacagaat aaggcatttc tgcaagaatt acaagaggca 780 tcggatcgtg
gtatcgtggt agtcaacctg acacagtgca tgtcaggtaa agttaatatg 840
ggtggatacg caaccgggaa tgcattagct catgcagggg taattggagg cgctgatatg
900 acggtcgaag ctaccctgac gaagcttcat tatctgttat cccaggagtt
ggacaccgag 960 accattcgca aagctatgtc tcagaacctt cgcggtgagc
ttactcccga tgac 1014 <210> SEQ ID NO 29 <211> LENGTH:
1461 <212> TYPE: DNA <213> ORGANISM: Mycobacterium
bovis <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1461) <223> OTHER INFORMATION:
Asparagine transporter ansp2 (Mycobacterium bovis) <400>
SEQUENCE: 29 atgccgcctc tggacatcac cgacgaacgc ttgactcgcg aagatacagg
atatcacaaa 60 ggccttcact cccgtcagct tcagatgatc gctcttggag
gtgctattgg gaccggactt 120 tttctggggg caggcggacg tctggcttct
gccggaccgg gattattctt ggtttatggt 180 atctgtggca tttttgtctt
tcttattctt cgtgccttgg gagaacttgt gcttcaccgc 240 cctagttcag
gatcatttgt atcctacgcg ggggaatttt atggtgaaaa ggtcgcgttc 300
gtcgcggggt ggatgtattt tttgaattgg gcaatgactg ggattgtgga cactacagcc
360 atcgcccact attgccacta ttggcgcgct tttcaaccaa ttccacagtg
gacgttggcc 420 cttattgcgt tgttagttgt attatccatg aatctgatct
ccgtccgctt attcggggaa 480 cttgagtttt gggcctcgct tattaaagta
attgcgcttg ttacgttcct gattgtaggg 540 actgtattcc tggcggggcg
ttacaagatt gacgggcaag aaactggtgt atcattatgg 600 tcatctcatg
gcggaatcgt tcctacgggg ttactgccca ttgtccttgt gacctctgga 660
gttgtgttcg catacgcagc catcgagctg gtaggaatcg cagccgggga gacggccgaa
720 ccagccaaaa tcatgccccg cgcaatcaat tcggtcgtcc ttcgtattgc
gtgtttttat 780 gtgggatcta cggtgcttct ggcgttgctt ttaccataca
cggcttataa ggagcacgta 840 agtcccttcg taacattttt cagcaaaatt
ggaattgatg ccgcggggag tgtaatgaac 900 ttggtagtgc ttacggcagc
gttatctagt ttgaacgctg gtttgtattc cacaggacgc 960 atcctgcgct
caatggcgat caacggcagc ggaccacgct ttacggcacc catgagtaaa 1020
accggtgttc cttatggcgg tatcttgctt acagcaggta tcggtttatt gggaatcatt
1080 cttaatgcga tcaaaccctc gcaggcgttc gaaatcgttt tacacatcgc
tgctaccggc 1140 gtaatcgcag cctgggctac gatcgtggct tgtcagttgc
gcttacatcg catggccaac 1200 gctggccaac ttcagcgccc taagttccgt
atgcccttgt caccttttag cgggtacctg 1260 actttggcgt ttctggcggg
cgtgctgatt ctgatgtatt tcgatgagca gcacgggccc 1320 tggatgatcg
ccgcgacagt aattggggtt cctgccctta ttgggggttg gtacttggtt 1380
cgtaaccgtg tgactgccgt cgctcatcac gctattgacc acactaagag tgtagctgtg
1440 gttcattcgg cagatcccat t 1461 <210> SEQ ID NO 30
<211> LENGTH: 1344 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1344)
<223> OTHER INFORMATION: Serine ammonia lyase (Bacillus
subtilis) <400> SEQUENCE: 30 atgtccatta atcaggaggc gcttcacgta
ctgttgaagg atccttttat tcatcgtctt 60 attgatgctg agccagtgtt
ttgggcaaat ccaggtatga aggaggggct gctttttcac 120 gctgacgagt
gggaaagtga gattgccgaa gcagagaaac gcttgcgtcg ttttgcgcct 180
tatatcgcgg aggttttccc agagaccaaa gatgcaaagg gcatgatcga gtctccactg
240 tttgagatgc aacatatgaa aaagaaactg gaggcagcat accaacaacc
tttccccgga 300 cgttggctgc ttaagtgtga ccatgaactt cccatttccg
ggtcgattaa ggcccgcgga 360 ggtatctatg aagtcttgaa acatgccgaa
aagctggctc ttcaggaggg gatgcttcaa 420 gagtcggacg attatcgtat
gttgcaagaa gatcgtttcg cggccttctt cagccgctat 480 tctatcgcag
tgggctccac gggcaattta ggtttaagta tcgggattat tggcgctgct 540
cttggtttcc gcgtaacagt tcacatgagt gctgacgcta agcaatggaa gaaagatctg
600 ttacgtcaga aaggggtaac cgtaatggaa tatgagagcg attattcaga
agcagttaaa 660 gaaggtcgtc gccaagcaga acaagaccca ttctgttact
tcattgatga tgaacatagc 720 cgtcaattgt tcctgggcta cgcagttgcc
gcgtcccgcc ttaagacaca actggattgc 780 atggaaatcc aacctggtcc
cgaaacaccc ctgttcgtgt atcttccctg tggcgtaggt 840 ggggggccag
gaggtgtcgc tttcgggttg aaactgctgt atggagatca cgtccatgta 900
tttttcggag aaccgacgca atccccgtgc atgcttttag gcttatattc tggcttacac
960 gagcagattt cagttcaaga cattggattg gacaaccgta ccgcggcgga
tggcttggcg 1020 gtagggcgtc cctcaggatt cgtaggaaaa ttaatcgaac
cactgctgtc gggctgctat 1080 actgtagaag acgatacact gtatgcttta
ctgcacatgc tggcagcttc ggaatccaag 1140 tatcttgaac ctagcgcctt
ggcggggatg ttcggcccga tccagctgtt cagcacagaa 1200 gaaggacgtc
gctattctca gaaacataaa atggagcatg cggtgcacgt tatctggggg 1260
acggggggta gcatggtgcc aaaggaggag atggccgcat acaaccgcat cggggcggat
1320 ctgttaaaaa atgaaatgaa gaag 1344 <210> SEQ ID NO 31
<211> LENGTH: 1374 <212> TYPE: DNA <213>
ORGANISM: Pseudomonas fluorescens F113 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1374) <223> OTHER INFORMATION: SdaA (Pseudomonas
fluorescens F113) <400> SEQUENCE: 31 atgtcccttt cagtgtttga
tctttttaag atcggaattg gtccctcgtc ctctcatacc 60 gtaggaccta
tgcgtgcggc cgctcgtttt gccgaaggtc tgcgccgcga cgacctgctg 120
aactgtacta ctagcgtgaa agtcgagctg tacggatctc tgggcgcgac tggtaaaggg
180 cacggttcgg acaaagcagt gttactggga ttggagggag aacaccctga
cactgtcgac 240 accgagacgg ttgacgctcg tttacaggcg atccgcagtt
caggccgcct gaatttattg 300 ggggagcata gcattgagtt taatgaaaag
ctgcacttgg caatgattcg caagccgtta 360 gctttccatc cgaatggcat
gattttccgt gcgtttgatg ctgcgggctt acaggtacgt 420 tcccgtgagt
attactccgt cggcggaggg ttcgttgtag acgaggacgc agcgggtgcc 480
gaccgtatcg tcgaggatgc aacacctttg acattcccct tcaagagcgc gaaggatctt
540 ttaggtcatt gttctactta tggtttaagc atcagccaag tcatgcttac
aaacgagtct 600 gcgtggcgtc cggaagcgga gacccgcgca gggcttctta
aaatttggca ggtgatgcaa 660 gactgcgttg ccgcggggtg tcgcaatgag
ggcatccttc caggaggtct taaagtaaag 720 cgccgcgcgg ctgcgttgca
tcgtcaattg tgtaagaacc ccgaggctgc cctgcgcgat 780 ccgttaagtg
tattagattg ggtgaatttg tatgcgttag cggtaaatga agagaacgcc 840
tacggtggac gcgtggtcac ggcgcccact aatggagccg caggaatcat tcctgccgta
900 ttgcattact acatgcgctt tattccgggg gcatctgagg acggagtagt
ccgcttcctt 960 cttacagcgg cggcaatcgg gatcttgtat aaagagaacg
cctctattag tggggctgag 1020 gttggctgtc agggcgaagt aggagtggca
tgctccatgg cagcgggggc gttgtgcgaa 1080 gtcttgggag gctcggtcca
acaagtagaa aacgcagcag aaatcggaat ggagcataac 1140 cttggcttga
catgtgatcc tatcggcggg ttagtacagg tcccgtgtat cgagcgtaac 1200
gcaatgggat ctgttaaagc cattaacgca gtacgcatgg ctatgcgcgg ggacggtcac
1260 catttcgtct cccttgacaa agtaattcgt accatgcgtc aaactggggc
cgacatgaaa 1320 agcaagtaca aggaaaccgc gcgtggtgga cttgctgtca
acatcatcga gtgt 1374 <210> SEQ ID NO 32 <211> LENGTH:
1365 <212> TYPE: DNA <213> ORGANISM: Klebsiella
pneumoniae <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1365) <223> OTHER INFORMATION:
sdaB(Klebsiella pneumoniae) <400> SEQUENCE: 32 atgattagtg
tgtttgacat ctttaaaatc ggtatcggtc cgtcttcttc ccatacggtt 60
ggtcccatga aagcagggaa gcagtttacc gacgacttaa ttgctcgtgg actgctggca
120 gaggtcagta aggtcgtggt tgatgtttat ggctcccttt cattgacggg
caaaggtcac 180 catactgaca ttgctatcat tatgggtctg gcgggaaact
tgccagacac cgttgacatc 240 gacgccatcc ccggcttcat ccaagatgtt
aacactcacg gacgtctgat gttagcgaat 300 gggcagcatg aagttgattt
cccggtagac cagtgtatga attttcacgc tgacaacctg 360 tccttgcacg
agaatggaat gcgtattacg gctcttgcgg gagacaaagt gttgtactct 420
cagacttact actcaatcgg cggcggattc attgttgatg aggaacattt tggccaaaca
480 acggaggctc ctgtagccgt cccatatcca tacaaaaacg ccgctgattt
gcagcgtcat 540 tgccgtgaaa ctggtttgag tttatctgga cttatgatgc
aaaacgaact tgcattgcat 600 agcaaagaag ctctggaaca gcactttgct
gcagtttggg aggttatgtc tgccggcatt 660 gagcgcggca ttacaactga
aggtgtgttg cctggcaaat tacgtgtacc ccgccgcgcc 720 gcggcactgc
gtcgtatgtt agtctcgcaa gacacgacga actcggaccc tatggctgtt 780
gtagattgga tcaatatgtt cgcgttggcc gtcaacgagg agaacgcggc gggcggtcgc
840 gttgttacag cccccacaaa tggcgcgtgc ggaattgttc cggccgtgct
ggcatattat 900 gacaaattta tccgcaaagt caactccaac agtctggcgc
gttatatgct ggtggcaagt 960 gcaatcggct cactttataa gatgaatgcg
agcatctccg gcgcagaagt tggctgccaa 1020 ggtgaagtgg gggtcgcctg
ctctatggca gcggctggct tggcagagct gttgggcggg 1080 tcgccagggc
aagtgtgcat tgcggctgaa attgcgatgg agcataactt gggccttacg 1140
tgcgatcccg tagctggcca agtgcaggta ccgtgtatcg aacgcaatgc aattgcagcc
1200 gtaaaagcag taaatgcggc tcgcatggcc ttacgtcgta cttccgagcc
ccgtgtgtgc 1260 ttggataagg tgatcgaaac catgtatgag acaggtaagg
acatgaatgc aaagtatcgt 1320 gaaacgtctc gtggaggcct ggccatgaag
atcgtcgcgt gtgac 1365 <210> SEQ ID NO 33 <211> LENGTH:
1362 <212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1362) <223> OTHER INFORMATION: tdcG L-serine
dehydratase (Escherichia coli O157:H7 str. SS17) <400>
SEQUENCE: 33 atgattagtg cattcgatat tttcaagatt ggaatcggcc cctcgtcatc
gcacacggtg 60 ggcccaatga acgcaggtaa gtccttcatt gatcgccttg
agtcgagtgg cttattgaca 120 gcgacaagcc acattgtcgt ggacctgtac
gggagtctgt cgttgacggg caaaggccat 180 gcgaccgatg ttgctattat
catgggattg gccgggaatt caccgcagga cgtagtaatc 240 gatgaaatcc
cggccttcat tgagctggta actcgttcgg gccgtctgcc agtcgcaagc 300
ggagctcata tcgttgactt cccagttgcc aagaacatta tttttcaccc tgaaatgtta
360 cctcgccatg agaacggaat gcgtatcaca gcatggaaag ctcaggaaga
attattgagt 420 aagacgtatt actcggttgg tggcgggttc atcgtcgagg
aagagcactt cggtttatct 480 catgacgtag aaacaccagt accatacgac
ttccattcag caggtgagtt gttgaaaatg 540 tgcgattaca atggccttag
tatttcggga cttatgatgc ataacgaatt agcgcttcgt 600 tcgaaggccg
aaattgacgc cggcttcgca cgtatctggc aagttatgca tgatggcatc 660
gaacgtggta tgaacaccga aggtgtgtta ccaggaccct tgaatgttcc gcgtcgtgca
720 gtcgcactgc gtcgtcaact tgttagtagt gacaacattt ccaatgatcc
aatgaacgtg 780 attgactgga tcaacatgta cgcgctggcg gtctcggagg
aaaacgccgc tgggggtcgc 840 gtggtaacag cacctacgaa tggggcttgc
gggatcatcc ctgcggtatt ggcctattac 900 gataagtttc gccgtccagt
caatgagcgc tcaatcgctc gttacttcct ggcggcgggg 960 gctatcggcg
ctttatacaa gatgaacgcc tctatttcag gggcggaggt cggttgtcaa 1020
ggagagattg gggtcgcgtg ctctatggca gctgcaggtt tgacagaatt attaggcggc
1080 agcccagccc aagtttgcaa cgcggctgaa atcgcaatgg aacataatct
tggtctgacc 1140 tgtgaccctg tcgcaggtca ggtacagatt ccttgcattg
agcgtaatgc aatcaacgca 1200 gtaaaagctg ttaatgcggc gcgtatggct
atgcgtcgca catcagcccc gcgtgtgagc 1260 ctggataagg taatcgagac
catgtacgaa accggtaaag acatgaatga caaataccgc 1320 gaaacctctc
gcgggggtct tgcaattaaa gtcgtgtgtg gc 1362 <210> SEQ ID NO 34
<211> LENGTH: 1251 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1251)
<223> OTHER INFORMATION: glyA(Escherichia coliEPEC C342-62)
<400> SEQUENCE: 34 atgttgaaac gtgagatgaa tattgccgac
tatgatgcag aattatggca agctatggaa 60 caagagaaag tccgccagga
agaacatatt gaattaatcg cctctgaaaa ttacactagt 120 ccccgcgtta
tgcaagccca aggcagccaa ttaactaaca aatatgccga gggatatcct 180
gggaaacgct actatggagg ttgcgagtat gtagatattg tcgaacagtt agcaatcgac
240 cgcgcgaaag agcttttcgg cgcagactat gcaaacgtgc agccccattc
gggtagccaa 300 gcgaattttg cggtctatac cgcactgctg gaaccgggag
acacggtact gggtatgaat 360 ttagctcatg gtggtcactt aacgcacggg
tcccccgtta atttctctgg aaaactgtac 420 aacatcgtcc cctatggaat
cgatgctacc ggccacattg attacgcgga tcttgagaag 480 caagctaagg
aacataaacc aaagatgatc attggcggtt tttcagctta tagtggtgtc 540
gtcgactggg ctaagatgcg tgaaattgca gactctattg gcgcgtacct ttttgtcgac
600 atggcccacg tggctggctt ggtggcggca ggggtctacc cgaaccccgt
tccccatgcg 660 catgtcgtga ccaccacgac acataagaca ctggctgggc
ctcgtggtgg cttaatcttg 720 gccaaggggg ggtctgagga attatacaaa
aaacttaact cagccgtttt tccaggcgga 780 cagggtggtc cgttgatgca
cgtgattgct ggaaaggcgg tcgctcttaa ggaagccatg 840 gaacctgaat
tcaaaacgta ccaacagcag gttgcaaaaa acgccaaagc gatggttgag 900
gttttcctgg aacgtggtta caaagtcgtt agtgggggta ccgataatca tcttttctta
960 gttgacctgg tagataaaaa tttgaccgga aaggaggcgg acgctgcctt
aggccgtgcg 1020 aatattaccg tcaataaaaa ctcggtgcca aatgatccca
agtcgccttt cgtgacttca 1080 ggaatccgcg taggaactcc cgcaattaca
cgccgcgggt tcaaggaagc tgaggcgaag 1140 gagttagcag gatggatgtg
tgatgtttta gactcgatta acgatgaggc ggtgatcgaa 1200 cgtatcaaag
gtaaagtatt agatatttgc gcccgttatc cagtttatgc c 1251 <210> SEQ
ID NO 35 <211> LENGTH: 1287 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1287)
<223> OTHER INFORMATION: SdaCserine STP transporter
(Escherichia coliBL21(DE3) <400> SEQUENCE: 35 atggagacca
cgcagacttc tacaattgcg agcaaagata gccgttctgc ttggcgcaaa 60
actgatacta tgtggatgtt gggcctgtat ggaacagcta ttggggccgg ggtactgttt
120 ttgccaatca atgctggagt ggggggtatg atcccgctga tcattatggc
gattcttgct 180 ttcccaatga cattttttgc acatcgcggt cttacacgct
ttgtcctttc aggaaagaat 240 cctggggagg acattacgga ggttgtagaa
gaacattttg gcattggggc tgggaaactt 300 atcacattgc tgtatttttt
tgcaatctat cccattttgc ttgtctatag cgtagcaatc 360 acgaacaccg
tagaatcatt catgtcgcac cagttaggca tgacacctcc gccacgtgcg 420
attctgtcat tgatcttgat cgtgggaatg atgacaattg ttcgtttcgg agagcaaatg
480 atcgtgaaag ccatgtcaat tttggtattt ccgttcgtgg gagtcttaat
gttgctggca 540 ttgtatttaa ttccccagtg gaatggtgcc gctctggaga
ccttgtcgtt ggatacggcg 600 tcagcgaccg gtaatggtct ttggatgacg
ctttggttgg ccattccggt catggttttt 660 tcatttaacc actcaccgat
cattagctcg ttcgctgtgg cgaaacgcga agaatacggt 720 gatatggctg
aacaaaagtg ctcgaagatt ttggcattcg cccacatcat gatggtactt 780
acggtcatgt tcttcgtgtt ttcttgcgtc cttagtttaa ccccagcgga cctggcggct
840 gcaaaggaac aaaatatcag catcttaagc tatttggcga atcatttcaa
cgcgcctgtt 900 atcgcatgga tggcacccat tatcgctatc attgcaatta
ccaaatcttt cttagggcac 960 tacttgggtg cgcgcgaagg atttaacggg
atggttatca agtcgcttcg tgggaaagga 1020 aagagtatcg agatcaataa
acttaatcgc atcaccgcct tgttcatgtt agtaacaacg 1080 tggatcgtcg
ctacacttaa tccctccatt ctggggatga ttgaaacgct tgggggtcca 1140
atcatcgcaa tgatcttgtt tctgatgccg atgtacgcta tccagaaggt acccgcaatg
1200 cgtaaatact ctgggcatat ctccaacgtg tttgttgttg ttatgggatt
aatcgctatt 1260 tctgctatct tctatagtct gttctcc 1287 <210> SEQ
ID NO 36 <211> LENGTH: 438 <212> TYPE: DNA <213>
ORGANISM: Lactobacillus saniviri <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(438) <223>
OTHER INFORMATION: threonine Serine Exporter (Lactobacillus
saniviri JCM 17471 = DSM 24301) <400> SEQUENCE: 36 atggcgtatt
ctgtccagtt cctgatccaa ctgtccttct cgtaccttgc cactgtggct 60
tttgctatct gcatcaacgt tccacgtcgt gcgttaaatt ttgccggatg ggccggtgcc
120 atcgggtgga tctgctactg gctgctgaac acacatggca cgggccgcat
gttcgctaac 180 ctgattggcg ctgtcgcagt tggggtatgt ggtatcattt
tcgctcgcat caagaagatg 240 cccgtgatta ttttcaatat tccggggctg
gtgccattag tgcctggagc aaccgcctac 300 caggcagttc gcgctcttgc
gttgggaaat atggaccttg ctatccagct tggagttcgt 360 gttattatgg
tcgcaggggc aatcgcggtg ggattcatgg ttagtcagct tctgtcagag 420
ttgacttacc gcttgcac 438 <210> SEQ ID NO 37 <211>
LENGTH: 930 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(930) <223> OTHER INFORMATION:
glutaminase YbaS (Escherichia coli ST131) <400> SEQUENCE: 37
atgctggatg ctaataagct gcagcaggct gtcgatcagg cttatactca atttcattct
60 ttgaatggtg ggcagaatgc cgattacatt cctttcttgg ctaatgtccc
agggcaatta 120 gcagccgtag ctattgtaac atccgatggc aacgtgtatt
ctgccgggga ctcggactac 180 cgcttcgcac ttgagtctat cagtaaagtc
tgcactttgg cactggcgct ggaggacgtt 240 gggcctcagg ccgtgcagga
caaggttggg gctgatccta cagggctgcc attcaactca 300 gtaattgctt
tggaattaca cggtggaaaa ccactgtcac cgctggtgaa cgcgggggca 360
atcgctacca cgtctttgat taatgcagaa aatacggaac agcgttggca acgtattttg
420 catattcagc agcagcttgc tggtgagcaa gtcgcacttt ctgatgaagt
gaaccaaagt 480 gaacaaacta ctaattttca caaccgtgca attgcttggt
tactgtacag tgctggctac 540 ttgtactgtg acgcaatgga agcctgtgat
gtttatacac gtcagtgcag tactttgatc 600 aacacaatcg aattggcaac
attgggagct acgttagccg ctgggggcgt gaatccgttg 660 acacataaac
gcgttctgca agcggacaat gtgccctata ttttggctga aatgatgatg 720
gaagggcttt atggccgctc tggggactgg gcctaccgtg taggcttgcc aggaaagtcg
780 ggggtcggag gagggattct ggccgtggtg cccggcgtaa tgggaattgc
cgcgttttcg 840 cctcccttag acgaagaagg taacagcgtg cgcggacaaa
agatggttgc gagcgttgca 900 aagcagcttg ggtataacgt atttaaaggg 930
<210> SEQ ID NO 38 <211> LENGTH: 924 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(924)
<223> OTHER INFORMATION: Glutaminase (Escherichia coli
O145:H28 str. RM12581) <400> SEQUENCE: 38 atggccgtcg
caatggataa cgccatttta gagaatatcc tgcgccaagt gcgcccatta 60
atcggacaag gcaaggttgc ggattacatt ccggccttag ctacagtgga tgggagtcgc
120 ctgggaatcg ctatttgcac tgttgacggc caattgtttc aggcaggcga
cgcacaagag 180 cgcttctcca tccagagcat ttctaaagtg ttgtcattgg
ttgttgctat gcgtcactac 240 tctgaggagg aaatttggca gcgcgtgggg
aaggacccgt ccggcagtcc atttaattcg 300 ttggtacagt tggagatgga
acaaggaatc cctcgtaatc ccttcatcaa tgcaggtgct 360 cttgtagtct
gcgacatgtt acaaggtcgt ttatctgccc ctcgccaacg catgttggaa 420
gttgtgcgtg gtttgtctgg agttagcgat atcagctacg acacggtcgt ggctcgcagt
480 gaatttgaac actcagcacg caatgcagcg attgcgtggt taatgaagtc
gtttgggaat 540 tttcatcacg atgtgacgac agtccttcaa aattatttcc
actactgcgc attgaagatg 600 tcgtgcgtag agcttgcccg tacgttcgtc
tttcttgcga accagggcaa ggccatccat 660 atcgacgagc ccgtcgtaac
cccgatgcag gcgcgtcaaa tcaatgcgct gatggcgaca 720 tcgggaatgt
atcagaatgc gggggagttc gcctggcgtg tcggattacc agctaaatcc 780
ggtgtaggcg gtggaatcgt tgccattgtg ccccatgaaa tggctatcgc tgtgtggtcc
840 ccagaattag atgacgcagg aaattcgtta gcaggtattg cggttttaga
acaacttacg 900 aaacaattag gacgctcggt gtat 924 <210> SEQ ID NO
39 <211> LENGTH: 927 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(927) <223>
OTHER INFORMATION: ylaM (Bacillus subtilis subsp. subtilis
str. 168) <400> SEQUENCE: 39 atggtgtgtc agcataatga tgaattagag
gctcttgtca agaaggcaaa aaaggttacg 60 gataaggggg aggtggctag
ttacattcca gctctggcta aggcggacaa acacgactta 120 agtgtcgcaa
tctactatag caataatgtg tgcctgtccg caggggacgt tgaaaagacg 180
ttcactctgc aatccatcag caaagttctg tcgttagctc tggtacttat ggagtatggg
240 aaggataagg tattcagtta tgttgggcag gaacctacag gtgatccctt
taacagcatc 300 attaaactgg agacagtcaa cccctctaag ccattaaatc
cgatgatcaa tgcgggcgcg 360 ttagtagtga ccagtcttat ccgcggacgt
acggtgaagg agcgtcttga ctatcttctt 420 agctttatcc gtcgtctgac
taataatcaa gaaattacat actgccgcga ggtagcggaa 480 agcgaatatt
ctacttcaat gattaaccgt gcgatgtgct attatatgaa acagtatgga 540
attttcgaag atgacgttga agcggttatg gacctttata caaagcaatg cgctattgaa
600 atgaactcac ttgatttggc taagatcggt tcggttttcg ccttgaacgg
acgccatcct 660 gaaaccgggg agcaagtgat ttcgaaggat gtagcccgta
tctgtaagac gtttatggtg 720 acgtgtggaa tgtataatgc ctctggtgaa
tttgcgatca aagttggtat ccctgcgaaa 780 tcgggagtgt caggtgggat
tatgggtatc tccccttacg atttcggaat cgggatcttt 840 ggacccgcgc
tggacgagaa ggggaatagt attgctggtg tgaagctttt agaaatcatg 900
agcgagatgt accgtcttag tatcttt 927 <210> SEQ ID NO 40
<211> LENGTH: 981 <212> TYPE: DNA <213> ORGANISM:
Bacillus subtilis <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(981) <223> OTHER
INFORMATION: ybgJ(Bacillus subtilis) <400> SEQUENCE: 40
atgaaagagt tgattaaaga gcatcaaaag gatatcaatc ctgcattaca actgcatgac
60 tgggtagaat actaccgtcc atttgcggca aatggccaaa gtgcaaacta
tatccccgct 120 ttagggaagg tgaacgacag ccagttaggg atctgcgtac
tggaaccgga tggcaccatg 180 attcacgctg gggattggaa tgtgtccttt
accatgcagt cgatttcaaa agtaattagc 240 ttcattgctg cctgcatgtc
gcgtggaatc ccgtatgtct tggatcgtgt agacgtggaa 300 cccacaggag
atgcttttaa tagtatcatc cgtttagaga tcaacaaacc aggaaagcct 360
ttcaatccta tgattaatgc cggagctttg actatcgcta gcattcttcc aggagagtcc
420 gcttacgaaa aacttgagtt tttgtatagc gtgatggaga ctttaatcgg
taaacgcccc 480 cgtattcacg aagaagtatt ccgttctgaa tgggagaccg
ctcatcgcaa tcgcgcctta 540 gcctactatc ttaaagaaac aaacttctta
gaggccgagg tcgaagagac actggaagta 600 tatttgaaac aatgcgcgat
ggaatcgacc acggaagaca tcgccctgat cgggttgatc 660 ctggcccacg
atgggtatca tcctatccgt catgagcagg tcattcccaa ggatgttgcc 720
aagttggcta aagcgttaat gttgacctgt ggcatgtata acgcttctgg aaagtatgcg
780 gctttcgttg gagtacccgc aaaatctgga gtttcgggtg gtattatggc
cttggtgcct 840 ccaagtgcgc gtcgcgaaca gccgttccag agcgggtgcg
gtatcgggat ttatggacct 900 gcaattgatg agtacgggaa tagcctgacg
ggcggcatgc ttttaaaaca catggcccaa 960 gagtgggaac tgagtatttt c 981
<210> SEQ ID NO 41 <211> LENGTH: 3436 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(3436) <223> OTHER INFORMATION: Glutamine permease
glnHPQ operon (Escherichia coli) <400> SEQUENCE: 41
ccatggcaga acgtgcagtg cagctgggcg gtgtagctct ggggaccact caagttatca
60 acagcaaaac cccgctgaaa agttacccgc tggacatcca caacgttcag
gatcacctga 120 aagaactggc tgaccgttac gcaatcgtcg ctaatgacgt
acgcaaagcg attggcgaag 180 cgaaagatga cgacaccgca gatatcctga
ccgccgcgtc tcgcgacctg gataaattcc 240 tgtggtttat cgagtctaac
atcgaataaa tccatcgctg atggtgcaga actttagtac 300 ccgataaaag
cggcttcctg acaggaggcc gttttgtttt gcagcccacc tcaacgcact 360
tatttagtgc atccatctgc tatctccagc tgattaagta aattttttgt atccacatca
420 tcacacaatc gttacataaa gattgttttt tcatcaggtt ttacgctaaa
taatcactgt 480 gttgagtgca caattttagc gcaccagatt ggtgccccag
aatggtgcat cttcagggta 540 ttgccctata aatcgtgcat cacgtttttg
ccgcatctcg aaaaatcaag gagttgcaaa 600 actggcacga ttttttcata
tatgtgaatg tcacgcaggg gatcgtcccg tggatagaaa 660 aaaggaaatg
ctatgaagtc tgtattaaaa gtttcactgg ctgcactgac cctggctttt 720
gcggtttctt ctcatgccgc ggataaaaaa ttagttgtcg cgacggatac cgccttcgtt
780 ccgtttgaat ttaaacaggg cgataaatat gtgggctttg acgttgatct
gtgggctgcc 840 atcgctaaag agctgaagct ggattacgaa ctgaagccga
tggatttcag tgggatcatt 900 ccggcactgc aaaccaaaaa cgtcgatctg
gcgctggcgg gcattaccat caccgacgag 960 cgtaaaaaag cgatcgattt
ctctgacggc tactacaaaa gcggcctgtt agtgatggtg 1020 aaagctaaca
ataacgatgt gaaaagcgtg aaagatctcg acgggaaagt ggttgctgtg 1080
aagagcggta ctggctccgt tgattacgcg aaagcaaaca tcaaaactaa agatctgcgt
1140 cagttcccga acatcgataa cgcctatatg gaactgggca ccaaccgcgc
agacgccgtt 1200 ctgcacgata cgccaaacat tctgtacttc atcaaaaccg
ccggtaacgg tcagttcaaa 1260 gcggtaggtg actctctgga agcgcagcaa
tacggtattg cgttcccgaa aggtagcgac 1320 gagctgcgtg acaaagtcaa
cggcgcgttg aaaaccctgc gcgagaacgg aacttacaac 1380 gaaatctaca
aaaaatggtt cggtactgaa ccgaaataat aacgctacac ctgtaaaacg 1440
cactggcagt tccctctccc ctatggggag aggattaggg tgaggggcgc aaacccgctc
1500 cggggccatt aattaccctg aatttgatta tttacaccac ggtaacagga
acaacatatg 1560 cagtttgact ggagtgccat ctggcctgcc attccgcttc
tgattgaagg tgccaaaatg 1620 accctgtgga tttcggtcct cggtctggca
ggcggtctgg taatcggatt gctggcaggt 1680 tttgcacgca ccttcggagg
ttggatagcc aaccacgtcg cgctggtctt tattgaagtg 1740 atccgcggca
cacctatcgt cgtccaggtg atgtttattt atttcgccct gccgatggcg 1800
tttaacgact tacgcatcga cccatttact gcggcggtgg tcaccatcat gatcaactcc
1860 ggcgcgtata ttgcggaaat cacgcgtggt gcggtgctgt ctatccacaa
aggttttcgt 1920 gaagcaggac tggcgctcgg tctttcacgt tgggaaacca
ttcgctacgt cattttaccg 1980 ctggcactgc gtcgtatgct gccgccgctg
ggtaaccagt ggatcatcag cattaaagac 2040 acctcgctgt ttattgtgat
cggcgtggcg gaactgaccc gtcaggggca agaaattatt 2100 gccggtaact
tccgcgccct tgagatctgg agcgccgtgg cggtgttcta tctgattatt 2160
accctggtgc tgagctttat tctgcgtcgt ctggaaagaa ggatgaaaat cctgtgattg
2220 aatttaaaaa cgtctccaag cactttggcc caacccaggt gctgcacaat
atcgatttga 2280 acattgccca gggcgaagtc gtggtgatta tcgggccgtc
cggttccggt aaatcgaccc 2340 tgctgcgctg catcaacaaa ctggaagaaa
tcacctccgg cgatctgatt gtcgatggcc 2400 tgaaggttaa cgatccgaaa
gttgacgagc gcctgattcg ccaggaagca ggtatggtgt 2460 tccagcagtt
ttacctcttc ccgcatctga cagcgctgga aaacgtcatg tttggcccgc 2520
tacgcgtgcg tggcgcgaac aaagaagagg cggaaaaact ggcacgtgag ctgctggcga
2580 aagtcggtct ggcagaacgt gcacatcact acccttccga actttctggt
ggtcaacagc 2640 agcgtgtggc gattgcccgc gcgctggcgg tgaagccgaa
aatgatgctg tttgatgaac 2700 cgacttccgc tcttgacccg gaactgcgcc
atgaagtgct gaaggttatg caggatctgg 2760 ctgaagaagg gatgacgatg
gtgatcgtga cccacgaaat cggttttgcc gagaaagtag 2820 cttcgcggct
gatctttatc gacaaaggcc ggattgcgga agatggcaat ccgcaggtgt 2880
tgatcaagaa cccgccgagc cagcgcttgc aggaattttt gcagcacgtc tcttaataag
2940 acacattgcc tgatcgtacg cttatcaggc ctacaggata tctggcaact
tattaaaatt 3000 gcatgaactt gtaggacgga taaggcgttc acgcgcatcc
ggcaaaaaag cccgcacgtt 3060 gtcagcaacc tgcttaatat cccttcctcc
ctttcacccg aaagggaggc acaccagatt 3120 cctctcattt aaaatcgccc
ctcctccagc atctatactt atctttttgc tctattttct 3180 cactggagga
gtcatgcggt ggatcctgtt catcctcttc tgcctgctgg gcgcacctgc 3240
ccacgcggta tccatacccg gcgttacaac cacaacgaca acggactcaa cgactgaacc
3300 ggccccggaa ccggatatcg aacaaaaaaa agcggcctat gcgcactggc
ggatgtgctg 3360 gataatgaca cctcgcgtaa agagttgatc gaccagttgc
gcaccgttgc cgctacgccc 3420 ctgctgaacc ggtacc 3436 <210> SEQ
ID NO 42 <211> LENGTH: 744 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(744) <223>
OTHER INFORMATION: Glutamine permease H glnH (Escherichia coliEPEC
C342-62) <400> SEQUENCE: 42 atgaaaagtg tacttaaagt gtcattggca
gcactgacac ttgcatttgc agtctccagt 60 catgctgcgg acaaaaagtt
agtcgtagcg actgacactg cgtttgttcc tttcgaattc 120 aagcaggggg
acaagtacgt cggctttgac gtagaccttt gggccgccat tgcaaaagag 180
cttaagttgg attacgagtt aaagcctatg gacttcagtg gtatcattcc cgccctgcaa
240 acgaaaaacg tggatcttgc gcttgcaggc attactatta ccgacgaacg
caagaaggcg 300 attgacttca gcgacggcta ttataagtcg ggtcttttag
ttatggtaaa agccaacaat 360 aatgatgtga aaagcgtgaa agatttggac
gggaaagtag tggcagttaa atcaggtaca 420 gggagtgtgg attacgcgaa
agctaatatc aaaaccaaag acttacgtca attcccgaat 480 atcgacaatg
cgtatatgga actggggacg aaccgtgcgg atgcggtgct gcacgataca 540
cccaacatcc tttatttcat taaaacagct ggtaatggtc aatttaaagc tgtaggcgac
600 agcctggaag cccagcaata cgggatcgcg ttccctaagg gctctgatga
gcttcgtgac 660 aaggtaaacg gggcgcttaa aacgctgcgt gaaaacggaa
cgtacaatga aatctataag 720
aagtggttcg gaaccgagcc caaa 744 <210> SEQ ID NO 43 <211>
LENGTH: 657 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(657) <223> OTHER INFORMATION:
Glutamine permease P glnP (Escherichia coliB354) <400>
SEQUENCE: 43 atgcaattcg attggagtgc gatttggcct gccattcccc ttctgattga
gggtgcaaaa 60 atgactctgt ggatttcagt gctggggtta gccggaggtc
ttgttattgg gttattagca 120 gggtttgcac gcactttcgg gggatggatt
gcaaatcatg ttgcgctggt cttcatcgaa 180 gtcattcgtg gcacccccat
cgtggtccaa gtgatgttta tttacttcgc gttgccaatg 240 gcatttaacg
atcttcgtat tgatccattt actgcggcag tggtgactat catgattaat 300
agtggggcgt acattgcgga gattactcgc ggcgctgttc tttccattca caaaggtttt
360 cgtgaggccg gtttagctct tgggctttcc cgctgggaaa caattcgtta
tgttatcttg 420 ccgcttgcct tgcgccgtat gttgccgccg ctgggtaacc
aatggatcat ttctatcaaa 480 gatacttcgc ttttcattgt tattggagtg
gctgaattaa cacgccaagg tcaagaaatc 540 atcgcgggga atttccgtgc
attagagatc tggagtgctg tcgccgtttt ctacttgatc 600 attacgctgg
tgctgtcctt tattttgcgc cgcttggagc gtcgcatgaa gattctt 657 <210>
SEQ ID NO 44 <211> LENGTH: 720 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(720)
<223> OTHER INFORMATION: Glutamine Permease Q glnQ
(Escherichia coliEPEC C342-62) <400> SEQUENCE: 44 atgattgaat
ttaagaatgt gtcgaagcat ttcggcccca cccaagtact tcacaacatt 60
gaccttaaca tcgcccaggg cgaggttgta gtaatcatcg gtccatctgg tagtggcaag
120 tccaccttgc tgcgttgtat caataaactt gaggaaatca ccagcggaga
cttaattgtg 180 gacggtctta aagtcaacga tccaaaagtg gacgaacgct
tgattcgtca ggaagcgggt 240 atggttttcc agcagttcta cttgtttccg
caccttacgg ctcttgagaa cgtcatgttc 300 ggaccgttac gcgtgcgcgg
ggccaataag gaggaggcgg agaagttggc acgcgagctg 360 ttagcaaaag
ttggcttggc tgaacgtgca catcattacc cttctgagct gtcaggtggg 420
caacagcaac gtgtcgccat cgcacgcgcg cttgctgtaa aaccaaagat gatgctgttc
480 gatgagccaa cgtcggcgct tgacccggag ttgcgccatg aggtccttaa
ggttatgcaa 540 gacttagctg aagagggaat gacgatggta atcgtgacgc
acgagattgg attcgcagag 600 aaggtagcat ctcgtttgat cttcatcgac
aaaggtcgca ttgcagaaga cggcgaccca 660 caagttctga ttaagaaccc
cccttcacag cgcctgcaag aatttctgca acatgtctcc 720 <210> SEQ ID
NO 45 <211> LENGTH: 1437 <212> TYPE: DNA <213>
ORGANISM: Ustilago maydis 521 <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1437)
<223> OTHER INFORMATION: tryptophanamino transferase
(transaminase) (Ustilago maydis 521) <400> SEQUENCE: 45
atgagttccg ccacaagtcc ggcactggat tatgcattgc tgttgtcttc ttctgctcgt
60 aaccgtatgc cttctgcaat ccgttccctg ttcccggcag aattaattcc
aggcatggtc 120 tctcttttgt caggtaaacc gaattcggag acctttccct
ttcagcgcat cagtttggaa 180 cttaaaccct ccatccatct ggagggacag
accgagacag tgagcatcga aggtagcgat 240 ttagacatcg ctcttcagta
ttcagcaacg agtgggttgc caaagttggt agactggatc 300 attaaatttc
aatctcgcgt tcacgctcgt aagcaggtcg atgagggcaa taagccgggt 360
gaagtatggc gctgtagctt tggcaacgga tctcaagacc tgctgaccaa gacatttgag
420 gctttagttg acgccggtga ttcagtagtc ctggaaagtc cggcttacag
tggaattttg 480 ccgtcgttgg ttgcgcataa agccaacctt ttcgaggcag
aaactgacgc cgagggcgtt 540 gagcccacgg ctttagacac attgctgact
aactggaaga ctgacagtgc aacacgtgac 600 tctcgttttc ccaagttttt
atatactacc ccgactggtg caaatccgtc cgggacatca 660 gcctctgata
atcgcaagcg tgcgatcctt gatattatcc gcaagcacaa tttacttctg 720
ctggaggatg atccttacta ttttttgtca ttccaagggt tggaaccggg ggctgacgcg
780 gtcaaacgca ctcgtgggaa gagctatttt cagttggaag ctcaggacga
ctatggcgtc 840 ggccgtgttg ttcgctttga ttcatttagt aagatcttgt
ctgccggatt acgcctgggt 900 ttcgttacag gacccaaaga gattctggac
gccatcgacc tggacacttc ctcccgcaat 960 ttgcagacaa gtggcacttc
ccaggcaatc gcctatgctt tgttgtctaa gtggggaatt 1020 gacggttttt
tacatcatgc ggacaatgtc gcacgttttt accaaaatcg cttagaacgc 1080
tttgaagcca gtgcccaggc aatcttaacc ggaagcccta gcatcgcctc gtgggttcgt
1140 ccttcggcag ggatgttcct gtggatcaag ttaaagttgc ctccgtcgcc
cgactcggcg 1200 gagggtgata gttttgacct gatctctaat aaagctaagg
cagctggggt attggcttta 1260 cccggtgtgg ccttcaaacc accgagcagt
tcaagtacgg gtggcaaacg taagacatcg 1320 gcatatgtcc gcacgtcatt
ctcccaggtg cctctggacc aagtggatac cgcattcaca 1380 cgcctgcgtc
aggtggtaga ggaggcctgg cgtgaggctg gacttcaaat ccccgcg 1437
<210> SEQ ID NO 46 <211> LENGTH: 1242 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1242) <223> OTHER INFORMATION: Mtrtryptophan ArAAP
transporter (Escherichia coliBL21(DE3)) <400> SEQUENCE: 46
atggctaccc ttactactac tcaaacttcc ccatcgcttc ttggaggagt cgttatcatc
60 ggtggaacta tcatcggagc agggatgttt tcactgccgg ttgtgatgtc
gggagcatgg 120 ttcttttggt caatggcggc tcttatcttc acgtggttct
gtatgttgca tagtggcctg 180 atgatcctgg aagcaaatct gaactaccgt
attgggtcct cttttgatac aattacaaag 240 gaccttctgg ggaaaggatg
gaatgtagtt aatggaatta gtatcgcgtt cgtcctttac 300 atcttgacct
acgcgtatat ctctgcctca gggagcatct tgcatcacac ttttgccgag 360
atgtcattga acgtgcccgc acgcgctgct ggctttggtt ttgcactgct tgtggcattc
420 gtagtctggt taagtacgaa ggctgtgagc cgtatgaccg ctatcgtcct
tggggctaaa 480 gtaattacct tctttttaac attcggctcg ctgttaggac
acgtgcagcc tgccactttg 540 ttcaatgtgg ctgaatcaaa cgcctcgtat
gccccctatt tacttatgac tttgccgttt 600 tgtctggctt ccttcggtta
tcacggaaac gtgccatcac tgatgaaata ttatggtaag 660 gatcctaaaa
caattgtgaa gtgcttggta tacgggacct taatggcact tgccctttac 720
acgatctggc ttcttgcaac gatgggcaat attcctcgcc ctgaatttat cgggatcgca
780 gaaaaagggg ggaatattga cgtgctggtc caggctttat cgggtgtctt
gaatagccgc 840 tctttggatc ttttgttagt tgtcttttcc aattttgccg
tggcatcgag tttcttaggt 900 gtgacgctgg gtctttttga ttacctggcc
gatctgttcg gattcgacga cagcgcggtg 960 ggccgtctta aaactgcttt
attaacattt gcgccccctg tagtgggagg tcttctgttt 1020 cctaacggat
tcttatacgc catcggctac gccggattgg cggccacgat ttgggcagct 1080
atcgtcccgg ctttattggc acgtgcctca cgcaaacgct tcgggagtcc taaattccgt
1140 gtttggggcg ggaagcctat gattgccctt attttagtgt ttggagtcgg
taatgcactt 1200 gtgcacatct tgtcatcgtt caatctgctt cccgtttatc aa 1242
<210> SEQ ID NO 47 <211> LENGTH: 1245 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1245) <223> OTHER INFORMATION: tryptophan permeaseTnaB
(Escherichia colistr. K-12 substr. MC4100) <400> SEQUENCE: 47
atgaccgacc aagctgaaaa gaagcattcg gcattctggg gagtaatggt cattgccggt
60 accgtgatcg gcggtgggat gtttgcttta cctgtggact tagcaggcgc
gtggtttttt 120 tggggggcgt tcattctgat tattgcttgg ttttccatgc
tgcatagtgg cttgctgctt 180 cttgaagcga atcttaacta tccggtgggg
tcaagtttca ataccattac aaaggacctg 240 attggtaaca catggaatat
catttcgggg atcacggtag catttgtatt gtatattctt 300 acatatgctt
atatcagtgc gaatggcgca atcatttccg agacgatctc catgaacctg 360
gggtatcacg cgaatccccg tattgtcggc atctgcacag cgatttttgt tgcgagcgta
420 ttatggctga gttcgttggc agcttcgcgt attacttccc ttttccttgg
tttgaaaatc 480 atcagcttcg taattgtgtt tgggagtttt tttttccagg
tcgactactc cattcttcgc 540 gatgcaacaa gtagcacagc aggcaccagt
tacttcccat atatctttat ggccttaccg 600 gtttgtttag cgtcttttgg
ttttcatggt aatatcccct cattaattat ttgctacggc 660 aagcgcaagg
acaaattaat taagtctgtt gttttcggct ccttgttggc gcttgtaatc 720
tatttatttt ggctttattg tacgatgggg aacatccctc gcgaatcctt taaggctatt
780 atttcttcag gaggcaacgt agacagtttg gtaaaaagtt ttttgggtac
gaagcagcat 840 ggtatcatcg agttttgttt acttgttttc agtaatcttg
ccgttgcttc ctcattcttt 900 ggcgtgactc tggggctttt tgattatctg
gcagatttat tcaagatcga caactcgcat 960 ggcgggcgct tcaaaacggt
tctgcttaca tttcttcctc cagctttact ttacctgatc 1020 tttccgaatg
gttttatcta tggtattggg ggggcaggcc tgtgcgccac tatctgggca 1080
gttatcattc ctgctgtatt ggctatcaag gcacgcaaaa agtttcccaa ccagatgttc
1140 accgtgtggg gcggcaattt gattccggca atcgtgatct tatttggtat
cacggttatt 1200 ctttgctggt tcggcaatgt gtttaacgtc ctgcctaagt ttgga
1245 <210> SEQ ID NO 48 <211> LENGTH: 1368
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1368) <223> OTHER INFORMATION: aroP
(Escherichia coliO104:H4 str. C227-11) <400> SEQUENCE: 48
atggaagggc agcagcatgg cgaacagctt aagcgtggcc tgaagaatcg tcatatccag
60 cttatcgcat taggcggagc tattgggacc ggcttgttct taggctctgc
ttcagtcatt 120 cagtctgcgg ggccaggcat tattttaggc tacgcgattg
cgggcttcat cgccttttta 180 attatgcgcc agcttggcga gatggtggtg
gaggaacccg tggcaggcag tttctctcac 240 tttgcataca agtattgggg
aagttttgca ggctttgcga gcggttggaa ctactgggtt 300 ctgtacgttc
tggtggccat ggcggaactg acagcagtcg gtaaatatat tcaattctgg 360
taccctgaaa ttcccacttg ggtctctgcc gctgtcttct ttgtcgtcat taatgcaatc
420 aacttgacca acgtcaaagt attcggcgag atggagtttt ggttcgctat
tatcaaagtc 480 attgctgttg tggccatgat cattttcgga ggctggctgc
ttttcagcgg caacggaggt 540 ccccaggcaa ctgtatcgaa tctttgggac
cagggtggtt tcttgccaca tgggttcacg 600 gggttagtta tgatgatggc
cattattatg ttctcgtttg gagggcttga attggtgggc 660 atcactgctg
ctgaagctga taacccggag caaagcattc ctaaggccac aaatcaagtg 720
atctatcgca tccttatctt ttacattgga tcgttggcag tattgctgag tttgatgccc
780 tggacccgtg tcaccgctga tacaagccct tttgttttga tttttcatga
attaggggat 840 acttttgtgg caaatgcgtt aaacatcgtc gtattaactg
ctgccttgtc agtatataac 900 tcctgcgtat actgtaatag ccgtatgctg
ttcggcttgg ctcagcaggg gaacgctccg 960 aaagcactgg ccagtgtcga
caagcgtgga gtacctgtga atacgatttt agtttctgct 1020 ctggtcactg
cactttgtgt attgatcaac tacctggcgc ctgagtcggc gtttggcctg 1080
ctgatggcgc tggtggttag cgcattggtc atcaattggg cgatgatctc cttggcacac
1140 atgaaattcc gccgtgctaa acaagaacag ggtgtggtta cacacttccc
agcattatta 1200 taccctctgg gcaactggat ttgcttactt tttatggcag
cggttctggt catcatgctg 1260 atgacgcctg gtatggctat ttctgtttat
ctgattccgg tttggttaat cgtattaggg 1320 attggctatt tattcaagga
aaaaactgca aaggctgtca aagcgcat 1368 <210> SEQ ID NO 49
<211> LENGTH: 879 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(879) <223> OTHER
INFORMATION: Aromatic amino acid exporterYddG (Escherichia
coliTW10598) <400> SEQUENCE: 49 atgacccgcc agaaggcgac
tctgatcggt ttgattgcta tcgtattatg gtccacaatg 60 gttggtttaa
ttcgtggggt ttctgagggg cttggcccgg tgggcggagc agcagctatc 120
tactccctga gcggtctgtt attgatcttt acagttgggt ttccgcgtat ccgtcaaatc
180 cccaagggat acttattggc ggggagttta ctttttgtga gctatgaaat
ttgccttgcc 240 ttgtctctgg gctacgcagc gacacgccat caagcaattg
aggtagggat ggttaattac 300 ctttggccgt cattgacgat tcttttcgca
atcttattta acggtcagaa gactaattgg 360 ttgattgtac cgggtttatt
attagcgttg gtgggagtat gctgggtgtt gggaggtgac 420 aatggtctgc
attatgacga gattattaat aatatcacaa catcgccctt atcctacttt 480
ctggctttca ttggtgcctt tatctgggcc gcctattgca ccgtgacgaa taagtacgct
540 cgtggcttca acggaattac agtatttgtc ttgcttactg gtgcatcttt
gtgggtatat 600 tatttcttga cccctcaacc agagatgatc ttctccaccc
cggttatgat caaattaatt 660 tcagcagctt tcactttggg attcgcatac
gcagcttgga atgtcggcat tcttcatggg 720 aatgtgacga ttatggcagt
cggttcctac ttcacgcccg tacttagttc cgctttagca 780 gcggtactgc
tgtcggcgcc tttgagtttt agtttctggc agggtgccct gatggtgtgt 840
gggggctccc ttttgtgctg gcttgctacc cgccgtggt 879 <210> SEQ ID
NO 50 <211> LENGTH: 1152 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1152)
<223> OTHER INFORMATION: S-adenosylmethioninesynthase
Escherichia coli (strain K12) <400> SEQUENCE: 50 atggcaaagc
accttttcac gtcggaatct gtatctgaag ggcatcccga caaaattgca 60
gatcaaatct ccgacgcggt acttgatgct attctggaac aagatcccaa agcccgcgtc
120 gcttgcgaaa cttatgtcaa gacaggcatg gtgttagtcg gcggcgagat
cactacctct 180 gcgtgggtgg atatcgagga aatcacgcgc aatacggtgc
gtgagattgg ctatgtacac 240 tcggacatgg ggttcgacgc caacagttgt
gcggttttaa gtgccattgg gaaacagtca 300 cctgatatta atcagggggt
ggatcgtgcg gaccctcttg aacaaggtgc tggtgaccaa 360 ggtctgatgt
tcggttatgc tacgaacgaa accgatgtgt tgatgcccgc cccgatcaca 420
tacgcccacc gtctggtcca acgccaggcg gaggtccgta aaaacggcac gcttccttgg
480 cttcgtccag atgctaagtc gcaggtcact ttccaatacg acgacgggaa
gattgtcgga 540 atcgacgccg tggtcttgtc aactcagcat tcagaggaga
tcgatcaaaa gagccttcag 600 gaagccgtca tggaagagat catcaagccg
attctgcctg cagaatggtt aacttccgcg 660 accaagttct ttattaaccc
caccgggcgt tttgtcattg gcggtcctat gggcgactgt 720 gggttgaccg
gccgtaaaat tattgtcgac acttatggcg gaatggctcg tcatggcggt 780
ggggcattca gtggcaagga cccgtcaaag gtagatcgtt cagccgccta tgccgcccgt
840 tacgtagcca agaacattgt tgctgcagga cttgctgacc gctgtgaaat
ccaagtgagc 900 tacgcgatcg gcgtagcaga acccacctcc attatggtgg
aaacttttgg caccgaaaaa 960 gtccccagtg agcaactgac cttattggtt
cgtgagtttt ttgatttgcg cccttacgga 1020 cttatccaaa tgttagacct
tttgcaccca atctacaaag aaactgcagc atacggtcac 1080 tttggacgcg
agcattttcc ctgggagaag acagacaaag cacagctgtt acgtgacgcg 1140
gccggattga aa 1152 <210> SEQ ID NO 51 <211> LENGTH:
1275 <212> TYPE: DNA <213> ORGANISM: Anabaena
cylindrica PCC 7122 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1275) <223> OTHER
INFORMATION: adenosylhomocysteinase (Anabaena cylindrica PCC 7122)
<400> SEQUENCE: 51 atgacggcta cgacgccacg cctgaaacat
gaagtgaagg accttgcgct tgcgccttta 60 ggtcgtcagc gtattgagtg
ggcggggcgc gaaatgcctg ttttaaagca aatccgcgac 120 cgctttgaaa
aagaaaagcc cttcgcgggc ctgcgtatct cggcttgtgc gcatgttaca 180
acagagacgg ctcatttagc aattgccctg aaggccgggg gagctgatgc cgtattgatc
240 gcaagcaacc cactgtctac gcaggatgac gtagcagcct cgcttgtcgc
tgatcatgag 300 atctctgtgt ttgcacaaaa gggcgaagac gccgcgacgt
actcgcgtca cgtccaaatt 360 gcgttggacc accgccccaa tatcatcgtt
gatgacggtt ccgacgtagt agctgaatta 420 gtacagcacc gtcagaatca
gatcgcggat cttattggat ccactgaaga aactacaact 480 gggattgttc
gccttcgcgc tatgttcaac gagggggttt tgacgtttcc cgcgatgaat 540
gtcaacgacg cagacacaaa acattttttt gacaaccgct acggtacagg acaatctacc
600 ttggacggga tcattcgtgc aaccaacatc ttgcttgccg gcaaaactat
cgtagttgta 660 ggctatggct ggtgcggaaa ggggaccgca ttacgcgccc
gcgggatggg agctaatgtc 720 attgttaccg agatcgatca cattaaggca
attgaggcgg tgatggatgg gtttcgcgtt 780 ctgcccatgg ctgaagccgc
accgcatggt gatatcttta tcactgtaac gggtaataaa 840 cacgtagttc
gtggtgaaca ctttgatgtc atgaaagacg gcgccattgt ttgcaactca 900
ggtcacttcg atttggagtt ggatttaaaa tatttagcag caaatgccaa ggaaatcaaa
960 gatgtgcgcc cattcacaca agaatataaa ttaaccaacg gcaaaagcgt
agtggtatta 1020 ggagaggggc gtttgattaa tcttgcagcg gcagaaggtc
atccgtcggc agttatggac 1080 atgtctttcg ccaatcaagc cttagcagtc
gagtatttag tgaaaaataa aggctccttg 1140 gcggctggat tacattcgat
cccccgcgag gttgatgagg aaatcgctcg tttaaaattg 1200 caagcgatgg
ggatttttat cgattccctg acagcagatc aaatcgatta tattaattct 1260
tggcagtcag ggacg 1275 <210> SEQ ID NO 52 <211> LENGTH:
1374 <212> TYPE: DNA <213> ORGANISM: Klebsiella
quasipneumoniae subsp. Quasipneumoniae <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1374) <223> OTHER INFORMATION:
Cystathionine-beta-synthase (Klebsiella quasipneumoniae subsp.
Quasipneumoniae) <400> SEQUENCE: 52 atggtaatgt cgttattcca
cagtgttagc gatttaatcg gtcacacacc tttattacaa 60 ttgcataagc
ttgatacagg accctgtagt ttgttcttga aacttgagaa tcaaaaccca 120
ggagggtcaa ttaaagatcg tgtagcgctt agcatgatta acgaagcgga acgtcaggga
180 aaacttgcgc caggaggaac tatcatcgag gctacggcgg gaaatactgg
gttggggctt 240 gctttgatcg cagcccagaa aaactaccgt cttatccttg
tagttcccga caagatgtca 300 cgtgaaaaaa ttttccactt gcgtgcctta
ggcgcaaccg tgcttttgac ccgttcagac 360 gtgaacaagg ggcacccggc
atattatcag gactatgctc gccgcttggc agatgagact 420 ccaggggcgt
tctacattga ccaattcaat aatgatgcca atcctttagc acatgcaaca 480
agcacggccc ctgagctgtt ccaacaatta gaaggggaca tcgatgccat tgtggttggt
540 gttgggtcgg gtggaacgtt gggcggcttg caggcctggt tcgcagaaca
ctctcccaaa 600 acagagttca tcttggctga tccagctggg tcgattcttg
ccgaccaggt agacacaggc 660 cgctacgggg aaacgggaag ctggcttgta
gagggtattg gcgaggattt tatcccacca 720 cttgctcgcc tggaaggagt
tcataccgca tatcgtgtat ctgatcgcga agcctttctt 780
acagcccgtc aactgcttca ggtagagggt gtattagcgg gctcgtcaac gggaacattg
840 ttatctgcgg ccttgcgcta ttgccgtgcc cagtctcgcc caaagcgtgt
ggttaccttc 900 gcatgtgact ctggaaataa gtacttgagt aagatgttca
atgacgactg gatgcgccaa 960 cagggactta ttgcgcgccc ggaacaggga
gatctgagtg atttcatcgc cttacgtcac 1020 gacgaggggg ccacggtcac
cgccgcgccc gacgacacac tggcggctgt atttactcgc 1080 atgcgcttgt
acgatatctc ccagcttccg gtcttggaag acggtcgtgt cgttggcatt 1140
gtggacgaat gggatttaat tcgccatgta cgtggcgacc gtcaacgctt ttccctgcca
1200 gtcagcgagg ctatgtcccg tcacgtagaa acgttagaca aacgcgcccc
cgaatccgaa 1260 ttgcaagcta tcttagaccg tggactggta gcagtcattg
cagacaatgc gcgctttctg 1320 ggactggtta cacgttcaga tgtcttaacg
gcatggcgca atcgtgtggc gcaa 1374 <210> SEQ ID NO 53
<211> LENGTH: 1146 <212> TYPE: DNA <213>
ORGANISM: Klebsiella pneumoniae subsp. pneumoniae HS11286
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1146) <223> OTHER INFORMATION:
cystathionine-gamma-lyase (Klebsiella pneumoniae subsp. pneumoniae
HS11286) <400> SEQUENCE: 53 atgtcgtcta ttcacaccct gtctgttcat
agtggcacct tcacggactc acatggcgcg 60 gtgatgcccc caatctatgc
cacctccacg ttcgcgcaac ctgcgcccgg acagcacacc 120 ggatatgaat
actcgcgcag tggaaatcct actcgtcatg ccttagagac tgcgatcgca 180
gacctggaga atggaacgcg cgggtacgca tttgcctcgg gcttggcagc gatctcgact
240 gtccttgaat tgttggataa ggacagccat ttagttgcag tggatgatgt
ctatggtggg 300 acctaccgtt tacttgaaaa cgttcgtcgt cgttctgctg
ggctgcaagt gtcgtgggtc 360 aagccagacg atttagcggg gattgaggcg
gctatccgtc ctgacacccg tatgatctgg 420 gtcgaaacac ctactaatcc
tttgctgaaa ttagccgatt tgagcgccat cgcagctatc 480 gcacgccgtc
acaatcttat ttcagttgcg gataacacgt tcgcttcacc agccatccac 540
cgtcctcttg aacacggttt cgacattgtg gtgcattctg cgacaaaata cttaaatgga
600 cattccgatg tggttgcggg gttagctgtc gtcggagata actccggctt
agccgagaaa 660 ttaggttatt tacaaaatgc agttggcggg gtattagacc
ccttttcctc gttccttaca 720 ttgcgcggca tccgcactct ggcactgcgt
atggaacgtc atagcgcgaa tgcactgcag 780 ttagccgaat ggttggaaca
acagcccgaa gtagagcgtg tatggtttcc ttggctggcc 840 tcccatcctc
atcatcaatt ggcacgtcag cagatggcat tacctggcgg gatgattagc 900
gtagtagtca aaggagatga gggatatgct gagcgcatca tcagtaaact gcgttggttc
960 actcttgccg agtctttagg cggcgtcgag tcgttagttt cccagccgtt
ctcaatgaca 1020 catgcttcga tcccacttga aaagcgtctt gcgaacggca
ttacgcccca gcttattcgc 1080 cttagtgtgg ggatcgaaga cccacatgat
cttatcgcgg attggcaaca agccctgcgt 1140 gccgaa 1146 <210> SEQ
ID NO 54 <211> LENGTH: 483 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(483) <223>
OTHER INFORMATION: cysteinedioxygenase (Bacillus subtilissubsp.
subtilis str. BAB-1) <400> SEQUENCE: 54 atggagttat acgagtgcat
ccaggacatc ttctcggggt tgaaaaaccc ttccgtgaaa 60 gatctggcaa
catccctgaa acaaatcccg aatgcagcta aattatctca gccttacatt 120
aaagagcctg accagtatgc atacggtcgc aatgccatct accgtaacaa cgagttggag
180 attattgtta tcaacattcc tcccaacaaa gagacaaccg tacacgatca
cggacaatcc 240 attggatgcg caatggttct ggaaggtaaa ttacttaata
gcatttatcg ttctgctggt 300 gagcacgccg agctgtccaa ctcttacttt
gttcacgagg gggaatgcct tatctcgact 360 aaaggcttga ttcacaaaat
gagcaacccc acaagcgagc gcatggtatc gttgcatgtt 420 tattcgccac
cgcttgagga catgacagta tttgaggaac agaaagaggt gttaaagaac 480 tct 483
<210> SEQ ID NO 55 <211> LENGTH: 891 <212> TYPE:
DNA <213> ORGANISM: Caenorhabditis elegans <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(891) <223> OTHER INFORMATION: Glutamate Oxaloacetate
Transaminase (Caenorhabditis elegans) <400> SEQUENCE: 55
atgagcgtta gtaaaaaact gttctctacg gctgtgcgtg gtaagagctg gtggtcacac
60 gtcgagatgg gccctcctga tgcgattttg ggggtgactg aagctttcaa
agctgattct 120 aaccccaaga agatcaattt gggcgtggga gcgtaccgtg
atgaccaagg aaaaccgttc 180 gtacttccta gcgtcaagga agccgaacgt
caagttattg cagcaaatct tgacaaggag 240 tacgccggga tcgttggcct
gcctgaattc acgaaactta gtgctcagtt agcattaggg 300 gaaaacagtg
acgtaatcaa aaacaagcgt atttttacga cgcaaagtat ttctgggact 360
ggtgcgctgc gtattggaag tgagttcctg agtaaatatg caaagactaa ggttatctat
420 caacccacgc ctacatgggg aaaccacgtg cctatcttca agttcgcggg
cgtggatgtg 480 aaacagtatc gttattatga caagtctaca tgtggatttg
atgagacggg ggcattggct 540 gatattgcgc aaatccccga aggtagcact
attttgctgc acgcgtgcgc acataaccca 600 acgggggtcg accctagtcg
tgaccaatgg aaaaagattt cagatattgt taagaaacgc 660 aatttgttcg
tgttttttga catggtgaat gagtcagtcc tgagtccgtt actgcctcgc 720
acgcttatgc gcctgcttgt gttgttactg aaatcccgca gtcttttcgc ccactcaaca
780 cccacccatc agtcgatgga attagctctt ttgccggcct cgtcgcgtat
ccaactttct 840 acctccaatg ggtcagaaat gtccagctct tggcttatcg
tcagcagccc t 891 <210> SEQ ID NO 56 <211> LENGTH: 1179
<212> TYPE: DNA <213> ORGANISM: Bacillus halodurans
C-125 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1179) <223> OTHER INFORMATION:
methionine gamma lyase (Bacillus halodurans C-125) <400>
SEQUENCE: 56 atgaaacgcg accaacattt tgaaacacgc gcgatccata ctggttacaa
gccgaacgag 60 cattttgata gcttgactcc ccctatttac caaaccagca
cgttcacatt tgcatcaatg 120 gagcaaggtg gcaaccgttt cgcaggcgag
gaagcaggat atgtttattc acgcctgggg 180 aaccccaccg tgcaaatttt
ggaacaacgc attgctgagt tggagggtgg ggaggcagct 240 cttgcctttg
gatctggcat ggctgctgtc agtgcgattt tggtggggct tacgaaggcc 300
aacgaccaca tcttagtgag caatggagtg tatggttgta cgtttgggtt gttaacgatg
360 ttaaaggaaa aatacaacat cgacgccact ttcagtccga tggacagcgt
agaggaaatc 420 ctggcaaaca tccaggataa taccacgtgc atttatgtgg
aaacacctat caaccccacc 480 atgcagttaa tcgatttgga actggttgtg
cgcgtagcga aggaaaaggg tattaaggta 540 atcgttgata acacgtttgc
cacaccatac ttacaacaac cgattgctct gggatgtgac 600 ttcgttgtcc
attcggccac gaaatacatc gggggtcatg gggacgtggt cgccggagtg 660
ctgattggag acaaggaaac aattcagttg atccgtaaga ccacccagaa ggatatgggg
720 ggcgtaattt ctccatttga tgcgtggctg ctgttgcgcg gattgaaaac
acttgcagta 780 cgtatggatc gccattgcga gaatgctgaa aaattggccg
agaaactgaa agagcatcca 840 aaagtaagta cggttctgta cccgggagac
tttgagcatc ccgatcactc catcgtcgcc 900 aaacagatga aaaagggagg
cggtttatta agctttgaga tcaaggggac tgaggcggac 960 atcgccaaag
ttgtaaatca gttaaaactg attcgtattg ctgttagttt gggtgacgca 1020
gagaccttga ttcagcatcc tgcaaccatg acccatgcag tagtacccga aaagcgccgc
1080 actcaaatgg gtattagtaa aaagttgtta cgcatgtcgg ccgggttaga
ggcctggcaa 1140 gatgtctggg ctgacttaga gcaggcgtta aatcaactg 1179
<210> SEQ ID NO 57 <211> LENGTH: 1269 <212> TYPE:
DNA <213> ORGANISM: Methylobacterium aquaticum <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1269) <223> OTHER INFORMATION: Methionine
aminotransferase (Methylobacterium aquaticum) <400> SEQUENCE:
57 atgaccgcga ttccggcctt ggcagacctg caggctcgtt atgccgactt
acaagggcgt 60 ggtctgaagt tagatatgac gcgcggtaaa ccggcgccag
agcagttgga tttatcggac 120 gatcttttca ctttaccagg taaccgcgat
caccgcacag agagcggaga agacgcgcgt 180 aattacggcg gagtacaggg
cctggctgag gtccgtgcct tattcgcccc tgtgcttggt 240 gcgtcacccg
atcgcattgc cgtaggtaat aactcatcgt tggcattgat gcatgactgc 300
attgcctatg cattgcttaa gggtgtaccc ggcggcgctc gtccttgggc aaaggaagag
360 gagattcgtt ttttatgccc agtcccaggg tacgaccgtc acttcgctct
gtgcgagacc 420 tacgggattg gaatgattcc agtccctatg accgctgacg
ggcctgatat ggaaatggtt 480 gaacgtgagg tacgcgatcc acgcgtcaaa
ggtatgtggg cggtgccgca gtatagtaac 540 ccaggcggtg agacatactc
cgacgcgact gttgagcgcc tggctcgtat ggaaaccggt 600 gcccctgact
tccgtctttt ttgggacaac gcgtatgcac ttcaccattt gaccgaacgt 660
cgcccaaccc ttcgtaatgt gttagatgcc tgtgcggaag ccgggtcacc ggatcgtgct
720 attgtgtttg ctagtacgtc gaaagttaca ctggcggggg caggccttgc
gatgcttgcg 780 tccagcgagg gcaatattcg ctggtattta gctaacgccg
gcaaacgctc aattggtcca 840 gataagctta accagttgcg ccatgttcgc
tttctgcgtg accagggcgg acttgatgca 900 ttaatggacg gccaccgccg
tcttttagct cctaagttcc gcgctgtaac ggaaaccctt 960 gctcgtcatc
tgggcgggac tggagtagcg cgctggagcg agccggaagg ggggtacttt 1020
atcctgctgg aagtccctga gggctgtgcg acacgcgtag ttaagcttgc tgctgcttgc
1080
ggactggctc tgacgcccgc aggggcgacg cacccatacg ggcgtgaccc tcaagataag
1140 ctgttacgtc ttgccccgtc atacccgaaa ccagcggagg tcgaggcagc
cgctgaggta 1200 gtcgctgtgt gcgttttact tgcggcagct gaaagccgcg
aagctggcgg ttcggggcag 1260 gttgctgca 1269 <210> SEQ ID NO 58
<211> LENGTH: 1902 <212> TYPE: DNA <213>
ORGANISM: Saccharomyces cerevisiae YJM1615 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1902) <223> OTHER INFORMATION: Aro10p decarboxylase
(Saccharomyces cerevisiae YJM1615) <400> SEQUENCE: 58
atggcacccg tcactattga gaaattcgtg aatcaagaag agcgtcattt agtgagcaat
60 cgttccgcca cgatcccttt tggagaatat attttcaagc gccttctttc
cattgacacc 120 aaaagcgtct tcggggttcc cggcgacttc aatttatctt
tattggaata tttatactcg 180 ccctccgtgg aatctgcggg tcttcgttgg
gttggcacct gtaacgagtt aaatgcagcc 240 tacgctgcag atggatattc
ccgctactct aataaaattg gatgcttaat caccacatac 300 ggcgtaggag
aactgagtgc gcttaatgga atcgcggggt cattcgctga aaatgtaaag 360
gttctgcata tcgtaggggt cgccaagtcc attgattccc gttcgtctaa cttctcggat
420 cgtaacttac atcacttggt cccgcagtta catgattcga actttaaagg
acccaaccat 480 aaggtctatc acgacatggt taaagatcgt gtcgcatgtt
ccgtcgccta cctggaggat 540 attgagacgg cctgtgacca agttgataac
gtgatccgtg acatttataa gtattcaaaa 600 cctggttaca ttttcgtccc
agccgacttt gccgacatgt ccgtaacctg cgacaacttg 660 gtcaatgtac
cgcgtatcag ccaacaagat tgtattgtct accccagcga gaaccaactg 720
tcagacatca ttaataaaat cactagctgg atctactcgt ctaagactcc agcaatcctt
780 ggagacgtct taactgatcg ttatggggta tcaaactttc tgaacaaact
gatctgcaaa 840 accggtatct ggaacttctc caccgtgatg ggaaaatcag
tcattgacga gagtaaccca 900 acttatatgg gtcaatacaa cggcaaagaa
ggtcttaaac aggtctatga acatttcgag 960 ctgtgtgatt tggttttaca
cttcggagta gatattaacg agatcaataa tggtcactac 1020 acgttcactt
acaagccaaa tgcgaaaatt attcaattcc accctaatta tattcgttta 1080
gtagacactc gtcaggggaa tgaacaaatg ttcaaaggca tcaattttgc gccaatcttg
1140 aaagagttgt ataagcgtat cgacgtctct aaattatcgt tgcaatacga
ttccaatgta 1200 acacaataca ccaatgagac tatgcgtctg gaggacccaa
cgaatggtca atcgagcatc 1260 attacccaag tacacctgca aaagaccatg
ccgaaatttt tgaatcccgg cgacgtcgtc 1320 gtgtgtgaga ctggtagttt
ccaattcagt gtacgcgact tcgcattccc cagtcagttg 1380 aaatatatca
gccagggttt ctttttatcc attggtatgg ccttgcctgc cgcgttgggg 1440
gttgggatcg caatgcagga tcattccaac gcgcatatta acggagggaa cgtcaaagaa
1500 gactacaagc cccgcttaat tttgtttgaa ggtgacggcg ccgcgcagat
gaccatccag 1560 gagcttagca cgatccttaa atgcaatatc cctttggagg
tcattatctg gaataacaat 1620 ggatacacta tcgagcgtgc catcatgggt
ccaacacgtt catataacga tgtgatgtcg 1680 tggaaatgga caaagttgtt
cgaagccttt ggggatttcg atggtaagta tacgaattcg 1740 actttaattc
agtgtcctag caaattagcg ttaaaacttg aagaattgaa gaattctaat 1800
aagcgttcgg ggatcgaact gttagaagtg aagctgggtg agcttgactt cccagagcaa
1860 ttgaagtgta tggtagaggc cgcagctctt aaacgtaata ag 1902
<210> SEQ ID NO 59 <211> LENGTH: 666 <212> TYPE:
DNA <213> ORGANISM: Bacillus subtilis <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(666)
<223> OTHER INFORMATION: Methionine import system permease
proteinMetP (Bacillus subtilis) <400> SEQUENCE: 59 atgtttgaga
agtattttcc aaatgttgac ttgaccgagt tatggaatgc cacatatgaa 60
actctgtata tgacattgat ttccttactg tttgccttcg taatcggcgt catcctggga
120 ttgctgttat tcttaacatc taaggggtct ctttggcaaa ataaagcagt
aaattccgtt 180 atcgcagccg ttgtcaacat ctttcgttca attcccttcc
ttattttaat catcctgctt 240 cttggtttca ctaaattctt agtgggaaca
attttgggac caaatgcggc tcttcccgcg 300 ttagtcatcg gtagtgctcc
cttttatgct cgtctggtcg aaatcgcact tcgtgaagtg 360 gacaaaggag
tgattgaggc ggcgaaatcg atgggggcta agacgagcac tattattttt 420
aaggttctta tccccgagtc catgcccgcg ctgatttccg gaattacagt gactgcgatt
480 gcattgatcg ggtcaaccgc catcgcagga gctattggtt ctggtggatt
gggaaactta 540 gcatacgttg aaggctatca atcgaataat gcggatgtga
ccttcgtggc cacagttttc 600 atcctgatta ttgttttcat cattcagatc
attggtgacc ttattaccaa catcatcgat 660 aaacgc 666 <210> SEQ ID
NO 60 <211> LENGTH: 1029 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1029)
<223> OTHER INFORMATION: DL-methionine transporter subunit
MetN (Escherichia coli K-12]) <400> SEQUENCE: 60 atgattaaac
tgagcaacat tactaaggtg ttccaccaag gtacacgtac gatccaggct 60
cttaataatg tgtcactgca cgttcctgct ggtcagattt atggggttat cggtgccagt
120 ggggctggga agagcactct gatccgctgc gtcaatctgt tagagcgccc
tacagagggc 180 tcggtactgg tggacggtca agagttgact actctgtcgg
agtccgagtt gacaaaagca 240 cgccgccaga ttggcatgat tttccaacat
ttcaatttgt tatcgagccg tacagttttc 300 gggaacgtgg ccttaccact
ggagttggac aatactccca aagacgaagt caaacgtcgt 360 gtgaccgaat
tattgtcctt ggtgggtctt ggtgacaaac acgacagtta tcccagtaat 420
ttgagtggcg ggcaaaaaca gcgtgttgcc atcgcacgcg cattagcttc gaatcccaag
480 gtgctgttat gtgatgaagc gaccagcgcc cttgacccag ccacaactcg
tagcatcctg 540 gagcttttga aagatatcaa tcgtcgcctg ggtttgacca
tcttattgat tacgcacgag 600 atggacgttg taaagcgtat ctgtgactgt
gtagcggtga tctccaacgg tgaattaatc 660 gaacaggaca ccgtatcgga
ggtcttctca catcctaaga caccccttgc acaaaaattc 720 atccaaagca
cgctgcattt agatattcct gaagattatc aggaacgcct gcaggctgaa 780
ccgtttactg attgcgttcc aatgcttcgc ttagagttca cagggcaatc ggttgacgct
840 cccttattga gtgaaaccgc ccgccgtttc aatgttaata acaacatcat
ttccgcgcaa 900 atggactacg cggggggtgt taaatttgga atcatgttaa
ccgaaatgca cggcacacag 960 caggatacac aggcggcgat cgcatggctg
caggaacatc atgttaaagt agaagtcctt 1020 gggtatgtg 1029 <210>
SEQ ID NO 61 <211> LENGTH: 654 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(654)
<223> OTHER INFORMATION: metI (Escherichia coli) <400>
SEQUENCE: 61 atgtctgagc cgatgatgtg gctgctggtt cgtggcgtat gggaaacgct
ggcaatgacc 60 ttcgtatccg gtttttttgg ctttgtgatt ggtctgccgg
ttggcgttct gctttatgtc 120 acgcgtccgg ggcaaattat tgctaacgcg
aagctgtatc gtaccgtttc tgcgattgtg 180 aacattttcc gttccatccc
gttcattatc ttgcttgtat ggatgattcc gtttacccgc 240 gttattgtcg
gtacatcgat tggtttgcag gcagcgattg ttccgttaac cgttggtgca 300
gcaccgttta ttgcccgtat ggtcgagaac gctctgctgg agatcccaac cgggttaatt
360 gaagcttccc gcgcaatggg tgccacgccg atgcagatcg tccgtaaggt
gctgttaccg 420 gaagcgctgc cgggtctggt gaatgcggca actatcaccc
tgattaccct ggtcggttat 480 tccgcgatgg gtggtgcagt cggtgccggt
ggtttaggtc agattggcta tcagtatggc 540 tacatcggct ataacgcgac
ggtgatgaat acggtactgg tattgctggt cattctggtt 600 tatttaattc
agttcgcagg cgaccgcatc gtccgggctg tcactcgcaa gtaa 654 <210>
SEQ ID NO 62 <211> LENGTH: 816 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(816)
<223> OTHER INFORMATION: metQ (Escherichia coli) <400>
SEQUENCE: 62 atggcgttca aattcaaaac ctttgcggca gtgggagccc tgatcggatc
actggcactg 60 gtaggctgcg gtcaggatga aaaagatcca aaccacatta
aagtcggcgt gattgttggt 120 gccgaacagc aggttgcaga agtcgcgcag
aaagttgcga aagacaaata tggcctggac 180 gttgagctgg taaccttcaa
cgactatgtt ctgccaaacg aagcattgag caaaggcgat 240 atcgacgcca
acgccttcca gcataaaccg taccttgatc agcaactgaa agatcgtggc 300
tacaaactgg tcgcagtagg caacactttt gtttatccga ttgctggtta ctccaagaaa
360 atcaaatcac tggatgaact gcaggatggt tcgcaggttg ccgtgccaaa
cgacccaact 420 aaccttggtc gttcactgct gctgctgcaa aaagtgggct
tgatcaaact gaaagatggc 480 gttggcctgc tgccgaccgt tcttgatgtt
gttgagaacc ccaaaaatct gaaaattgtt 540 gaactggaag caccgcaact
gccgcgttct ctggacgacg cgcaaatcgc tctggcagtt 600 atcaatacca
cctatgccag ccagattggc ctgactccgg cgaaagacgg tatctttgtt 660
gaagataaag agtccccgta cgtaaacctg atcgtgacgc gtgaagataa caaagacgcc
720 gagaacgtga agaaattcgt ccaggcttat cagtctgacg aagtttacga
agcagcaaac 780 aaagtgttta acggcggagc tgttaaaggc tggtaa 816
<210> SEQ ID NO 63 <211> LENGTH: 2286
<212> TYPE: DNA <213> ORGANISM: Bacillus atrophaeus
UCMB-5137 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(2286) <223> OTHER INFORMATION:
MetE (Bacillus atrophaeus UCMB-5137) <400> SEQUENCE: 63
atgacgacta tcaaaacatc aaatctgggc ttccctcgca ttggacttaa tcgcgaatgg
60 aaaaaatcac tggaagcgtt ttggaaaggt aacagcgaca aagatacatt
tcttaagcag 120 atggatgagt tatttcttac tgccgtaaaa acccagattg
atcaaaaaat cgacatcgtg 180 cccgtgagcg acttcactca ctacgaccac
gttcttgaca cagctatctc ttttaattgg 240 attccagaac gctttaaaca
cattacggat gcgactgata catatttcgc gctggcacgt 300 ggcattaagg
atgctgttag ttcggaaatg actaagtggt ttaataccaa ttaccactat 360
atcgttccgg aatacaataa agacatcgaa ttccgtttaa cccgcaacaa gcagttagag
420 gactaccgcc gcgtcaaaca agcgtttggc gtcgaaacta aacccgtcat
tgtcggtcct 480 tacacattcg tgacgcttgc caagggctac gaacaaagtg
aggccaaaga aatccaaaag 540 cgtttagtcc cattgtatgt gcaattattg
aaagaattgg aacaagaggg cgtgcagtgg 600 gtacaaatcg atgagccagc
acttgtgaca gcctcatccg aggatgttag cgcggccaag 660 gagttatacc
aggccattac gaatgagtta tccggcttga atgtcctttt gcagacttac 720
ttcgattctg ttgatgctta tgaggagtta atcagctacc cggtacaggg tatcggcttg
780 gattttgtac acgataaagg gcgcaacttg gagcaattaa aagcgcatgg
atttccgaag 840 gataaggtat tagcagctgg tgttattgat ggtcgtaaca
tttggaagac ggatttagat 900 gagcgcttgg acgccatcct tgcgctgtta
tcttcgacgg acattgacga attatggatt 960 caaccaagca attcgcttct
tcatgtacca gtagcaaagc acccagacga gcacctggag 1020 aaggatctgt
tgaatggctt gagttacgca aaagaaaagc tggcagaact gtccgcttta 1080
aaagagggtt tgttatcggg taaagcggca atctcggccg acattcagca ggccaaagcg
1140 gatttacagg ccctgaagca attcgccacc ggggctaaca gtgagcagaa
agaggaatta 1200 aatcagttga ccgagaaaga ctttaagcgc ccgatcccct
tcgaagagcg cctgaaaatc 1260 cagaatgaat ccttggggct tcccctgctt
cctactacga ctattggttc ttttcctcaa 1320 agcgccgagg tgcgttcggc
gcgccaaaag tggcgcaaaa gtgagtggag cgacgagcaa 1380 tatcaagaat
ttatcaacgc ggaaacgaag cgctggatcg acattcagga agagcttgat 1440
cttgacgttt tagtacatgg agagttcgag cgcaccgaca tggtcgaata tttcggtgag
1500 aaactggctg gattcgcgtt tactaaatac gcatgggtcc agagctacgg
atcccgctgt 1560 gtacgccctc ccgtcatcta tggggacgtg gagtttattg
aacctatgac tgtcaaggac 1620 acagtgtacg ctcaatcttt aacgagtaag
caggttaaag ggatgttgac tggcccggtc 1680 acaatcttga attggagctt
cccgcgtaac gacattagcc gtaaggagat cgccttccaa 1740 atcgggttag
ctcttcgcaa agaggtcaag gcgttggaag atgctggtat tcaaatcatc 1800
caagttgacg aaccggccct gcgtgaaggg ctgcctctga aagaaaacga ttgggaagag
1860 tatttaacgt gggccgcgga ggcgttccgc ttaactactt cggctgtgaa
aaacgacact 1920 cagattcata cacacatgtg ttattccaat tttgaggaca
ttgtcgacac aattaatgac 1980 ttggatgcgg acgtcattac aatcgaacac
tcccgcagtc acggtgggtt cttggactac 2040 ttgcgcgatc atccgtatct
taaaggttta ggtcttggcg tgtacgatat tcacagccct 2100 cgtgtacccc
cgacagagga aatttataag atcattgacg aagccctgac cgtatgtcct 2160
actgaccgct tctgggtaaa cccagactgc gggctgaaga cccgtcacca ggaggaaacg
2220 attgccgcgt tgaagaacat ggtcgaggct gctaaacagg ctcgtgccaa
acagagtcaa 2280 cttgtc 2286 <210> SEQ ID NO 64 <211>
LENGTH: 753 <212> TYPE: DNA <213> ORGANISM:
Corynebacterium glutamicum <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(753) <223>
OTHER INFORMATION: BrnF (Corynebacterium glutamicum) <400>
SEQUENCE: 64 atgcagaaaa cacaggagat tcacagctcg ttagaggtta gccccagtaa
agctgctctg 60 gagcccgacg ataaggggta tcgtcgttac gaaatcgcac
aaggcctgaa gacctctctt 120 gctgcaggcc tgggaatgta tcctatcgga
attgcattcg gcttactggt gattcaatat 180 ggttatgaat ggtgggccgc
tccactgttc tccggcctga tttttgcggg gtctacggag 240 atgcttgtaa
ttgcacttgt ggtcggcgct gctccgctgg gtgccattgc ccttacgacc 300
ttacttgtta atttccgtca tgttttctat gccttttcct ttcccttgca cgttgttaaa
360 aaccctattg cgcgcttcta ttctgtattc gctcttattg atgaagcata
cgctgttaca 420 gccgctcgtc ccgccggttg gagtgcatgg cgtctgattt
caatgcagat tgcgttccac 480 tcctactggg tatttggagg cttgaccggt
gtagcaatcg cagagttaat tcctttcgag 540 atcaaaggcc tggagttcgc
actttgttcg ttatttgtaa ctcttacttt agacagttgt 600 cgcactaaga
aacaaattcc gagtttgtta ttggctggac tgagctttac tatcgcgtta 660
gtagtgatcc ccggccaagc tctgttcgct gcgttactta tctttctggg gcttctgaca
720 atccgttatt ttttcttagg gaaggcagcc aaa 753 <210> SEQ ID NO
65 <211> LENGTH: 324 <212> TYPE: DNA <213>
ORGANISM: Corynebacterium glutamicum <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(324)
<223> OTHER INFORMATION: BrnE (Corynebacterium glutamicum)
<400> SEQUENCE: 65 atgacgactg atttctcctg catcctgttg
gtggtcgcgg tatgtgcagt cattacattt 60 gcgcttcgtg ccgtaccttt
tctgatcttg aaacccttgc gtgaatcgca atttgtggga 120 aaaatggcca
tgtggatgcc tgcgggcatt ctggcaatcc tgacggcttc taccttccgt 180
tcaaacgcca tcgatttaaa gacgttgacg ttcggtctga ttgccgtggc aatcacagtc
240 gtagcccact tattaggagg ccgtcgcacc ttattatctg ttggcgctgg
aacaattgtg 300 tttgtaggtc ttgttaattt gttt 324 <210> SEQ ID NO
66 <211> LENGTH: 1023 <212> TYPE: DNA <213>
ORGANISM: Salmonella enterica subsp. enterica serovar Typhistr.
CT18 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1023) <223> OTHER INFORMATION:
threonine 3-dehydrogenase (Salmonella enterica subsp. enterica
serovar Typhistr. CT18) <400> SEQUENCE: 66 atgaaggccc
tgagcaaatt gaaagccgag gaggggatct ggatgaccga tgttcctgaa 60
ccagaagtgg ggcacaacga ccttttaatc aaaattcgca agactgcaat ctgcgggaca
120 gacgtacata tctataactg ggacgagtgg agtcaaaaaa ctattcccgt
ccctatggtg 180 gtcgggcacg agtatgtcgg agaggttgta ggaatcggac
aagaagtcaa aggatttaaa 240 atcggggatc gtgtgagtgg ggagggtcac
attacctgtg ggcattgccg caattgccgt 300 ggaggacgca cacatttgtg
ccgtaacact acaggcgtag gcgtgaatcg tcccggatgt 360 ttcgcggaat
accttgtcat tccagcgttt aacgccttta agatccctga caacatttca 420
gatgatttag catccatttt tgacccattc ggtaacgcgg tccatactgc gttgagcttc
480 gacttagttg gagaagatgt attagtttcc ggcgccggac cgattggcgt
catggcagct 540 gccgttgcga agcacgtggg cgcacgtcat gtggtaatta
cggacgtaaa tgagtatcgt 600 ctggagctgg cacgtaaaat gggggttaca
cgtgccgtaa acgttgcgaa agagtcttta 660 aacgatgtca tggctgaact
gggcatgacg gaagggtttg atgtcggact ggaaatgtcc 720 ggtgccccgc
cagccttccg taccatgttg gacaccatga accatggggg ccgtatcgca 780
atgttgggaa ttcccccgag cgacatgtct atcgactgga caaaggtaat ttttaaaggc
840 ctgttcatta aggggattta cggtcgtgag atgtttgaga cgtggtacaa
gatggctgcc 900 ttgattcaat cggggttgga tctgagccct atcatcacac
accgtttttc agtggatgac 960 tttcaaaaag ggtttgacgc catgtgcagc
ggtcaatcag ggaaagtaat tctttcttgg 1020 gac 1023 <210> SEQ ID
NO 67 <211> LENGTH: 999 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(999) <223>
OTHER INFORMATION: threonine aldolase (Escherichia coliO26:H11 str.
CVM10026) <400> SEQUENCE: 67 atgattgacc ttcgttcgga caccgtaacc
cgcccatctc acgcaatgtt ggaagctatg 60 atggccgcgc ctgtggggga
tgacgtttat ggggatgacc cgaccgtcaa cgctttacaa 120 gattacgctg
ctgaattgtc gggcaaagaa gcagcaatct tcttacctac aggtacacaa 180
gctaatcttg tcgccctgct tagtcactgt gagcgtggcg aagaatacat tgttggtcaa
240 gcagcgcata attacctgtt cgaagctgga ggggctgctg ttcttggtag
cattcagccc 300 caacccattg atgctgctgc cgatggtact cttcctctgg
ataaagtcgc tatgaaaatt 360 aagccagacg acattcactt cgcacgcaca
aagctgctgt cgcttgagaa tacacacaat 420 ggaaaagtcc tgccccgtga
gtacctgaaa gaggcttggg aatttacacg cgaacgcaac 480 ctggctctgc
acgtagacgg tgctcgcatc ttcaacgccg ttgtcgccta cggttgcgaa 540
ttgaaagaga ttacgcaata ctgtgactcc ttcacgattt gcttgtccaa aggcttaggc
600 accccggtgg gttcattgtt ggtaggaaac cgtgactata ttaagcgcgc
catccgctgg 660 cgtaaaatgg cagggggtgg aatgcgtcaa tcagggattc
ttgcggcagc tggcatgtac 720 gcgctgaaaa ataatgtggc tcgccttcaa
gaggatcacg ataatgctgc gtggatggct 780 gagcaattac gtgaggcggg
tgcagacgta atgcgccaag ataccaatat gctgttcgta 840 cgtgttgggg
aagaaaacgc tgcggcctta ggagaataca tgaaggcgcg taacgtgttg 900
atcaacgcat cccctattgt tcgccttgta actcaccttg atgtttcacg tgaacaattg
960 gcggaagttg ccgcccactg gcgtgccttt cttgctcgc 999 <210> SEQ
ID NO 68
<211> LENGTH: 1251 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1251)
<223> OTHER INFORMATION: serine hydroxymethyltransferase
(Escherichia coli) <400> SEQUENCE: 68 atgcttaaac gtgagatgaa
tatcgccgac tacgacgccg aattatggca ggcgatggag 60 caggagaaag
tccgccaaga ggaacacatt gagcttattg cgtcggagaa ctatacatcc 120
cctcgcgtta tgcaggcgca aggctcacag ttgacgaaca aatacgctga gggatatccg
180 ggaaagcgtt attatggcgg ttgcgagtac gttgacattg ttgaacagtt
agcgattgat 240 cgtgctaagg agttatttgg agcggattat gccaatgttc
aacctcactc gggcagccag 300 gctaactttg ctgtatacac cgcactttta
gaacctggtg acacggtcct gggtatgaat 360 ttggcccatg gaggccactt
aactcatgga agccctgtga attttagtgg gaagttgtat 420 aacatcgtgc
cctacgggat cgacgccaca ggacacattg attacgcaga tttggagaaa 480
caagccaagg aacataagcc taaaatgatc atcggcggat tttcagcata tagcggagtg
540 gtagactggg ccaaaatgcg cgagattgct gattcgattg gtgcttacct
gtttgtcgat 600 atggcgcatg tcgctggtct ggtcgctgcg ggagtttatc
ctaaccccgt gcctcacgct 660 cacgtcgtga cgactactac acataagact
ttagcgggtc ctcgtggggg tttgattctt 720 gcgaaggggg gctcagagga
actttataag aagcttaact ctgccgtatt tcccggcggt 780 caggggggcc
ctcttatgca cgtcatcgca ggaaaggcgg tggctctgaa ggaagcgatg 840
gaacccgaat tcaagactta ccaacagcaa gtagccaaaa acgccaaagc catggtggag
900 gtattcctgg agcgcggcta caaggtagtt agcgggggga cggacaacca
tttgttctta 960 gtcgatttag tggacaaaaa ccttactggt aaggaggctg
atgctgctct tgggcgtgca 1020 aatatcacag tcaataagaa tagcgtgccc
aatgacccaa agtcgccatt tgtgacttct 1080 ggcatccgcg ttgggactcc
ggcaatcacc cgtcgtggct ttaaggaggc agaggccaag 1140 gagctggcag
ggtggatgtg tgacgtactg gactctatta atgatgaggc agttatcgaa 1200
cgtattaaag gcaaagtgct tgacatttgt gcgcgctacc ccgtgtatgc c 1251
<210> SEQ ID NO 69 <211> LENGTH: 1293 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1293) <223> OTHER INFORMATION: tdcC(Escherichia coli)
<400> SEQUENCE: 69 atgtctactt cggactctat tgtttcatcg
caaacaaaac agtcatcctg gcgtaaatca 60 gataccacct ggactttggg
tctgtttggt accgcgatcg gggctggtgt attgtttttc 120 ccgatccgcg
ctggatttgg tggtttaatt cctatcctgc tgatgcttgt actggcatat 180
cctattgctt tttattgtca tcgcgcagcg cgcttgtgtt taagcggaag caacccctcg
240 ggtaatatca cagagacggt ggaggagcat ttcgggaaaa caggaggggt
cgtaatcaca 300 tttctgtact tttttgctat ttgtcccctg ttgtggattt
atggggttac gatcaccaat 360 acttttatga cgttttggga gaatcaactg
ggctttgcac cgcttaaccg cggattcgtg 420 gcgctgttcc ttttactgtt
gatggcgttt gtcatctggt tcggtaaaga cttaatggtg 480 aaagtcatgt
cttatttggt atggcctttc attgcttcac ttgtcttaat tagtctgtca 540
ttaatccctt attggaactc ggcagtaatc gatcaagtag atctgggtag cctgtctttg
600 accggacatg atgggatctt aattaccgta tggctgggca tttctattat
ggtctttagt 660 tttaactttt cacctatcgt gtcctccttt gtggtgtcca
agcgcgagga atatgagaag 720 gattttggtc gtgattttac ggaacgtaag
tgctcacaaa ttattagccg cgcgtctatg 780 cttatggtgg ctgtcgttat
gttctttgct ttctcctgct tatttacctt gtcaccggcg 840 aacatggcgg
aagcgaaggc gcaaaacatt ccagttttat catatcttgc taatcatttc 900
gcttctatga cagggaccaa aactactttt gccatcacat tggagtatgc ggcgtctatc
960 attgcattag tggccatttt taagtcgttc tttggccatt atttaggtac
tttagaaggg 1020 ttgaatggct tagtcttgaa attcggatac aagggggaca
aaactaaagt ttccttgggt 1080 aagttgaaca caatctcgat gatctttatt
atggggagta catgggtcgt tgcgtatgca 1140 aatccaaaca ttctggattt
aattgaggcg atgggagcac cgattatcgc gtcattgttg 1200 tgccttttgc
cgatgtacgc catccgtaag gcgccttcac tggccaaata tcgtgggcgc 1260
ttggataacg tgttcgtaac cgtcatcgtt tgc 1293 <210> SEQ ID NO 70
<211> LENGTH: 885 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(885) <223> OTHER
INFORMATION: Threonine/ homoserine exporterRhtA, Escherichia coli
(strain K12) <400> SEQUENCE: 70 atgcctggtt ccttgcgtaa
aatgccggtt tggttgccga ttgttattct tctggttgca 60 atggctagca
tccaaggagg cgctagttta gcaaaaagtc tgtttccttt ggtgggggca 120
ccgggtgtga ccgcgctgcg tttggctttg ggcactttaa ttttgattgc cttctttaag
180 ccctggcgcc ttcgttttgc taaagaacaa cgtttgccgc ttttgttcta
cggcgtctca 240 cttggtggca tgaactatct tttttattta agcatccaaa
ccgtacccct gggtattgcg 300 gtggctttgg agttcacggg tccattggca
gttgcccttt tcagctcgcg tcgcccagtc 360 gatttcgtct gggtagtgct
tgcggtactt ggactgtggt tcttactgcc cttaggccaa 420 gacgtgagtc
acgtagacct taccgggtgt gcgctggctt tgggagccgg tgcttgttgg 480
gcaatttaca tcctgtcggg acagcgtgcg ggagcagagc acgggcctgc gacagtagcg
540 attgggtcgc tgatcgcagc cctgattttc gtccccattg gtgccttaca
ggcaggagag 600 gcgttgtggc actggtcagt gattccctta ggtttggcgg
tagcaatcct gtctaccgca 660 cttccttatt ctttagagat gattgcctta
acccgtctgc cgacacgtac gtttggcacc 720 ttaatgtcga tggaaccggc
attggctgcc gtttcaggta tgatcttcct gggagagacg 780 ttaactccca
ttcagttgtt agctcttggg gcaatcatcg ctgcgagtat gggatcgacc 840
cttacggttc gtaaagagtc gaagattaaa gaattggaca tcaat 885 <210>
SEQ ID NO 71 <211> LENGTH: 618 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(618)
<223> OTHER INFORMATION: rhtB(Escherichia coliFVEC1302)
<400> SEQUENCE: 71 atgacgctgg agtggtggtt cgcatacttg
ctgacatcca tcatcctgag tttaagcccc 60 ggatctggtg caatcaacac
gatgactacg tctttgaatc acggctatcg tggtgctgtt 120 gcatccattg
ccggcttgca gacgggatta gccatccata ttgttttagt gggtgtagga 180
cttggaacat tattcagtcg ctcggttatc gcctttgagg tcttaaagtg ggctggtgcc
240 gcttatttga tttggctggg aattcagcaa tggcgtgcag ccggtgcgat
tgacttgaag 300 agccttgcgt ccacacagag ccgccgtcac ttgtttcaac
gtgcagtatt cgtcaatttg 360 accaacccca aaagtatcgt ctttctggcg
gcactgtttc cccagttcat tatgcctcaa 420 cagccgcagt tgatgcagta
catcgtcttg ggcgtcacca ccatcgtagt ggacattatt 480 gtaatgattg
gatacgccac tctggcccaa cgtattgcgc tgtggatcaa gggcccgaaa 540
cagatgaagg cactgaacaa aatttttggt tctttgttta tgttggttgg ggcacttctt
600 gccagtgcac gtcacgcg 618 <210> SEQ ID NO 72 <211>
LENGTH: 618 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(618) <223> OTHER INFORMATION:
RhtCthreonine Rht Transporter (Escherichia coliBL21(DE3))
<400> SEQUENCE: 72 atgctgatgc tttttttaac agtagcaatg
gtgcatatcg tcgcattgat gtcaccggga 60 cctgactttt tttttgtttc
acaaacagca gtatcacgct cacgtaagga ggcaatgatg 120 ggtgtcttag
ggatcacttg cggcgtaatg gtatgggccg gtattgcact tctgggactg 180
catttaatta ttgagaagat ggcctggctt cacacattaa tcatggtagg cggtgggctt
240 tatttatgtt ggatgggcta tcaaatgctg cgtggagctc ttaagaaaga
agccgtgtcc 300 gcaccggctc cccaagtgga acttgcgaaa tcaggtcgct
ccttcttgaa ggggttgttg 360 actaatcttg cgaaccctaa ggccatcatt
tatttcggtt ctgtgtttag tttgttcgtt 420 ggggataatg tgggaaccac
ggaacgctgg ggaatcttcg cattaatcat tatcgagacg 480 ttagcttggt
tcaccgtcgt ggcctccctt tttgctctgc cgcaaatgcg ccgtggttac 540
caacgtttag caaagtggat cgacggtttt gctggagctt tatttgcggg tttcggcatt
600 catctgatta ttagccgt 618 <210> SEQ ID NO 73 <211>
LENGTH: 984 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(984) <223> OTHER INFORMATION:
cysteine desulfhydrase (Escherichia coli) <400> SEQUENCE: 73
atgcccctgc acaacttaac acgttttcca cgcctggaat tcattggtgc accgactccc
60 ttggaatatc tgcctcgctt ttcggactac ttaggccgcg agattttcat
taagcgcgat 120 gatgttacac cgatggctat ggggggtaac aaattgcgta
aattggaatt tcttgcagcg 180 gatgcactgc gtgaaggcgc ggacacttta
attaccgctg gtgcaattca gtcaaatcac 240 gtacgccaaa ctgcggcagt
tgctgcgaag ttaggtcttc attgtgtcgc ccttttggaa 300 aatccaattg
gcacaacggc agaaaattac cttaccaacg ggaaccgttt gttgcttgac 360
ctttttaaca cacagatcga aatgtgcgac gctttaactg atcccaacgc tcaattggag
420 gagcttgcga ctcgcgtgga agctcaaggc ttccgtccgt atgttattcc
ggtcggcggc 480 agcaatgctc ttggggcatt agggtatgta gagtccgctc
tggagatcgc gcaacaatgt 540
gagggcgcgg ttaacatttc gagtgtagtt gtggcctctg gaagtgcggg cacccacgcc
600 gggctggctg tgggtcttga gcacttaatg cctgaatctg aactgatcgg
ggtcacagtc 660 tcgcgttccg tcgcagatca gttacctaag gtagtaaact
tacagcaagc cattgcgaaa 720 gaattagaat taaccgctag tgcagaaatc
ttattatggg atgattactt tgcgcctggg 780 tacggtgtcc ccaatgatga
aggtatggaa gcagtcaagc ttttagctcg tttggagggg 840 atcttgctgg
accctgttta caccggcaaa gcaatggcag gcttaattga cggtatcagt 900
cagaaacgct tcaaagacga gggaccaatt ctgttcatcc ataccggcgg cgctcctgcc
960 ctttttgcct accaccctca cgtt 984 <210> SEQ ID NO 74
<211> LENGTH: 1413 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1413)
<223> OTHER INFORMATION: tnaA (Escherichia coliDH1)
<400> SEQUENCE: 74 atggagaatt tcaagcattt gcccgagccg
ttccgcattc gtgtcattga gcctgtcaag 60 cgtactactc gcgcgtatcg
cgaagaggcg attatcaaat cgggtatgaa tccattttta 120 cttgattcag
aagatgtgtt catcgattta cttacagatt ctgggacagg cgcggtaacg 180
caatcgatgc aagcagcgat gatgcgcggt gacgaagcct attctggctc gcgctcctat
240 tatgctctgg ccgaatcagt caaaaacatt tttggttacc aatatacgat
tcccacgcat 300 cagggacgcg gagcagagca aatctatatc ccagtcttaa
tcaaaaagcg cgagcaagaa 360 aagggattgg accgctcgaa aatggtagcc
ttctcaaatt acttcttcga cactactcag 420 gggcactcgc aaatcaacgg
ctgcactgtt cgcaatgtgt atatcaagga agcctttgat 480 acaggcgtac
gttacgattt caaggggaac tttgacctgg aaggtcttga acgtggcatt 540
gaagaagtag gacccaacaa cgtaccctat atcgtcgcca cgatcacatc taatagcgca
600 ggaggtcagc ctgtgtcttt ggcgaatctg aaagcgatgt attcgatcgc
caaaaagtat 660 gatatccccg tcgtaatgga ttctgcacgt tttgcagaga
acgcctactt cattaaacag 720 cgtgaagcgg agtacaaaga ttggaccatc
gaacagatca ctcgtgagac ttataaatat 780 gctgacatgc tggctatgtc
ggctaagaag gacgctatgg tcccaatggg aggcctttta 840 tgcatgaagg
acgatagttt ttttgacgtt tatacggaat gtcgcaccct ttgtgtagtg 900
caggaaggat tccccactta tggcggcctt gaaggtggag cgatggaacg tttagctgtt
960 ggactgtatg atggtatgaa tctggattgg ctggcatatc gtattgcgca
ggtgcagtac 1020 ctggtagacg ggttagagga gatcggggtt gtgtgccagc
aggccggggg ccatgcggcg 1080 ttcgtggacg caggaaaact gcttccccac
attcccgccg atcagttccc tgcgcaggca 1140 cttgcttgcg agttatacaa
ggtggccggt atccgtgcgg tagagatcgg ctcgtttctt 1200 ttggggcgcg
accctaaaac aggaaaacaa ttgccctgcc ctgccgaact tcttcgcctt 1260
actatccctc gtgcgaccta cactcaaacc cacatggact ttattatcga ggccttcaaa
1320 catgtgaagg agaatgctgc taatatcaag ggcctgacct ttacctacga
gccaaaggtt 1380 ttgcgccact ttacagcaaa acttaaagaa gtt 1413
<210> SEQ ID NO 75 <211> LENGTH: 969 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(969)
<223> OTHER INFORMATION: cysK (Escherichia coliO104:H4 str.
C227-11) <400> SEQUENCE: 75 atgtcaaaaa ttttcgagga taactcgtta
acgatcggcc acactccctt ggttcgtctg 60 aatcgtatcg gtaacgggcg
cattctggca aaggttgaat cacgcaatcc gtccttctca 120 gttaagtgcc
gtattggagc gaatatgatt tgggatgctg agaagcgcgg agtcctgaag 180
cctggggtgg agttggtgga gccaacctct gggaatacag gtatcgcgct ggcttatgta
240 gctgcagcgc gtggctacaa attaacactt accatgcccg agaccatgtc
aatcgaacgt 300 cgtaagttgt tgaaggcatt aggagcgaat ctggtactga
ccgaaggagc taagggaatg 360 aagggcgcta ttcaaaaagc ggaagaaatt
gtcgcaagta accccgaaaa gtatctttta 420 ctgcaacagt tttctaaccc
tgcaaatcct gagatccacg aaaaaacaac aggtcccgaa 480 atctgggaag
acaccgacgg tcaagttgac gtatttatcg ccggggtagg aactggagga 540
accttaacgg gggtcagtcg ttatattaag ggtacgaagg gaaagactga tttgattagc
600 gtagcagtgg agccaacgga tagtcctgtt attgcccaag ccctggcggg
ggaggaaatc 660 aaaccgggac ctcacaaaat ccaagggatt ggtgcgggtt
ttatcccagc caatctggat 720 ctgaaacttg tcgacaaggt cattggaatt
actaatgaag aggcgatctc cactgcgcgc 780 cgtttgatgg aggaagaagg
gattttggca gggatttcaa gcggtgcggc ggtggcagca 840 gctttgaaat
tgcaagaaga cgagtcattc actaataaga atattgttgt tattttacca 900
agcagcggtg agcgctactt atcaaccgct ttgttcgctg atttatttac ggaaaaagag
960 ttacaacaa 969 <210> SEQ ID NO 76 <211> LENGTH: 909
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(909) <223> OTHER INFORMATION: cysM
(Escherichia coliFVEC1412) <400> SEQUENCE: 76 atgtcaacat
tagaacagac aattggtaat acccccctgg tcaaattgca gcgcatgggg 60
ccaaacaatg gaagcgaggt ttggctgaaa ttggaaggca acaacccggc gggatctgtg
120 aaagaccgtg ccgcactgtc catgatcgta gaagctgaga aacgtggcga
gattaaacct 180 ggggatgttt taatcgaggc tacaagtggg aacactggaa
tcgcccttgc catgattgcg 240 gctttaaagg gttatcgtat gaagttactt
atgcccgata acatgagcca ggagcgccgt 300 gccgctatgc gtgcctatgg
tgctgaactt atcttagtta ccaaggagca aggcatggaa 360 ggtgcgcgtg
acttggcatt agaaatggcg aatcgtggcg aagggaagct gcttgaccaa 420
tttaataatc cagataaccc ttatgcacac tataccacga ccggcccgga aatctggcaa
480 caaaccggcg ggcgcatcac ccactttgta tcatccatgg gcacaactgg
tacaattacg 540 ggcgtttctc gtttcatgcg cgagcagagt aaacctgtta
caatcgtggg acttcaacct 600 gaggagggat cttcgatccc aggcattcgt
cgttggcctg ctgagtactt acctggcatt 660 ttcaacgcat ccttagtgga
tgaagttctt gacattcatc agcgcgaagc agagaatacc 720 atgcgcgagt
tggcagtacg tgagggcatt ttctgcgggg tttcttctgg gggggccgtg 780
gcgggtgctt tacgtgtcgc caaagcaaac cccggagcag tagttgttgc cattatttgt
840 gatcgtggtg accgctactt atctacggga gtcttcggag aggaacactt
ttcacaaggg 900 gccggaatt 909 <210> SEQ ID NO 77 <211>
LENGTH: 1170 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1170) <223> OTHER
INFORMATION: malY (Escherichia coli) <400> SEQUENCE: 77
atgttcgatt tttcgaaagt cgtcgatcgt catgggacct ggtgcactca atgggactac
60 gtggcggacc gctttgggac agcagatttg ttaccgttca ctattagcga
catggatttt 120 gccacagcac cttgcattat cgaggcactg aatcagcgct
taatgcatgg ggttttcggt 180 tatagccgtt ggaagaacga tgagttcctt
gcagcaattg cacattggtt cagtacccaa 240 cattataccg ctatcgattc
ccagacggtt gtgtacggcc ccagcgttat ttacatggtg 300 agcgaattga
tccgtcagtg gtctgaaaca ggagaaggtg tagtaatcca tactcccgcc 360
tatgacgcgt tctacaaagc cattgagggg aatcaacgta cagtaatgcc cgttgcctta
420 gaaaaacagg cagacggatg gttttgcgat atgggaaaat tagaggcggt
acttgcaaaa 480 cccgagtgca aaatcatgct tttatgcagt ccgcaaaacc
caacaggcaa ggtctggacc 540 tgtgatgaat tagagattat ggcggatttg
tgcgagcgtc acggagtccg tgtcatctct 600 gacgagattc acatggacat
ggtctggggg gaacagccgc acattccttg gtctaatgtc 660 gcacgtggtg
attgggccct tttgacatcg ggttcgaaaa gctttaacat tccagccctg 720
accggggcat atggaattat cgaaaactcg tcgagccgtg acgcgtattt atctgccctt
780 aagggacgtg atggactttc gagcccgtcg gttcttgcct tgacggcaca
cattgctgct 840 taccaacagg gagcgccgtg gctggacgct cttcgcattt
acctgaagga taaccttact 900 tacattgcgg ataagatgaa tgcggccttc
ccagaactta actggcagat tccccagtca 960 acgtatttag cctggcttga
ccttcgtccc ttaaacattg atgacaacgc actgcaaaag 1020 gcactgatcg
aacaggaaaa ggtagccatc atgcctggct atacctacgg cgaggagggc 1080
cgtgggttcg tccgcctgaa cgcaggatgt ccccgctcga aacttgaaaa aggggtagct
1140 ggtcttatta atgctattcg cgctgtgcgc 1170 <210> SEQ ID NO 78
<211> LENGTH: 1185 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1185)
<223> OTHER INFORMATION: MetC (Escherichia coli) <400>
SEQUENCE: 78 atggccgaca agaagttgga tactcaactg gtgaacgccg ggcgttccaa
aaaatatacc 60 ttgggagctg ttaatagcgt tatccaacgt gcatcaagtt
tagttttcga tagtgtcgaa 120 gcaaagaagc atgcgacacg caatcgcgca
aatggggaat tattttatgg acgccgcggg 180 accttgaccc acttctcttt
acagcaggcc atgtgtgagc tggaaggggg agccggttgt 240 gtattgttcc
cctgcggagc cgcggcggtg gctaacagta tcctggcgtt cgtggagcag 300
ggtgatcacg tcctgatgac gaacaccgcg tacgaaccct cgcaagactt ctgcagtaaa
360 atcttatcca aattaggtgt gactacctcg tggtttgacc cgttgatcgg
ggcggacatt 420 gtgaaacatc tgcagcccaa cacgaaaatt gtttttttgg
agtctcccgg ttcgattact 480 atggaggtac acgacgtgcc agctatcgtt
gcagcagttc gttccgtggc gcccgacgca 540 attatcatga tcgacaatac
atgggccgca ggcgtccttt ttaaagcctt agattttggc 600
attgatgtaa gtatccaagc ggctaccaag tacttggtcg gacattccga tgcgatgatt
660 ggtacagcag tatgcaatgc acgctgctgg gagcaattgc gtgaaaacgc
ttacctgatg 720 gggcaaatgg tagacgcaga taccgcttat attaccagtc
gtgggttgcg tacattagga 780 gtgcgtttgc gtcaacacca cgagtcatcc
ctgaaagtgg ctgaatggct ggctgaacat 840 ccccaggttg ctcgcgtaaa
ccaccccgca cttccgggat caaagggcca tgaattttgg 900 aagcgcgact
tcacgggctc cagtggattg ttttctttcg tacttaagaa aaagttgtct 960
aatgaagaat tggcgaatta ccttgataac tttagcttgt ttagtatggc atatagttgg
1020 gggggatatg aatcactgat tttggcaaat caaccagaac atattgctgc
gattcgtcct 1080 caaggcgaaa ttgattttag cggaacgtta attcgtctgc
acatcgggct tgaggatgtg 1140 gacgatttaa ttgcagattt ggatgcggga
tttgcacgta ttgtg 1185 <210> SEQ ID NO 79 <211> LENGTH:
1230 <212> TYPE: DNA <213> ORGANISM: Trypanosoma grayi
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1230) <223> OTHER INFORMATION: cystathione
gamma lyase (Trypanosoma grayi) <400> SEQUENCE: 79 atgtcaggtg
cccagcactt gttcgcagat ttcagcgaag gatcaggatc gtggcaaccc 60
caggcccaag ggtttgagac gcttctggta catggtggcg taaagccaga tcccgtcacg
120 ggggcaatcc tgacccccgt ctaccagtct acgacgttcg tgcaagagag
tatcgaacgt 180 tatcaagcaa agggctatag ctatacccgt tcagccaatc
ctaccgtatc tgcattggaa 240 gagaaattgt gcgcaatcga gcacggcgaa
tatgccactg tgtatagcac cggcatgtcc 300 gctacgacaa cggccatcag
tagttttatg tctgctggcg accacgctat tgtgaccgaa 360 tgtagctatg
gcggaaccaa tcgtgcctgc cgtgtcttct tcacgcgctt aggtatgtct 420
tttacattcg tagatatgcg cgacgttaaa aatgtagagg ctgccatcaa acccaatacc
480 aagctggtta tctcagaatc gccagcaaac cctacactga cgcttactga
tattgacgca 540 cttagctcgc tttgcaaggc taagggtatt attcacatgt
gtgacaacac tttcgcaacc 600 gctttcatta tgcgtccgct tgatcacgga
gcagacgtga ccctgatctc cacgactaag 660 tttgttgatg gccacaatat
gaccgtcgga ggggccttgg tcactaaatc caaggaatta 720 gacggaaagg
tacgtttaac gcaaaatatc ttaggtaact gtatgagtcc atttgttgcg 780
ttccttcaat tacaaacggt gaagacgatg agccttcgca tttctcgtca atcagaaaac
840 gcccagaaag tagcggaatt tcttgagacc caccccgcag tggaacgcgt
aatgtatcca 900 ggtcttaaat ctttcccaca gaaggcctta gcggatcgtc
agcacgcaaa caatttacat 960 ggcggtatgt tatggtttga agtgcgcgga
ggaacagcgg cagggcgtcg cttgatggac 1020 accgttcagc gcccgtggag
cttatgcgag aatctgggtg cgacggaatc catcattact 1080 tgcccgagtg
tcatgaccca cgcgaacatg actactgagg accgtatgaa ggtcggtatc 1140
accgacggat ttgtacgtgt cagctgcggg atcgaagatg cagccgatct tatctcagct
1200 ttgaaggccg cactggatgc cttgggcaag 1230 <210> SEQ ID NO 80
<211> LENGTH: 915 <212> TYPE: DNA <213> ORGANISM:
Helicobacter pylori 2017 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(915) <223> OTHER
INFORMATION: Cystathione beta-synthase (Helicobacter pylori 2017)
<400> SEQUENCE: 80 atgatcttaa cagcaatgca agatgcaatc
gggcgtacac ctatcttcaa gtttacacgt 60 aaagattacc caattccatt
gaagtcggca atttacgcga aattggaaca cttaaacccg 120 gggggatccg
tgaaagatcg ccttgggcag tatcttatta aggaggcctt ccgtacacac 180
aagattacct ctactaccac tatcatcgaa cctactgctg ggaatactgg catcgccctt
240 gcccttgtag ctatcaaaca tcatcttaaa acgatctttg ttgttcccga
aaaattttcg 300 gttgagaaac aacagatcat gcgtgctctt ggtgccttag
taatcaatac gcctacctca 360 gagggtatct caggggccat taaaaaaagc
aaagagttag ccgagtctat cccggacagc 420 tacttgcctc ttcaatttga
gaatcccgac aatccggctg cttattacca cactcttgct 480 cctgaaattg
taaaggaact ggggacgaat tttacctctt ttgtagcggg catcggttct 540
ggaggaactt tcgcaggcac cgccaagtac cttaaagaac gtatcccgaa catccgcttg
600 attggagttg aaccagaagg ttctatttta aatgggggtg aaccggggcc
ccacgaaatc 660 gaaggaattg gagtagagtt catcccacca ttcttcgcta
atttggatat tgatgggttt 720 gagacgattt cagacgaaga gggcttcagt
tatacgcgca aattagccaa aaagaacgga 780 ttattagtgg gtagttcgtc
cggagcagcg ttcgccgcgg ctcttaagga agtacaacgt 840 ctgcccgaag
ggtcacaagt gttgacgatt ttcccagata tggctgatcg ctaccttagt 900
aaaggcattt attcc 915 <210> SEQ ID NO 81 <211> LENGTH:
1320 <212> TYPE: DNA <213> ORGANISM: Helicobacter
pylori 2017 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1320) <223> OTHER INFORMATION:
putativeamino transferase (Helicobacter pylori 2017) <400>
SEQUENCE: 81 atgcaagctt tcttgaaccg ttcgttcgcg ccccttttaa acccaaatga
gaacctgctg 60 gatcaagtta agagttcgat tattttgaag aaaggtgtta
gctactttga ctggggtgct 120 agtgggctgg ccagtgcatt ggtcgagaaa
cgtgttaagt ccctgcttcc atattatgcc 180 aatgcccaca gcgtagcaag
taaacatgcc atcttaatgg gcatgttact taaagaatgc 240 caagagaagc
tgaaacgctc gttaaacctt agtactaacc attgcgtgct tagcgccggg 300
tatggcgcga gctcagcgat caagaaattc caagagatcc tgggagtttg catcccctct
360 aaaaccaaaa agaatctgga accttattta aaagacatgg cgctgaaacg
cgtaatcgta 420 ggtccttatg aacatcactc taacgaggtc tcttggcgcg
agtctctttg tgaggtggtg 480 cgcattccac ttaacgaaca tggactgctg
gatttggaga ttttagagca gatcttaaag 540 aaatccccca attctctggt
ctccgtctcg gccgcaagta atgtaacggg gattctgaca 600 cccctgaaag
aaattagctc actgtgcaag gagtatcgcg cgatcctggc gcttgatctg 660
gccaactttt ccgcacacgc gaacccgaaa gactgcgagt accagacggg gttctatgca
720 ccacacaagt tgttgggtgg tattggggga tgcgggcttc ttggaatctc
caaagacttg 780 atcgatacac agatcccacc tagtttttca gccggaggag
tcattaagta cgcaaaccgc 840 acgcgtcacg aatttattga tgagctgccg
ttgcgtgagg agttcggaac tccgggactg 900 ctgcaatttt atcgctcagt
gttagcctac cagttacgtg acgaatgcgg tttggatttc 960 attcataaga
aggagaataa tctgcttcgt gtgttaatgc atggcttgaa agatctgcca 1020
gctatcaaca tttacggcaa tttaaccgca agccgcgtag gagtagtcgc gtttaacatc
1080 ggaggcatta gtccatacga tcttgcccgt gtcctgagtt acgaatatgc
tattgagact 1140 cgcgcagggt gctcttgtgc cggcccgtat ggacatgact
tactgaattt gaatgcacaa 1200 aagtcttccg atttcaatgc aaaacctgga
tggttgcgcg tctcacttca ttttacacac 1260 agtattaatg acattgacta
tctgttggac tctctgaaga aagctgttaa gaaactgcgt 1320 <210> SEQ ID
NO 82 <211> LENGTH: 918 <212> TYPE: DNA <213>
ORGANISM: Bacillus atrophaeus UCMB-5137 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(918)
<223> OTHER INFORMATION: YdeD (Bacillus atrophaeus UCMB-5137)
<400> SEQUENCE: 82 atgaacggtg aacacgccgc gttggcccac
tcccgcacaa aagggattgc tttggtttta 60 acgggcagta tcttatgggg
cgtttcaggg acagttgcgc agtacttatt ccaacaacaa 120 cattttaacg
tagagtggtt gaccgtcgtt cgcttgttgc tgtctggtat cttgctgctt 180
ggccttgcct atcgtaagga aaagcaacgc atctgggctg tctggaaaga caagacagat
240 ggtctgaatc tggttctgtt cgggattttg gggatgttgt ccgtccagta
cacatacttt 300 gcggctatcc agcatggtaa tgcggcgacg gcaactgtac
ttcagtatct ggccccggca 360 cttattacct gctacgtagc cattcgctct
aagcgtcttc caaccgtcaa agagttgatc 420 gcagttttcc tggctattat
tggaacgttt tttttagtca cccatgggga catccacagt 480 cttagtatct
cagggtgggc tttattctgg ggattaagtt cggcgtttgc cctggcgttt 540
tacactttgc accctcataa acttctggcc aagtgggggg cggctatcgt tgttggctgg
600 ggtatgctta tcggagggct tggtctttcc ttaatccatc ctccatggaa
atttgaggga 660 cagtggtcgg tctcggctta tgccgccgtt attttcattg
tcctgtttgg gaccctgact 720 gccttctact gctacctgga atctttaaag
tacttaactg ccagcgaaac ttcattaatc 780 gcctgcgcgg agcccttaag
tgctgcgttc ttaagcgtga tttggttgca tgtgactttt 840 ggtatcagcg
agtggcttgg tacttgttgt attttatcta cgattatgat cttatcgatt 900
aaggagaaga agctgaag 918 <210> SEQ ID NO 83 <211>
LENGTH: 585 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(585) <223> OTHER INFORMATION:
proteinYfiK (Escherichia coli) <400> SEQUENCE: 83 atgacaccca
cgttgcttag cgccttctgg acgtacaccc ttattacagc catgacgcct 60
gggccaaata atatccttgc cttatcatcc gcaacgtcgc atgggttccg ccagtccacc
120 cgtgtgcttg caggtatgtc tcttggcttt ttaatcgtta tgctgttgtg
cgcgggaatc 180 agtttctcct tggcggtaat cgaccccgcc gccgtacatt
tattgtcttg ggctggtgcc 240 gcgtatattg tttggctggc ttggaaaatt
gccacgtctc cgactaagga agatggttta 300 caagcaaaac ccatctcgtt
ttgggcttca tttgcacttc agttcgtgaa tgtcaagatt 360 attctttacg
gggtaacagc cctgtccact ttcgttttac cccagacgca ggcgttgtca 420
tgggtagtcg gagtgtccgt cttattagcc atgatcggta cgtttgggaa tgtgtgctgg
480
gcgctggcgg gccacttgtt tcaacaatta ttccgtcagt acggtcgcca gttaaatatc
540 gttcttgctt tattactggt gtattgtgca gtccgcatct tctat 585
<210> SEQ ID NO 84 <211> LENGTH: 1188 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1188) <223> OTHER INFORMATION: multidrug efflux
transporterBcr(Escherichia coli) <400> SEQUENCE: 84
atgacgaccc gccagcatag ctcgttcgca atcgtattta ttcttggatt gcttgctatg
60 ttgatgccat tatcaatcga catgtactta ccagccctgc ctgttatttc
ggcccaattt 120 ggagtacccg ctgggtcaac ccaaatgaca ttatcaacat
acattctggg gttcgcttta 180 ggacagttga tttatggtcc aatggctgac
tcgtttgggc gcaaaccagt ggtcttgggc 240 gggacactgg tctttgcggc
cgcagccgtt gcgtgtgcct tggctaacac gatcgaccag 300 cttattgtaa
tgcgtttctt ccatggctta gctgcggcgg ctgccagtgt agtgattaat 360
gcgcttatgc gtgacatcta tccgaaggag gaattcagcc gcatgatgag cttcgtaatg
420 ttggtaacga ccatcgctcc attaatggcc cctattgttg ggggttgggt
cttagtctgg 480 ctttcatggc attacatttt ttggatcctt gccctggcgg
ctattctggc ctcagcgatg 540 attttcttcc tgattaaaga aacccttcct
ccggagcgcc gtcagccttt ccatattcgc 600 actactatcg gtaattttgc
ggccttgttt cgccataaac gcgtgctgtc atacatgttg 660 gcaagcggct
tttctttcgc gggtatgttc tcgtttttaa gtgctggtcc cttcgtgtat 720
atcgaaatca atcacgtagc cccggagaac ttcggctatt acttcgcatt aaatatcgtg
780 tttcttttcg tcatgaccat cttcaactct cgcttcgtcc gtcgtatcgg
tgccttaaat 840 atgtttcgtt cggggctgtg gatccaattt atcatggctg
cgtggatggt gatctccgca 900 ctgttggggc ttgggttttg gtcgcttgtg
gtgggcgtgg ctgcattcgt tggatgtgtc 960 agcatggtat cttctaacgc
gatggctgta attttggatg agttcccaca tatggcaggg 1020 actgcttcct
ctctggctgg cacatttcgc ttcggaattg gtgcaatcgt aggcgcgttg 1080
ctgagcttag cgacattcaa ttcggcgtgg cccatgattt ggtccattgc gttttgtgcg
1140 accagcagca tcctgttctg cctttatgct tcccgtccaa agaagcgt 1188
<210> SEQ ID NO 85 <211> LENGTH: 1356 <212> TYPE:
DNA <213> ORGANISM: Pseudomonas fluorescens R124 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1356) <223> OTHER INFORMATION: TolC (Pseudomonas
fluorescens R124) <400> SEQUENCE: 85 atgaacaaac ttagtatgct
gggagctgcc ttcgcgttgt tggcagggaa ctcagcattg 60 gcagcaatgg
ggcctttcga aatctacgaa caggctcttc gcaatgaccc agttttctta 120
ggggccatta aggagcgtga cgccggattg gaaaaccgca tcatcggccg cgcaggattg
180 ttaccacgct tggggtacaa ctacaatcgt ggccataaca cctctaaagc
gacccagttg 240 acaaatcgtg gctctctgac tgaagaccgt aactataatt
cgtatggttc aactcttaca 300 ttacagcaac ccttattaga ctatgaggcc
tatgccgcct accgtaaggg agtagcgcaa 360 agcttgttcg ccgatgaagc
ctttcgcggt aagtcacagg aattattggt tcgcgtctta 420 gataattaca
cgaaagcgtt gttcgcacaa gaccaaatcg atatcgcaca ggcgaaaaaa 480
aaagcttatg aacaacaatt tcagcagaac gaacatatgt tcaaacaagg cgaggggacg
540 cgcactgaca ttttggaagc tgaaagtcgt tatgaacttg ccacggcaga
agaaatcgag 600 gcgcgtaacg aacaggatgc cgctcttcgc gagcttggtg
cgcttgtcgg tgtcccaact 660 gtcgacattt ctgaacttgc acccttagac
cagaattttc aaacgttcgc gctgatgcct 720 gctaactatg atacgtggca
cgagttagca atttctaata atccgaacct ggcatcacag 780 cgtcaggccg
tggaagtagc aaaatacgaa gttgaacgta accgtgcagg acatttaccc 840
aaggtctcag catatgccag cattcgtcag actgagtctg acagtggtaa tacctacaat
900 caacgttatg atacgaacac cattggcttt gaggtaaacg tccctctgta
tgcaggagga 960 ggagtctcag caagtacacg ccaagcatca cgcacgatgg
agcaggcgga gtatgaatta 1020 gatggaaaga cgcgtgagac gttaattgaa
ttacgtcgtc agttcagcgc gtgccttagt 1080 ggagttaata agttacgcgc
ctatcagaaa gccctggcct cggccgaagc actggtggtc 1140 tcaaccaagc
agagcattct tggcggcgaa cgcaccaact tggacgcgct taacgcggaa 1200
cagcagctgt tcaccacgcg tcgcgacctt gcacaggccc gctatgacta cttgatggcg
1260 tggacgaaac tgcattatta cgcaggaacc ctgaacgaac aagatttagc
gcgtgtggac 1320 gaggcatttg gccaagggcc caaatcaaat cctcgc 1356
<210> SEQ ID NO 86 <211> LENGTH: 1167 <212> TYPE:
DNA <213> ORGANISM: Sinorhizobium meliloti AK83 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1167) <223> OTHER INFORMATION: Tyrosine transaminase
(Sinorhizobium meliloti AK83) <400> SEQUENCE: 86 atgttcgatg
cgctggcgcg tcaagcggat gatccgcttt tggcgctgat cggactgttt 60
cgcaaagacg agcgccccgg taaagtggac ttaggtgtgg gagtttaccg cgacgaaact
120 ggccgcactc cgatctttcg cgcggttaaa gcagccgaaa aacgcttgct
tgagactcag 180 gactcgaagg cctacatcgg cccggaagga gacctggttt
ttcttgaccg tttgtgggaa 240 cttgttgggg gggataccat tgaacgttct
cacgtagctg gtgtacaaac acctggcggg 300 agcggcgcac ttcgtttggc
ggcagattta atcgcccgca tgggcggtcg cgggatttgg 360 ttggggttgc
catcctggcc gaatcacgct cccattttca aagcggctgg actggatatc 420
gcgacttacg atttctttga tatcccgagt caatccgtta tttttgataa cctggtgtct
480 gccctggaag gtgcagcatc tggcgatgcc gtcttattgc atgctagctg
ccacaatcca 540 actggagggg tattatccga ggcacagtgg atggaaattg
ccgcgctggt cgccgaacgc 600 ggactgttac cacttgttga tcttgcgtat
caagggttcg gacgtgggct ggatcaagac 660 gtcgcgggct tacgccattt
attaggtgta gttcccgaag cccttgtcgc cgttagctgc 720 tctaaatcgt
tcggcttgta ccgcgaacgc gctggagcca tcttcgcccg tacatcatct 780
accgcttcag ccgaccgcgt ccgcagtaac ttagctggcc ttgctcgcac atcgtatagt
840 atgccccccg atcacggggc cgcggttgtc cgtacgatct tagacgaccc
agagctgcgt 900 cgtgactgga ccgaggaatt agagacaatg cgcttgcgta
tgacgggtct tcgccgctct 960 cttgcagagg gcttgcgcac ccgttggcag
tctcttggcg ccgtagctga ccaagaaggg 1020 atgttctcga tgctgccgtt
gtccgaagca gaggttatgc gccttcgcac tgagcatgga 1080 atttacatgc
ccgcatcagg acgcattaac attgcggggt taaaaacggc ggaggctgcc 1140
gaaattgcag gtaaatttac gagtttg 1167 <210> SEQ ID NO 87
<211> LENGTH: 1209 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1209)
<223> OTHER INFORMATION: tyrosine transporterTyrP(Escherichia
coliW) <400> SEQUENCE: 87 atgaagaacc gcactcttgg atcagtattc
attgttgcgg ggaccaccat cggtgcaggt 60 atgcttgcca tgcccctggc
tgcagctggc gtcgggttca gcgttaccct gattttactg 120 attggtctgt
gggctctgat gtgttacacg gcattgcttt tgcttgaagt gtaccagcat 180
gtacccgcag acaccggtct tggcactctg gcgaaacgtt atttaggacg ttatggtcaa
240 tggctgaccg gtttctccat gatgtttctg atgtatgcgc tgacggccgc
atacattagt 300 ggtgcaggtg aactgctggc aagttcaatt tctgactgga
cgggcatctc tatgagcgcg 360 actgctgggg ttttattgtt tacatttgtg
gctggcggtg tagtgtgtgt agggacgtca 420 ttagttgatc tgtttaaccg
cttccttttc agtgcaaaaa tcattttcct tgtagtaatg 480 cttgtcttat
tattaccaca tattcataag gtaaatcttt tgacattacc attgcagcag 540
ggattggcgt tatcagccat ccctgtaatc ttcacatcct tcggattcca cgggtccgtc
600 ccatccatcg tgtcctacat ggacggcaat gtacgcaagt tacgttgggt
ctttatcaca 660 gggagcgcca ttccccttgt agcgtatatt ttttggcaag
ttgctactct ggggtcaatc 720 gactctacca ccttcatggg tttacttgcg
aaccacgcgg ggttgaacgg actgttacag 780 gctttgcgtg aaatggttgc
ctcgccacat gttgagttgg cggttcatct ttttgctgac 840 ttagccttag
ctacctcttt ccttggggtt gcgctgggat tattcgacta tctggctgat 900
ctttttcaac gctccaacac cgtaggtgga cgtttacaga ctggagccat tactttcttg
960 ccccctttag cctttgcgct gttttatcca cgtgggtttg ttatggcctt
ggggtatgct 1020 ggagtcgcct tagctgtact tgctcttatt attccatcgt
tattaacgtg gcaatcgcgt 1080 aaacacaacc cccaagcagg gtaccgcgtg
aagggaggac gccccgcgct ggtggttgtt 1140 tttctgtgcg ggattgccgt
catcggcgtg caatttttga ttgcagcagg tttgttgccg 1200 gaggtgggg 1209
<210> SEQ ID NO 88 <211> LENGTH: 1302 <212> TYPE:
DNA <213> ORGANISM: Variovorax paradoxus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1302) <223> OTHER INFORMATION: Beta-phenylalanine
transaminase (Aromatic beta-amino acid aminotransferase;
Beta-phenylalanineaminotransferase; VpAT) <400> SEQUENCE: 88
atgactcatg ctgcaattga ccaggcgttg gcagacgcct atcgtcgttt tactgacgca
60 aaccctgcca gccagcgtca gtttgaagcg caagcccgct atatgcccgg
ggctaactct 120 cgctctgttt tgttttatgc accctttcca ttgacgatcg
cacgtgggga aggcgccgct 180 ctttgggatg cggacggcca ccgttacgct
gactttatcg cggaatacac agctggggtg 240 tatggacaca gtgccccaga
gattcgtgac gcagtaatcg aagctatgca gggtgggatt 300 aatttgacgg
gtcataattt gttggaaggc cgcttagccc gccttatttg tgagcgtttc 360
ccacagatcg aacagttgcg tttcacgaat agcggaacag aggccaatct gatggccctt
420 accgcggcgc ttcattttac tggtcgccgc aaaatcgtcg tatttagtgg
aggttatcat 480 gggggggttc ttgggttcgg tgcccgtcct agccctacca
cagtaccatt tgacttcctt 540 gtgctgcctt acaacgatgc tcagacggct
cgtgctcaga tcgagcgcca cggcccggag 600 atcgcggtcg tgttagtcga
gcccatgcaa ggtgcttctg gctgcatccc aggtcagccc 660 gactttctgc
aagccctgcg cgaatccgct actcaggtag gggcgctgtt agtttttgac 720
gaagtgatga ctagtcgctt agcgccacat ggtttagcta acaaattggg gatccgttcg
780 gatttgacaa ccctgggtaa gtacattggc ggcggtatgt catttggggc
ctttggcggt 840 cgtgctgatg tcatggccct gttcgaccct cgcactggac
ctttggctca ttccggtacg 900 tttaacaaca atgtgatgac gatggctgcc
ggttatgctg gcttaacgaa attattcact 960 ccggaagcgg caggggcatt
ggcagagcgt ggagaagcgc ttcgcgcacg tcttaacgcc 1020 ctgtgtgcta
acgaaggagt agcaatgcag ttcactggca tcggctcgct gatgaatgcc 1080
cacttcgtcc agggagacgt tcgtagctct gaggatctgg ccgcagttga tgggcgttta
1140 cgtcagttgt tgttctttca tttattgaat gaagatattt actcttcacc
gcgtgggttt 1200 gttgtattat cgttgccatt gactgacgct gatattgacc
gctacgttgc tgcgatcggt 1260 tcatttattg gcggtcatgg ggcgttgtta
ccgcgcgcta ac 1302 <210> SEQ ID NO 89 <211> LENGTH:
1401 <212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1401) <223> OTHER INFORMATION: gadA glutamate
decarboxylase (Escherichia coli) <400> SEQUENCE: 89
atggaccaga agctgttaac ggatttccgc tcagaactac tcgattcacg ttttggcgca
60 aaggccattt ctactatcgc ggagtcaaaa cgatttccgc tgcacgaaat
gcgcgatgat 120 gtcgcatttc agattatcaa tgatgaatta tatcttgatg
gcaacgctcg tcagaacctg 180 gccactttct gccagacctg ggacgacgaa
aacgtccata aattgatgga tttgtcgatc 240 aataaaaact ggatcgacaa
agaagaatat ccgcaatccg cagccatcga cctgcgttgc 300 gtaaatatgg
ttgccgatct gtggcatgcg cctgcgccga aaaatggtca ggccgttggc 360
accaacacca ttggttcttc cgaggcctgt atgctcggcg ggatggcgat gaaatggcgt
420 tggcgcaagc gtatggaagc tgcaggcaaa ccaacggata aaccaaacct
ggtgtgcggt 480 ccggtacaaa tctgctggca taaattcgcc cgctactggg
atgtggagct gcgtgagatc 540 cctatgcgcc ccggtcagtt gtttatggac
ccgaaacgca tgattgaagc ctgtgacgaa 600 aacaccatcg gcgtggtgcc
gactttcggc gtgacctaca ccggtaacta tgagttccca 660 caaccgctgc
acgatgcgct ggataaattc caggccgaca ccggtatcga catcgacatg 720
cacatcgacg ctgccagcgg tggcttcctg gcaccgttcg tcgccccgga tatcgtctgg
780 gacttccgcc tgccgcgtgt gaaatcgatc agtgcttcag gccataaatt
cggtctggct 840 ccgctgggct gcggctgggt tatctggcgt gacgaagaag
cgctgccgca ggaactggtg 900 ttcaacgttg actacctggg tggtcaaatt
ggtacttttg ccatcaactt ctcccgcccg 960 gcgggtcagg taattgcaca
gtactatgaa ttcctgcgcc tcggtcgtga aggctatacc 1020 aaagtacaga
acgcctctta ccaggttgcc gcttatctgg cggatgaaat cgccaaactg 1080
gggccgtatg agttcatctg tacgggtcgc ccggacgaag gcatcccggc ggtttgcttc
1140 aaactgaaag atggtgaaga tccgggatac accctgtacg acctctctga
acgtctgcgt 1200 ctgcgcggct ggcaggttcc ggccttcact ctcggcggtg
aagccaccga catcgtggtg 1260 atgcgcatta tgtgtcgtcg cggcttcgaa
atggactttg ctgaactgtt gctggaagac 1320 tacaaagcct ccctgaaata
tctcagcgat cacccgaaac tgcagggtat tgcccagcag 1380 aacagcttta
aacacacctg a 1401 <210> SEQ ID NO 90 <211> LENGTH: 1398
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1398) <223> OTHER INFORMATION: glutamate
decarboxylase (Escherichia coliKO11FL) <400> SEQUENCE: 90
atggataaaa agcaagtgac ggacctgcgc tctgaacttc ttgacagtcg ttttggggca
60 aagagtatta gtaccattgc tgagtcaaag cgttttcctt tgcatgagat
gcgcgatgac 120 gtcgcattcc agattatcaa cgacgagctg tatttggacg
gcaatgcccg ccaaaacttg 180 gccacgtttt gtcagacttg ggatgacgag
aatgttcata aacttatgga cctttcaatt 240 aacaaaaatt ggattgacaa
agaagagtac ccccaatctg ccgcaattga tttacgttgt 300 gttaatatgg
tggccgactt atggcatgca ccagccccta aaaacggcca agcggtggga 360
accaacacga tcgggtctag tgaggcatgt atgttaggcg ggatggccat gaagtggcgt
420 tggcgtaaac gcatggaggc agcagggaaa ccaaccgata aacctaattt
agtctgcgga 480 ccggttcaga tctgttggca taaatttgcg cgctactggg
atgtggaatt acgcgaaatt 540 ccgatgcgtc cgggccaact gttcatggat
cccaaacgta tgatcgaagc atgtgacgaa 600 aacacgattg gggtggtacc
cacctttggg gtcacatata caggtaacta cgagtttcca 660 caaccgttgc
atgatgctct ggacaagttt caagctgaca ccgggatcga cattgatatg 720
cacattgacg ctgcctccgg cggattcttg gccccatttg tagcccctga cattgtctgg
780 gactttcgtc ttccccgtgt gaaatccatc agcgcatccg gtcacaagtt
tgggcttgcc 840 ccattagggt gtggatgggt catctggcgt gatgaggaag
cattacccca agaacttgtc 900 ttcaatgtag attaccttgg gggacagatt
ggcacttttg ccatcaactt ttctcgccca 960 gcgggtcaag tgatcgccca
gtattacgag tttctgcgcc tgggacgtga gggatataca 1020 aaagtgcaga
acgcatcgta ccaggtagcg gcttaccttg cggacgaaat tgcaaagctg 1080
ggaccatacg agtttatctg taccgggcgt ccagatgaag gtattccggc tgtgtgtttt
1140 aagctgaaag acggggaaga tcccggatat acgctgtatg atctgtctga
acgtttacgt 1200 ttgcgcggtt ggcaagttcc agccttcacg ttgggtggcg
aagccactga tattgtagtc 1260 atgcgtatca tgtgtcgtcg cggctttgaa
atggatttcg cagagttact tctggaagac 1320 tacaaagcga gcttaaaata
tttgtctgac catcccaagt tgcaagggat cgcacagcaa 1380 aattcgttta
aacacact 1398 <210> SEQ ID NO 91 <211> LENGTH: 1287
<212> TYPE: DNA <213> ORGANISM: Bacillus atrophaeus
UCMB-5137 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1287) <223> OTHER INFORMATION:
GltT (Bacillus atrophaeus UCMB-5137) <400> SEQUENCE: 91
atgaagaaat tacgcttcgg actggcgact caaatctttg tggggctgat tcttggggta
60 gtagtgggcg ttatctggta cggtaatccg gcggtggtaa cttatttgca
gccagttggg 120 gacctttttt tacgtttgat taaaatgatc gttattccta
tcgtggtgtc ttctttgatc 180 attggcgtcg cgggagctgg gtccggaaaa
caggtcggaa agctgggctt tcgtactatt 240 ctgtacttcg agatcatcac
tacctttgcc atcattctgg gacttgctct ggcgaatctt 300 ttccagcctg
gtacaggagt aaatatcgag agcgcgcaga aaagtgacat ttcccagtac 360
gtggagactg aaaaagagca atccaccaaa tccgtagctg agactttcct gcatatcgtg
420 cccaccaatt tctttcaatc acttgcggaa ggtgatcttc ttgctattat
ctgctttacc 480 gtacttttcg cccttggcat ttcggctatc ggtgaacgtg
gcaaaccggt gcttgctttc 540 tttgacggag tatcccacgc gatgtttcat
gtagtgaacc ttgtgatgaa ggttgctccg 600 ttcggcgtat ttgctctgat
tggagtaaca gtaagcaaat ttggactggg ttctttactg 660 agcctgggta
aacttgtggg gctggtatat gttgctctgg cattttttct tattgtaatc 720
tttggtattg ttggaaagct ggctggcgtg aatatcttca agtttttagc ttacatgaag
780 gatgaaatct tattagcgtt ctcgacctca tcgtccgaga ctgtgttgcc
ccgcatcatg 840 gagaaaatgg agaagatcgg gtgtccaaag ggaattgtaa
gctttgtagt ccccatcggt 900 tacacattca atcttgacgg ctcggtctta
taccaatcta ttgctgcgct gttcttggca 960 caggtttacg gaatcgacct
gactatttgg catcagatta ctctggtgtt agttctgatg 1020 gtcactagca
aaggcatggc agccgttcct ggaactagct ttgtagtcct gctggcaacc 1080
ttaggtacca ttggtgttcc agcggaaggg cttgcattca ttgcgggggt tgaccgcatt
1140 atggacatgg ctcgcactgt ggtcaattta acaggcaatg ctcttgcgag
tgtcgtaatg 1200 agcaagtggg agggtcagta cgacccggtg aaaggtgcag
agattatgag ccgcagcaag 1260 acggaacagg acgctactat ctccgga 1287
<210> SEQ ID NO 92 <211> LENGTH: 858 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(858)
<223> OTHER INFORMATION: mechanosensitive
channelMscS(Escherichia coli) <400> SEQUENCE: 92 atggaggact
tgaacgtagt agatagcatt aatggagcgg gctcatggtt agtagccaac 60
caagccctgt tgttatcgta tgctgtaaat atcgtcgcag ccttagccat cattatcgtt
120 gggttaatca tcgcccgtat gatttctaat gcggtgaatc gcttaatgat
ctcgcgcaag 180 atcgacgcca ctgtcgcgga tttcttgtcc gccctggtgc
gttacggtat catcgcgttc 240 acattgattg cggcattagg gcgcgtagga
gtccagacag cttctgtgat tgcggtatta 300 ggtgcagcag gattagctgt
gggattggcg ttacaggggt ctctttccaa tctggcggcc 360 ggcgtacttc
tggttatgtt tcgccccttt cgcgccggag agtatgtgga tttgggagga 420
gtggccggaa cagtgctgtc agtgcaaatc ttttctacca cgatgcgtac agcagatgga
480 aaaatcatcg tgatccccaa tggcaagatc atcgcgggta acattatcaa
cttctcccgc 540 gaacctgttc gccgcaacga atttatcatc ggtgttgcct
atgattcaga catcgatcag 600 gtcaaacaaa ttcttacgaa catcattcag
tcagaggacc gtattctgaa agaccgcgaa 660 atgacggtgc gtttgaatga
gttaggggct tcaagtatca acttcgtagt ccgcgtgtgg 720 agcaattccg
gtgatttgca aaacgtgtat tgggacgtcc ttgagcgcat taagcgtgaa 780
ttcgatgctg ccgggatctc ctttccgtat cctcagatgg atgtgaattt caagcgtgta
840
aaggaagata aggctgcc 858 <210> SEQ ID NO 93 <211>
LENGTH: 1539 <212> TYPE: DNA <213> ORGANISM: Bacillus
amyloliquefaciens subsp. plantarum str. FZB42 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1539) <223> OTHER INFORMATION: HutH (Bacillus
amyloliquefaciens subsp. plantarum str. FZB42) <400>
SEQUENCE: 93 atgatggtca ccttggatgg gtcttcatta acgacggctg atgcacaacg
tgtacttttc 60 gattttgaag aggtacaggc atcggctgaa tcgatggagc
gcgtaaaaaa gagccgtgcc 120 gccgtggaac gcattgtaca agaagaaaaa
actatctacg gaatcactac ggggtttggt 180 aagttttccg atgtgctgat
ccaaaaagag gacgctgcgg atttacaatt gaatttgatc 240 ttgtcacatg
catgtggagt cggcgatcct ttcccagagt cagtctcccg cgccatgctg 300
cttctgcgtg caaacgcatt gttaaaaggc ttctccggtg ttcgtacgga attaattgac
360 cagcttttag cgtacttaaa ccaccgtatc caccctgtta tcccccaaca
aggttcgctg 420 ggggcctccg gcgatttggc ccctcttagc caccttgcgt
tggcactgat cggacaaggg 480 gaagtgttct acgaaggagc acgtatgccc
actgctcatg cccttgaaca aaccaatctg 540 cagcccgcag tcctgacatc
gaaggaaggg ctggcgttga tcaatgggac tcaggctatg 600 accgcaatgg
gcttaatcgc ataccttgaa gccgaaaagt tggcatatca gagcgagcgc 660
atcgcttcat tgactatcga aggattgcaa ggtattattg acgcgtttga cgaagatatt
720 catgccgctc gtggatacca ggaacaaatg gatgtcgctg agcgcattcg
ctattatctt 780 tcggattcga agctgacaac cgtacaaggc gagctgcgtg
tgcaagatgc ttactccatt 840 cgctgcatcc ctcaagtcca cggagcttct
tggcagaccc tggcgtatgt gaaggagaag 900 ttagaaattg agatgaacgc
tgctactgat aaccctttaa tttttgaaga cggggccaaa 960 attatctcgg
gggggaactt tcacgggcaa ccgatcgcgt ttgcaatgga cttcttgaaa 1020
gtagctgctg ctgagttggc taatatcagc gagcgccgta ttgagcgtct tgtcaatcca
1080 cagctgaatg accttcctcc ttttctttcg ccgcaaccgg gtttacagtc
tggtgccatg 1140 attatgcagt acgccgctgc ctccttggtc tcggaaaaca
aaacacttgc gcatcccgcc 1200 tcagtcgact caatcccctc ctcggctaac
caggaggatc acgtctccat ggggacgatc 1260 gcttcacgtc atgcttacca
gattattgca aacactcgtc gcgtattagc cgtcgaggcc 1320 atttgcgctt
tacaagctgt agagtaccgt ggggaagagc actgcgctag ctacacgaaa 1380
caactttacc atgagatgcg taacatcgtg ccatcgattc aggaggaccg tgttttctcg
1440 tacgacatcg agcacttatc cgactggctt aaaaaggaat ccttcttacc
taatgaacac 1500 caccaaaagt taatgactaa tgagggcggg ttaactcgc 1539
<210> SEQ ID NO 94 <211> LENGTH: 780 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(780)
<223> OTHER INFORMATION: Histidine ABC transporter,
histidine-binding periplasmic protein precursorHisJ (Escherichia
coliO145:H28 str. RM12581]) <400> SEQUENCE: 94 atgaagaaac
ttgtcctttc attgtctctg gtattagcgt tcagttcagc aactgcagca 60
ttcgctgcta ttccgcaaaa tatccgcatc gggacggatc ccacgtatgc gccattcgag
120 tcaaagaatt cacaaggtga attggtcggg ttcgatattg acctggcgaa
agaattgtgt 180 aaacgtatca atacccaatg cacgttcgtg gaaaatccct
tggatgcatt aattccgtct 240 ttgaaagcga aaaaaatcga tgccatcatg
tcatcccttt ctatcacaga aaagcgccag 300 caggagattg ccttcacaga
caagttgtac gctgcagaca gccgcctggt cgttgcaaag 360 aattctgaca
ttcaacctac cgtggaatcg ctgaagggca agcgcgtagg ggtcttgcag 420
ggcactactc aggaaacatt tgggaacgaa cattgggcgc ctaagggaat tgagatcgtg
480 tcttatcagg gtcaggataa catctacagt gatctgacag ccggacgtat
tgacgccgct 540 tttcaggacg aggtggcggc atctgaaggg ttcttaaagc
agccagtcgg caaagactac 600 aaatttggtg ggccgagcgt gaaggacgag
aaattgtttg gggtaggaac agggatgggc 660 ttgcgtaagg aggacaatga
attacgtgaa gctcttaata aagcctttgc tgagatgcgt 720 gcggacggga
cttacgaaaa acttgcaaaa aagtatttcg actttgacgt ctacggcggt 780
<210> SEQ ID NO 95 <211> LENGTH: 684 <212> TYPE:
DNA <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(684)
<223> OTHER INFORMATION: Histidine ABC transporter, permease
proteinHisQ (Escherichia coliO145:H28 str. RM12581) <400>
SEQUENCE: 95 atgctgtatg gattcagtgg cgttatcttg cagggggctc ttgtcacttt
agagttagct 60 atctcgtccg ttgtgttagc tgtcattatt ggacttatcg
gggctggtgg caaattgagt 120 cagaaccgtt tgagcggcct tatttttgaa
gggtacacaa ccttaattcg cggagtccca 180 gacttagtgc tgatgttgct
tattttctat ggtttacaga tcgctttgaa tacggttacc 240 gaggcaatgg
gggtcggcca aatcgatatc gatcctatgg tggctggaat cattactttg 300
ggcttcattt acggggcata tttcacggag acgttccgcg gagctttcat ggccgtcccg
360 aagggccaca ttgaagcggc aacagctttt ggattcactc gtgggcaagt
tttccgtcgc 420 atcatgtttc cagcgatgat gcgctatgcg cttcctggga
tcgggaataa ctggcaggta 480 atcttaaaat cgacggcttt agtcagttta
ttggggttgg aagatgtcgt aaaagcgacc 540 cagttggctg ggaaatcgac
ttgggagccc ttttacttcg ctattgtgtg tggcgttatt 600 tacttagttt
tcactacagt atcaaacggt gtgttattgt ttttggaacg tcgctacagc 660
gtgggtgtaa agcgtgctga tttg 684 <210> SEQ ID NO 96 <211>
LENGTH: 771 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(771) <223> OTHER INFORMATION:
hisP (Escherichia coliEPEC C342-62) <400> SEQUENCE: 96
atgtccgaga acaaattaaa tgttatcgat ttgcataagc gttatggaga gcatgaagtg
60 ttgaaaggag tgtctcttca agcaaacgcg ggggacgtaa tttctatcat
cggatcgtct 120 ggttctggta agtcaacctt cctgcgttgt attaacttct
tagagaagcc gtctgagggt 180 tctattgtag ttaatgggca gaccatcaat
cttgtgcgcg ataaggacgg ccagttgaaa 240 gtggcagaca aaaaccaact
tcgtttgctt cgcacccgtc ttaccatggt attccaacac 300 ttcaacctgt
ggtcgcacat gacggtactt gagaacgtga tggaagcgcc aattcaggta 360
cttggattga gcaaacaaga agcccgcgaa cgtgcggtga aatatttggc caaggtgggt
420 atcgacgagc gtgcgcaggg caaatacccc gttcacttgt ccgggggtca
acaacagcgt 480 gtcagtattg cccgcgctct ggctatggaa ccagaggtgc
ttctgtttga cgagccgacg 540 tcagctttgg acccggaatt agtgggcgaa
gtattgcgca tcatgcagca gttagcagaa 600 gaaggcaaga ccatggttgt
tgtcacacac gaaatggggt ttgcgcgtca tgtctcgact 660 catgtaatct
tcttgcatca aggtaaaatc gaggaagaag gagcgccgga acagttattc 720
gggaatcctc aatccccccg tctgcagcag tttcttaaag ggtccttaaa g 771
<210> SEQ ID NO 97 <211> LENGTH: 660 <212> TYPE:
DNA <213> ORGANISM: Clostridium botulinum <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(660) <223> OTHER INFORMATION: proline reductase
(Clostridium botulinum) <400> SEQUENCE: 97 atgtcaatgt
ccgctgagca cgctgaggaa ttaaaaaatg aacctgcggt cgtttgttgt 60
cgcactgagg aggggaccat cttgtcagcc gataatttgg aagacccaaa catttttcca
120 gatatggtgg atagcggttt actgaacatt cctggggact gcttaaaagt
tggggaagta 180 atcggggcca aactgcttaa gacgattgac tctttgaccc
ctcttgccaa ggacatcatt 240 gagggggcca aatccttaga cggagacgta
cgcagtaaat cagagattca gatcgaatca 300 ccagaggaga aggcgatcct
taaaaacaat ttgaaggcgg gagatattat caaggttgag 360 gacctggaga
accctatgca cttcgccaag ttacaagatt cgcttcttat caagctggat 420
gagaaagtgc ttacgcgccg cgaagttgta gacgcgaaac ttacggaaga tgcaccggcg
480 atttcagggg tcactgcatc aatgttggaa ggcttcgagg aaaaggccct
ggagattacc 540 caagatagca aggatgtgga cttcaattca gtaattccac
tgaacggcaa tcgtgaattc 600 cttcgtttga aaatcgagga aggcacaggc
atttatatcg aaattccctt tacccaagtc 660 <210> SEQ ID NO 98
<211> LENGTH: 1347 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1347)
<223> OTHER INFORMATION: ProlineporterII (Escherichia coli
PMV-1) <400> SEQUENCE: 98 atgtcagaaa aacttccggc acctcgcgag
ggtttatccg gtaaagctat gcgtcgtgtt 60 gtcatgggta gctttgccgg
tgcattaatg gaatggtatg atttcttcat ctttgggacg 120 gcggcgggtc
ttgtttttgc accgctgttt tatcctgaca gtgatccgtt tattgggttg 180
atcgcgtcgt tcgctacatt tggagttggt tttttgaccc gcccgttagg aggtatcgtg
240 ttcggtcatt ttggtgacaa gatcgggcgt aagattacct taatctggac
attggcgatt 300 gtggggtgtt ctacattctt aatcggtttc attccaacgt
accaagaaat cggcatttgg 360 gcccctttgg tccttatggt tttgcgcctg
attcagggtt ttggcttggg aggagaatac 420 ggaggggcgg cgttaatgac
catcgaaagt gcccccgaaa gccgccgtgg ttttcttggg 480 tcattgccac
agacggccgc cagcgtcggc atcatgcttg caacgggtat tttcgcgctt 540
tgtaatcatt tccttacttc tgaacagttc ttatcatggg gctggcgtat tcccttctgg
600
ttgtccgcgg ttatgttaat cgtcggactt tttatccgtc tgcatactga agagacgctt
660 gactttcaga agcaaaaaac gactaacaat aaagaaaagt cggttccccc
ccttatcgag 720 ctttttaaga aacatccacg caatattttg ttggccttgg
gggcccgcct tgcggagtca 780 gtaagcagca acatcattaa tgcattcggc
atcgtctata tcagcagtca acttgcactg 840 agccgtgaca tccccttgac
tgggatgctg attgcaagcg ccatcggaat ttttagttgc 900 ccattggtag
gctggctttc ggaccgtatc ggacagaaat cattatattt gtcaggcgct 960
ggattttgtg tcctgtttgc cttcccgttt tttctgctgt tggactcgaa gagtacactg
1020 attatctggt gctcaatgat tttgggctat aacttgggtc caactatgat
gtttgctgta 1080 caaccaacat tgtttactcg tatgttcggc accaaggtcc
gctacacagg cttatcattt 1140 gcttaccagt tctcggctat cttaggcggc
ctgtccccac tgattgcatc ctcacttctt 1200 gcgttggggg gcggcaaacc
ctggtatgtc gccttgttcc ttttcgctgt gtccgtgtta 1260 tctttcgtct
gtgtatggtt aatcgagccc acagacgaac aagagacggc ttcataccgc 1320
tacatccgcg aacaaagtca tgagaac 1347 <210> SEQ ID NO 99
<211> LENGTH: 1377 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1377)
<223> OTHER INFORMATION: Escherichia coli PheP <400>
SEQUENCE: 99 atgaaaaacg cgtcaaccgt atcggaagat actgcgtcga atcaagagcc
gacgcttcat 60 cgcggattac ataaccgtca tattcaactg attgcgttgg
gtggcgcaat tggtactggt 120 ctgtttcttg gcattggccc ggcgattcag
atggcgggtc cggctgtatt gctgggctac 180 ggcgtcgccg ggatcatcgc
tttcctgatt atgcgccagc ttggcgaaat ggtggttgag 240 gagccggtat
ccggttcatt tgcccacttt gcctataaat actggggacc gtttgcgggc 300
ttcctctctg gctggaacta ctgggtaatg ttcgtgctgg tgggaatggc agagctgacc
360 gctgcgggca tctatatgca gtactggttc ccggatgttc caacgtggat
ttgggctgcc 420 gccttcttta ttatcatcaa cgccgttaac ctggtgaacg
tgcgcttata tggcgaaacc 480 gagttctggt ttgcgttgat taaagtgctg
gcaatcatcg gtatgatcgg ctttggcctg 540 tggctgctgt tttctggtca
cggcggcgag aaagccagta tcgacaacct ctggcgctac 600 ggtggtttct
tcgccaccgg ctggaatggg ctgattttgt cgctggcggt aattatgttc 660
tccttcggcg gtctggagct gattgggatt actgccgctg aagcgcgcga tccggaaaaa
720 agcattccaa aagcggtaaa tcaggtggtg tatcgcatcc tgctgtttta
catcggttca 780 ctggtggttt tactggcgct ctatccgtgg gtggaagtga
aatccaacag tagcccgttt 840 gtgatgattt tccataatct cgacagcaac
gtggtagctt ctgcgctgaa cttcgtcatt 900 ctggtagcat cgctgtcagt
gtataacagc ggggtttact ctaacagccg catgctgttt 960 ggcctttctg
tgcagggtaa tgcgccgaag tttttgactc gcgtcagccg tcgcggtgtg 1020
ccgattaact cgctgatgct ttccggagcg atcacttcgc tggtggtgtt aatcaactat
1080 ctgctgccgc aaaaagcgtt tggtctgctg atggcgctgg tggtagcaac
gctgctgttg 1140 aactggatta tgatctgtct ggcgcatctg cgttttcgtg
cagcgatgcg acgtcagggg 1200 cgtgaaacac agtttaaggc gctgctctat
ccgttcggca actatctctg cattgccttc 1260 ctcggcatga ttttgctgct
gatgtgcacg atggatgata tgcgcttgtc agcgatcctg 1320 ctgccggtgt
ggattgtatt cctgtttatg gcatttaaaa cgctgcgtcg gaaataa 1377
<210> SEQ ID NO 100 <211> LENGTH: 1704 <212>
TYPE: DNA <213> ORGANISM: Anabaena variabilis PAL1
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1704) <223> OTHER INFORMATION: Anabaena
variabilis PAL1 <400> SEQUENCE: 100 atgaaaacac tatcacaggc
ccaatctaaa acttcttcac agcaattcag ctttaccggg 60 aactcgtctg
cgaatgtaat tatcggcaat caaaagctga ccattaatga tgtagctcgc 120
gttgcccgga atggcacttt ggtgtcactg acgaacaata ccgacattct gcaaggtatt
180 caagctagct gcgattatat caataacgcc gttgaatctg gcgagccaat
ctacggggta 240 acaagcggtt ttggtgggat ggcgaacgtt gccattagcc
gtgaacaggc gagcgaactt 300 cagaccaacc tcgtttggtt cctaaagaca
ggagctggta ataagttacc tctggctgac 360 gtaagagccg cgatgctgct
tcgcgctaat agtcacatgc gcggcgccag tggtatccgt 420 cttgagctta
tcaagaggat ggaaatcttc ctcaacgcgg gtgtcacacc atatgtttat 480
gagtttggta gtatcggagc cagtggtgat cttgttcccc tgagttatat tacgggttca
540 ttgattggtt tagacccgtc ctttaaagtg gattttaacg ggaaagaaat
ggacgccccg 600 accgctttac gacagcttaa tctgagccca cttactttgc
tccctaaaga aggtcttgcc 660 atgatgaatg gcacctctgt gatgactgga
attgccgcga attgtgtgta tgacacgcag 720 atcctaacgg ccattgccat
gggtgttcac gcgttggaca ttcaagccct gaatggtaca 780 aaccagtcgt
ttcatccgtt tatccataat tcaaaacccc atccgggaca gctttgggct 840
gctgatcaga tgatctcact cctggccaat agtcaactgg ttcgggacga gctcgacggc
900 aaacatgatt atcgcgatca tgagctcatc caggaccggt attcacttcg
ttgtctccca 960 caatacctgg ggcctatcgt tgatggtata tctcaaattg
cgaagcaaat tgaaattgag 1020 atcaatagcg taaccgacaa cccgcttatc
gatgttgata atcaggcctc ttatcacggt 1080 ggcaattttc tgggccagta
tgttggtatg gggatggatc acctgcggta ctatattggg 1140 cttctggcta
aacatcttga tgtgcagatt gccttattag cttcaccaga attttcaaat 1200
ggactgccgc catcattgct cggtaacaga gaaaggaaag taaatatggg ccttaagggc
1260 cttcagatat gtggtaactc aatcatgccc ctcctgacct tttatgggaa
ctcaattgct 1320 gatcgttttc cgacacatgc tgaacagttt aaccaaaaca
ttaactcaca gggctataca 1380 tccgcgacgt tagcgcgtcg gtccgtggat
atcttccaga attatgttgc tatcgctctg 1440 atgttcggcg tacaggccgt
tgatttgcgc acttataaaa aaaccggtca ctacgatgct 1500 cgggcttgcc
tgtcgcctgc caccgagcgg ctttatagcg ccgtacgtca tgttgtgggt 1560
cagaaaccga cgtcggaccg cccctatatt tggaatgata atgaacaagg gctggatgaa
1620 cacatcgccc ggatatctgc cgatattgcc gccggaggtg tcatcgtcca
ggcggtacaa 1680 gacatacttc cttgcctgca ttaa 1704 <210> SEQ ID
NO 101 <211> LENGTH: 1599 <212> TYPE: DNA <213>
ORGANISM: Photorhabdus luminescens PAL3 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1599) <223> OTHER INFORMATION: Photorhabdus luminescens
PAL3 <400> SEQUENCE: 101 atgaaagcta aagatgttca gccaaccatt
attattaata aaaatggcct tatctctttg 60 gaagatatct atgacattgc
gataaaacaa aaaaaagtag aaatatcaac ggagatcact 120 gaacttttga
cgcatggtcg tgaaaaatta gaggaaaaat taaattcagg agaggttata 180
tatggaatca atacaggatt tggagggaat gccaatttag ttgtgccatt tgagaaaatc
240 gcagagcatc agcaaaatct gttaactttt ctttctgctg gtactgggga
ctatatgtcc 300 aaaccttgta ttaaagcgtc acaatttact atgttacttt
ctgtttgcaa aggttggtct 360 gcaaccagac caattgtcgc tcaagcaatt
gttgatcata ttaatcatga cattgttcct 420 ctggttcctc gctatggctc
agtgggtgca agcggtgatt taattccttt atcttatatt 480 gcacgagcat
tatgtggtat cggcaaagtt tattatatgg gcgcagaaat tgacgctgct 540
gaagcaatta aacgtgcagg gttgacacca ttatcgttaa aagccaaaga aggtcttgct
600 ctgattaacg gcacccgggt aatgtcagga atcagtgcaa tcaccgtcat
taaactggaa 660 aaactattta aagcctcaat ttctgcgatt gcccttgctg
ttgaagcatt acttgcatct 720 catgaacatt atgatgcccg gattcaacaa
gtaaaaaatc atcctggtca aaacgcggtg 780 gcaagtgcat tgcgtaattt
attggcaggt tcaacgcagg ttaatctatt atctggggtt 840 aaagaacaag
ccaataaagc ttgtcgtcat caagaaatta cccaactaaa tgatacctta 900
caggaagttt attcaattcg ctgtgcacca caagtattag gtatagtgcc agaatcttta
960 gctaccgctc ggaaaatatt ggaacgggaa gttatctcag ctaatgataa
tccattgata 1020 gatccagaaa atggcgatgt tctacacggt ggaaatttta
tggggcaata tgtcgcccga 1080 acaatggatg cattaaaact ggatattgct
ttaattgcca atcatcttca cgccattgtg 1140 gctcttatga tggataaccg
tttctctcgt ggattaccta attcactgag tccgacaccc 1200 ggcatgtatc
aaggttttaa aggcgtccaa ctttctcaaa ccgctttagt tgctgcaatt 1260
cgccatgatt gtgctgcatc aggtattcat accctcgcca cagaacaata caatcaagat
1320 attgtcagtt taggtctgca tgccgctcaa gatgttttag agatggagca
gaaattacgc 1380 aatattgttt caatgacaat tctggtagtt tgtcaggcca
ttcatcttcg cggcaatatt 1440 agtgaaattg cgcctgaaac tgctaaattt
taccatgcag tacgcgaaat cagttctcct 1500 ttgatcactg atcgtgcgtt
ggatgaagat ataatccgca ttgcggatgc aattattaat 1560 gatcaacttc
ctctgccaga aatcatgctg gaagaataa 1599 <210> SEQ ID NO 102
<211> LENGTH: 819 <212> TYPE: DNA <213> ORGANISM:
Legionella pneumophila phhA <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(819) <223>
OTHER INFORMATION: Legionella pneumophila phhA <400>
SEQUENCE: 102 atggagttta gtagccggta tgtcgcacat gtccctgatg
ctcagggttt agtcgattat 60 tcggcacaag aaaatagaat ttggaatatt
ttatttgaga ggcaactcaa gttattgcca 120 ggaagagctt gtgatgaatt
tctgtctgga ttacagactt taggacttaa ctcctcgact 180 attccacaac
ttccagaagt aagtgagcga ttaaaggcca aaacgggatg gcaagtagcg 240
ccagttgctg ctttaatttc agccagggaa ttttttgaat tattagcaga aaaatatttt
300 cctgcggcga cttttattcg aagtgaagaa gaattggatt atgttcaaga
acctgatatt 360 tttcatgagc tttttggtca ttgtcctatg ttaaccgata
gagtctatgc tgaatttgtc 420 catgattacg catgtaaggt attaactttt
cctgaacagg attggccttt attgcaaaga 480 atgttttggt ttactgtaga
gtttggattg attaaaacgc ctaaagggct tagagcatac 540
ggcgggggaa ttttatcttc tatcagtgaa acggtatatt gtgtggaaag tgatattcct
600 gtgcgaattt tatttgatcc agtggtggct tttcgaatgc cttatcggat
tgaccagcta 660 caacctgttt atttcgttat tgacagctat caaaatttat
atgatttcgt gctttctgac 720 atgggtaaat tcatggatcg tgcgcgagag
ttaggtgaat ttccaccgta ttttgatgtg 780 gatccggata atccaaatat
tcatataagg gcttgttaa 819 <210> SEQ ID NO 103 <211>
LENGTH: 717 <212> TYPE: DNA <213> ORGANISM: Escherichia
coli hisM <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(717) <223> OTHER INFORMATION:
Escherichia coli hisM <400> SEQUENCE: 103 gtgatcgaaa
tcttacatga atactggaaa ccgctgctgt ggaccgacgg ttatcgcttt 60
actggtgtgg cgatcactct gtggctgctt attttgtcgg tagtgatagg cggagtcctg
120 gcgctgtttc tggcgattgg tcgtgtctcc agtaataaat acatccagtt
tccaatctgg 180 ttatttacct atatttttcg cggtacgccg ctgtatgttc
agttgctggt gttctattcc 240 ggcatgtaca cgcttgagat tgttaaggga
accgaattcc ttaacgcttt cttccgcagt 300 ggcctgaact gtaccgtgct
ggcgctgacg cttaacacct gcgcttacac taccgagatt 360 tttgctgggg
caatccgttc ggttccgcat ggggaaattg aagccgccag agcctatggc 420
ttctcgactt ttaaaatgta tcgctgcatt attttgcctt ctgcgctgcg tattgcgtta
480 ccggcataca gcaacgaagt gatcctgatg ctgcactcta ctgcgttggc
atttactgcc 540 acggtgccgg atctgctgaa aatagcccgc gatattaacg
ccgccacgta tcaacctttt 600 accgccttcg gcattgccgc ggtgctctat
ttaatcatct cttatgtcct gatcagcctc 660 tttcgcagag cggaaaaacg
ctggttgcag catgtgaaac cttcttcaac gcactga 717 <210> SEQ ID NO
104 <211> LENGTH: 967 <212> TYPE: DNA <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: clbA (wild-type) <400> SEQUENCE: 104 caaatatcac
ataatcttaa catatcaata aacacagtaa agtttcatgt gaaaaacatc 60
aaacataaaa tacaagctcg gaatacgaat cacgctatac acattgctaa caggaatgag
120 attatctaaa tgaggattga tatattaatt ggacatacta gtttttttca
tcaaaccagt 180 agagataact tccttcacta tctcaatgag gaagaaataa
aacgctatga tcagtttcat 240 tttgtgagtg ataaagaact ctatatttta
agccgtatcc tgctcaaaac agcactaaaa 300 agatatcaac ctgatgtctc
attacaatca tggcaattta gtacgtgcaa atatggcaaa 360 ccatttatag
tttttcctca gttggcaaaa aagatttttt ttaacctttc ccatactata 420
gatacagtag ccgttgctat tagttctcac tgcgagcttg gtgtcgatat tgaacaaata
480 agagatttag acaactctta tctgaatatc agtcagcatt tttttactcc
acaggaagct 540 actaacatag tttcacttcc tcgttatgaa ggtcaattac
ttttttggaa aatgtggacg 600 ctcaaagaag cttacatcaa atatcgaggt
aaaggcctat ctttaggact ggattgtatt 660 gaatttcatt taacaaataa
aaaactaact tcaaaatata gaggttcacc tgtttatttc 720 tctcaatgga
aaatatgtaa ctcatttctc gcattagcct ctccactcat cacccctaaa 780
ataactattg agctatttcc tatgcagtcc caactttatc accacgacta tcagctaatt
840 cattcgtcaa atgggcagaa ttgaatcgcc acggataatc tagacacttc
tgagccgtcg 900 ataatattga ttttcatatt ccgtcggtgg tgtaagtatc
ccgcataatc gtgccattca 960 catttag 967 <210> SEQ ID NO 105
<211> LENGTH: 424 <212> TYPE: DNA <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION: clbA
knock-out <400> SEQUENCE: 105 ggatgggggg aaacatggat
aagttcaaag aaaaaaaccc gttatctctg cgtgaaagac 60 aagtattgcg
catgctggca caaggtgatg agtactctca aatatcacat aatcttaaca 120
tatcaataaa cacagtaaag tttcatgtga aaaacatcaa acataaaata caagctcgga
180 atacgaatca cgctatacac attgctaaca ggaatgagat tatctaaatg
aggattgatg 240 tgtaggctgg agctgcttcg aagttcctat actttctaga
gaataggaac ttcggaatag 300 gaacttcgga ataggaacta aggaggatat
tcatatgtcg tcaaatgggc agaattgaat 360 cgccacggat aatctagaca
cttctgagcc gtcgataata ttgattttca tattccgtcg 420 gtgg 424
<210> SEQ ID NO 106 <211> LENGTH: 1921 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic: Prp promoter
<400> SEQUENCE: 106 ttacccgtct ggattttcag tacgcgcttt
taaacgacgc cacagcgtgg tacggctgat 60 ccccaaataa cgtgcggcgg
cgcgcttatc gccattaaag cgtgcgagca cctcctgcaa 120 tggaagcgct
tctgctgacg agggcgtgat ttctgctgtg gtccccacca gttcaggtaa 180
taattgccgc ataaattgtc tgtccagtgt tggtgcggga tcgacgctta aaaaaagcgc
240 caggcgttcc atcatattcc gcagttcgcg aatattaccg ggccaatgat
agttcagtag 300 aagcggctga cactgcgtca gcccatgacg caccgattcg
gtaaaaggga tctccatcgc 360 ggccagcgat tgttttaaaa agttttccgc
cagaggcaga atatcaggct gtcgctcgcg 420 caagggggga agcggcagac
gcagaatgct caaacggtaa aacagatcgg tacgaaaacg 480 tccttgcgtt
atctcccgat ccagatcgca atgcgtggcg ctgatcaccc ggacatctac 540
cgggatcggc tgatgcccgc caacgcgggt gacggctttt tcctccagta cgcgtagaag
600 gcgggtttgt aacggcagcg gcatttcgcc aatttcgtca agaaacagcg
tgccgccgtg 660 ggcgacctca aacagccccg cacgtccacc tcgtcttgag
ccggtaaacg ctccctcctc 720 atagccaaac agttcagcct ccagcaacga
ctcggtaatc gcgccgcaat taacggcgac 780 aaagggcgga gaaggcttgt
tctgacggtg gggctgacgg ttaaacaacg cctgatgaat 840 cgcttgcgcc
gccagctctt tcccggtccc tgtttccccc tgaatcagca ctgccgcgcg 900
ggaacgggca tagagtgtaa tcgtatggcg aacctgctcc atttgtggtg aatcgccgag
960 gatatcgctc agcgcataac gggtctgtaa tcccttgctg gaggtatgct
ggctatactg 1020 acgccgtgtc aggcgggtca tatccagcgc atcatggaaa
gcctgacgta cggtggccgc 1080 tgaataaata aagatggcgg tcattcctgc
ctcttccgcc aggtcggtaa ttagtcctgc 1140 cccaattaca gcctcaatgc
cgttagcttt gagctcgtta atttgcccgc gagcatcctc 1200 ttcagtgata
tagcttcgct gttcaagacg gaggtgaaac gttttctgaa aggcgaccag 1260
agccggaatg gtctcctgat aggtcacgat tcccattgag gaagtcagct ttcccgcttt
1320 tgccagagcc tgtaatacat cgaatccgct gggtttgatg aggatgacag
gtaccgacag 1380 tcggcttttt aaataagcgc cgttggaacc tgccgcgata
atcgcgtcgc agcgttcggt 1440 tgccagtttt ttgcgaatgt aggctactgc
cttttcaaaa ccgagctgaa taggcgtgat 1500 cgtcgccaga tgatcaaact
ccaggctgat atcccgaaat agttcgaaca ggcgcgttac 1560 cgagaccgtc
cagatcaccg gtttatcgct attatcgcgc gaagcgctat gcacagtaac 1620
catcgtcgta gattcatgtt taaggaacga attcttgttt tatagatgtt tcgttaatgt
1680 tgcaatgaaa cacaggcctc cgtttcatga aacgttagct gactcgtttt
tcttgtgact 1740 cgtctgtcag tattaaaaaa gatttttcat ttaactgatt
gtttttaaat tgaattttat 1800 ttaatggttt ctcggttttt gggtctggca
tatcccttgc tttaatgagt gcatcttaat 1860 taacaattca ataacaagag
ggctgaatag taatttcaac aaaataacga gcattcgaat 1920 g 1921 <210>
SEQ ID NO 107 <211> LENGTH: 864 <212> TYPE: DNA
<213> ORGANISM: Salmonella enterica subsp. enterica serovar
Typhimurium LT2 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(864) <223> OTHER
INFORMATION: Tsx - Salmonella enterica subsp. enterica serovar
Typhimurium LT2 (STM0413) <400> SEQUENCE: 107 atgaaaaaaa
ctttactcgc agtcagcgca gcgctggcgc tcacctcatc ttttactgct 60
aacgcagcag aaaatgatca gccgcagtat ttgtccgact ggtggcacca gagcgtaaac
120 gtggtaggca gctaccatac ccgtttctcg ccgaaattga acaacgacgt
ctatctggaa 180 tatgaagcat ttgccaaaaa agactggttt gatttctacg
gctatatcga tattcccaaa 240 acctttgatt ggggtaacgg caacgataaa
ggtatctggt ccgacggttc tccgctgttc 300 atggaaatcg aaccgcgttt
ctcaattgat aagctgaccg gcgcagacct gagcttcggc 360 ccgtttaaag
agtggtattt cgccaacaac tacatctacg atatgggcga taacaaagcc 420
agccgccaga gcacgtggta tatgggtctg gggaccgata tcgacaccgg cctgccgatg
480 ggtctgtcgc tgaacgtgta tgcgaaatat cagtggcaaa actacggcgc
gtccaatgaa 540 aacgaatggg acggctaccg tttcaaagtg aaatacttcg
tccccatcac cgatctgtgg 600 ggcggtaaac tgagctatat cggctttacc
aactttgact ggggatctga tttaggcgac 660 gatccgaacc gtaccagcaa
ctccatcgct tccagccata tcctggcgct gaactacgat 720 cactggcact
actcggtcgt tgcgcgttac ttccataacg gcggacagtg gcagaatggc 780
gcaaaactga actggggcga cggcgatttc agcgcgaaat ctaccggctg gggcggctac
840 ctggtcgtgg gttacaactt ctaa 864 <210> SEQ ID NO 108
<211> LENGTH: 885 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(885) <223> OTHER
INFORMATION: Tsx - Escherichia coli K-12 MG1655 (b0411) <400>
SEQUENCE: 108 atgaaaaaaa cattactggc agccggtgcg gtactggcgc
tctcttcgtc ttttactgtc 60
aacgcagctg aaaacgacaa accgcagtat ctttccgact ggtggcacca gagcgttaac
120 gttgtcggaa gctatcacac ccgtttcgga ccgcagatcc gcaacgatac
ctaccttgag 180 tacgaagcat tcgctaaaaa agactggttc gacttctatg
gttatgcgga tgcgccggta 240 ttcttcggcg gtaactccga tgctaaaggt
atctggaacc acggttctcc gctgtttatg 300 gaaatcgaac cacgtttctc
catcgacaag ctgaccaata ctgaccttag cttcggtccg 360 ttcaaagagt
ggtacttcgc gaacaactac atttacgaca tgggtcgtaa taaagatggt 420
cgccagagca cctggtacat gggtctgggt accgatatcg acactggcct gccgatgagc
480 ctgtccatga acgtctatgc gaaataccag tggcagaact atggcgcagc
gaacgaaaac 540 gagtgggacg gttaccgttt caaaattaaa tactttgtgc
cgattaccga tctgtggggc 600 ggtcagctga gctacatcgg cttcaccaac
ttcgactggg gttccgattt aggggatgac 660 agcggtaacg caatcaacgg
tattaagacc cgtactaata actctatcgc ttccagccat 720 attctggctc
tgaactacga tcactggcac tactctgtcg tagctcgtta ctggcacgac 780
ggtggtcagt ggaacgacga tgcagaactg aacttcggca acggcaactt caacgttcgc
840 tctaccggct ggggtggtta cctggtagta ggttacaact tctga 885
<210> SEQ ID NO 109 <211> LENGTH: 1221 <212>
TYPE: DNA <213> ORGANISM: Bacillus halodurans <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1221) <223> OTHER INFORMATION: BH1446 - Bacillus
halodurans (BAB05165) <400> SEQUENCE: 109 atgaatattt
tgtggggttt attaggaatc gtcgttgttt ttctaatcgc ttttgcattt 60
tccacaaatc gtcgtgcaat taaaccacga acgatattag gtggtctcgc gattcagcta
120 ttatttgcga ttattgtatt aaaaattcca gctggacaag cgttacttga
gagcttaacc 180 aatgtagttt tgaacattat tagttatgcg aatgaaggga
tcgacttcgt atttggtgga 240 tttttcgaag aaggttcagg cgtaggcttc
gtttttgcaa ttaacgtttt gtctgtcgtc 300 attttcttct cagcactaat
ctcgatcctt tattatttag ggatcatgca atttgtcatt 360 aaaattatcg
gtggtgcgct gtcctggcta ctcggaacat caaaggcaga atcaatgtca 420
gcagcagcta acattttcgt tgggcaaacg gaagcgccac tcgttgttaa gccatactta
480 ccaaaaatga cgcaatccga gctctttgcg gttatgaccg ggggacttgc
ttctgttgct 540 ggttctgttt taatcggtta ttctctttta ggagtaccgc
tacaatattt attagcggca 600 agctttatgg ctgctcctgc gggcttgatt
atggcgaaaa tgatcatgcc tgaaacggag 660 aaaacaaccg atgcagaaga
tgactttaag ctcgcaaagg atgaagagtc cacgaacttg 720 attgacgcgg
ccgccaatgg ggcgagcact gggttaatgc tcgttctaaa tattgcggcg 780
atgttactag cgttcgttgc attgattgca ttaattaatg gaattcttgg atggatcgga
840 ggattgtttg gggcgtcgca attgtcttta gagttaatcc tcggatacgt
gtttgctccg 900 cttgcgtttg tcatcggaat tccttgggct gaagcgcttc
aagcgggaag ctacatcgga 960 cagaaactcg tagtgaacga atttgttgcc
tacttaagct ttgcaccaga aattgaaaac 1020 ctttcagata aagcggtgat
ggtgattagt tttgcccttt gcggatttgc taacttctca 1080 tccctcggaa
tccttttagg aggattgggt aagcttgctc cgagccgtcg ccctgatatt 1140
gcccgtctcg gattacgcgc gatccttgca ggtacgctag cttctttact cagcgcctcc
1200 attgcgggaa tgttattcta a 1221 <210> SEQ ID NO 110
<211> LENGTH: 1182 <212> TYPE: DNA <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1182)
<223> OTHER INFORMATION: nupC Bacillus subtilis subsp.
subtilis 168 (BSU39410; CAA57663) <400> SEQUENCE: 110
atgaagtatt tgattgggat tatcggttta atcgtgtttt taggcctcgc gtggatcgcg
60 agcagcggca aaaaaagaat taagatccgc ccaattgttg ttatgctcat
tttgcaattt 120 attcttggct acattctcct caataccgga atagggaatt
tcctcgtggg aggatttgca 180 aaaggattcg gttacctgct tgaatacgcg
gcagagggaa ttaactttgt gtttggcggc 240 ttggtgaatg cggaccaaac
gacattcttt atgaatgttc tcttgccaat cgtgtttatt 300 tccgctctga
tcgggattct gcaaaagtgg aaagtcctcc cgtttatcat tagatatatc 360
ggccttgccc tcagcaaggt aaacggtatg ggaagattgg aatcgtataa cgcagtggct
420 tctgcgattt tagggcagtc agaagtattt atctccttga agaaagaact
cggtctttta 480 aatcagcagc gcttgtacac gctttgcgca tctgcgatgt
caactgtatc aatgtcgatt 540 gtcggtgcgt atatgacaat gctgaaaccg
gaatatgttg taacagcgct tgttttgaac 600 ttatttggcg gtttcattat
cgcttctatt atcaatccgt acgaggttgc aaaagaagag 660 gatatgcttc
gtgttgagga agaagaaaaa caatccttct tcgaagtgct cggagaatac 720
attcttgacg gtttcaaagt agcggttgtc gtcgctgcga tgctgattgg atttgtcgcg
780 attattgcat tgatcaatgg catttttaat gcagtattcg gtatttcgtt
ccaaggcatt 840 cttggatatg tgtttgctcc attcgctttt cttgtcggta
tcccatggaa tgaagctgtt 900 aatgcgggaa gcattatggc aacaaaaatg
gtatcgaatg aatttgtcgc catgacgtcg 960 cttacgcaaa acggtttcca
tttcagcggc cgtacaacag cgatcgtatc ggtattcctt 1020 gtgtcatttg
cgaacttctc ctcaatcgga atcattgccg gtgccgtaaa aggactgaat 1080
gaaaagcaag gaaatgtcgt cgctcgtttc ggcttgaaat tattatacgg tgctacgctt
1140 gtcagctttt tatcagcagc aattgtgggc ttgatttact ga 1182
<210> SEQ ID NO 111 <211> LENGTH: 1215 <212>
TYPE: DNA <213> ORGANISM: Bacillus subtilis <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1215) <223> OTHER INFORMATION: yutK - Bacillus subtilis
subsp. subtilis 168: BSU32180 <400> SEQUENCE: 111 atgaatgttc
tgtgggggct gctgggcgca gttgcgatca ttgctatcgc gtttttattt 60
tcagaaaaga aaagcaatat taagataaga accgtcatcg ttggtttatg cacacaggtg
120 gcgtttggat acatcgtgtt gaaatgggaa gcgggacgcg ctgttttttt
atggttttca 180 agccgtgtac agcttctgat tgactatgcg aatgaaggca
tcagttttat ttttggaccg 240 cttctaaagg tcggagacag tccggcattt
gcattaagtg tactgcccgt tatcattttc 300 ttctcagcac tgattgcagt
tttatatcat ttgaaaatca tgcagctcgt tttccgtgtc 360 attggcggcg
gattgtcgaa gctccttgga acaagcaaaa cggaatctct ggcggctgct 420
gccaatattt ttgtaggaca atcagaatct ccgttagtga tcaaacccct gattgccggg
480 ctgacgcgct ctgagttgtt tacgattatg acgagcggtc tatcggcagt
tgcgggatct 540 accttgtttg ggtacgcgct tctcggtatt ccgattgagt
acttgctggc ggccagcttt 600 atggctgctc cagctggact agtctttggt
aaattgatta tacccgaaac ggaaaaaacg 660 caaaccgtaa aaagcgattt
caaaatggat gaaggcgaag gcgcagccaa tgtcattgac 720 gcagctgcaa
agggagcgtc aacaggactg caaattgcgt taaatgttgg ggcgatgctg 780
cttgcgtttg ttgcgttaat cgctgtagta aacggtattc tcggcggggc tttcggcttg
840 ttcggtttaa aaggcgtaac attagaatcc attctcggct atgtgttttc
tcctatcgcc 900 tttttgattg gcgtgccttg gcatgaagca ttgcaggcgg
gaagctatat cggccagaaa 960 ttggtgctga atgagtttgt cgcttattct
aacttcggtt cgcacatcgg cgagttttct 1020 aagaaaactg ctaccattat
cagtttcgcg ttatgcggat tcgccaattt ttcatcaatt 1080 gcgattatgc
ttggtacgct tggcggttta gcgcccagcc gccgttcaga tatcgcacgt 1140
ctcggcctga aggctgttct tgcaggaaca ttagccaatc tgctcagcgc agccattgcc
1200 ggcatgttta tataa 1215 <210> SEQ ID NO 112 <211>
LENGTH: 1194 <212> TYPE: DNA <213> ORGANISM: Bacillus
subtilis <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1194) <223> OTHER INFORMATION:
yxjA - Bacillus subtilis subsp. spizizenii W23 (BSUW23_19355)
<400> SEQUENCE: 112 atgtactttt tattaaacct tgtcggtctc
attgtgatta tggcagttgt gttcctatgc 60 tccccgcaga aaaagaaaat
ccagtggcgt ccgatcatta cgttaattgt tctggaattg 120 ctgattactt
ggtttatgct gggaacaaag gtcgggagct gggccatcgg taaaattggt 180
gatttcttca cttggctgat tgcttgcgcc agtgacggta tcgcgtttgc cttcccgtca
240 gtcatggcga atgaaacagt agactttttc tttagtgcac ttcttccaat
tatctttatc 300 gtcacattct ttgatatttt aacatatttc ggcattttgc
cttggctgat tgataaaatc 360 ggatgggtga tttcaaaggc ttcccgcttg
ccgaaattag aaagcttttt ctctattcaa 420 atgatgttct tgggaaatac
tgaagcactt gcggtcatcc gccagcagct tacggtatta 480 aataacaacc
gcttgcttac atttggctta atgagcatga gcagcatcag cggctccatt 540
attggatctt acctgtcaat ggtgccggcg acatacgtgt ttacagcgat tccattgaac
600 tgcttaaacg cgctgattat tgcaaacctg ctgaaccctg ttcatgtgcc
ggaggatgaa 660 gatatcatct atacaccgcc taaagaagag aagaaagact
ttttctctac gatttctaac 720 agtatgcttg tcggcatgaa catggttatc
gttattttgg caatggtgat cggatatgta 780 gcattaacgt ctgcagtcaa
tggcattctt ggtgttttcg tacacggcct gaccatccag 840 acaatttttg
cttatctctt cagtccgttc gcattcctgc ttggtctgcc agtacatgat 900
gcaatgtatg tcgctcagct aatgggaatg aaattggcaa cgaacgagtt tgttgcgatg
960 cttgacttga aaaacaatct tacaacactt ccgcctcaca cagttgcggt
ggcgacgaca 1020 ttcctgacgt catttgccaa cttcagtact gtcggcatga
tttacggaac gtacaactcg 1080 atccttgacg gcgaaaagtc aacggtcatc
gggaaaaacg tgtggaaatt gctcgtcagc 1140 ggcattgcgg tatctttact
aagtgctgcg attgtcggcc tgtttgtgtg gtag 1194 <210> SEQ ID NO
113 <211> LENGTH: 1281 <212> TYPE: DNA <213>
ORGANISM: Caulobacter crescentus CB15 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1281) <223> OTHER INFORMATION: ccCNT (CC2089) -
Caulobacter crescentus CB15 (AAK24060) <400> SEQUENCE: 113
atgttccgtc ccgagaacgt tcaggccctc gcgggtctgg cgctcaccct gggcctgtgc
60 tggctcgttt ccgagaatcg caagcggttc ccctggggcc tggccatcgg
cgcggtcgtc 120 attcaggtcc tgctggtcct ggtcctgttc ggcctgccgc
aagcccagca gatgctgcgc 180 ggcgtcaacg gcgcggtgga gggccttgcc
gcctcgaccc aggccggcac cgccttcgtg 240 ttcggctttc tggccggcgg
cgaccagccc tatccggtca gcaatccggg cgcgggcttc 300 atcttcgcct
tccgcgtgct gccggtgatc ctggtggtct gcgccctgtc ggcgctgctg 360
tggcactgga agattctcaa gtggctggct cagggcttcg gctttgtgtt ccagaagacg
420 ctgggcctgc gcggcccgcc ggccctggcc accgccgcga ccatcttcat
gggtcaggtc 480 gaggggccga tcttcatccg cgcctatctc gacaagctga
gccgctcgga actcttcatg 540 ctgatcgcgg tcggcatggc ctgcgtgtcg
ggctcgacca tggtcgccta cgccaccatc 600 ctggccgacg tcctgcccaa
cgccgccgcc cacgtgctga ccgcctcgat catctcggct 660 ccggccggcg
tgctgctggc ccggatcatt gtgccgtccg atccgatgga gaagagcgcc 720
gatcttgatc tgtcgaccga ggacaagacc tatggcagct cgatcgacgc cgtgatgaag
780 ggcaccaccg acggcctgca gatcgcgctg aacgtcggcg ccaccctgat
cgtcttcgtg 840 gccctggcca ccatggtcga caaggtcctg ggcgccttcc
cgccggtggg cggcgagccg 900 ctgagcatcg cgcgcggcct gggcgtggtc
ttcgcgccgc tggcctggtc gatgggcatc 960 ccgtggaaag aagcgggcac
ggccggcggt ctgctgggcg tgaagctgat cctgaccgag 1020 ttcaccgcct
tcatccagct gtccaaggtg ggcgaagccc tgctggacga acgcacccgg 1080
atgatcatga cctacgctct gtgcggtttc gccaatatcg gctcggtcgg catgaacgtc
1140 gccggcttct cggtgctggt gccccagcgc cggcaggaag tgctgggcct
ggtctggaag 1200 gcgatgatgg ccggcttcct ggccacctgc ctgaccgcct
cgctggtcgg cctgatgccg 1260 cgaagcctgt ttgggctgta a 1281 <210>
SEQ ID NO 114 <211> LENGTH: 1251 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1251) <223> OTHER INFORMATION: yeiJ - Escherichia coli
K-12 W3110 (AAC75222; JW2148) <400> SEQUENCE: 114 atggatgtca
tgagaagtgt tctgggaatg gtggtattgc tgacgattgc gtttttactg 60
tcagtaaaca agaagaagat cagcctgcgt accgttggcg cggcgttagt gttacaggtc
120 gtgattggcg gcattatgct ttggttaccg ccagggcgtt gggtcgctga
aaaagtcgct 180 tttggcgtgc ataaagtgat ggcgtacagc gacgcgggta
gcgcatttat cttcggttct 240 ctggtcggac cgaaaatgga taccttattt
gatggtgcag gatttatctt tggtttcagg 300 gtgttaccgg caattatctt
cgtcaccgcg ctggtgagta ttctctacta catcggtgtg 360 atggggattt
taattcgaat tctcggcggt atcttccaga aagcattaaa tatcagcaag 420
atcgagtcat tcgtcgcggt caccaccatt ttcctcgggc aaaacgaaat tccggcaatc
480 gtcaaaccct ttatcgatcg tctgaatcgc aatgaattat ttacagcgat
ttgtagtggc 540 atggcctcga ttgctggttc gacaatgatt ggttacgccg
cactgggcgt gcctgtggaa 600 tatctgctgg cggcatcatt aatggcgatc
cctggcggga tcttgtttgc ccgcctgtta 660 agcccggcaa cggaatcttc
gcaggtttcc tttaataacc tctctttcac cgaaacaccg 720 ccaaaaagca
ttattgaagc cgctgcgaca ggggcaatga ccgggctgaa aatcgccgca 780
ggtgtggcaa cagtggtgat ggcatttgtt gcaataattg cgttgattaa cggtattatc
840 ggcggcgttg gtggctggtt tggttttgaa catgcctcgc tggagtccat
tttaggttac 900 ctgctggctc cactggcgtg ggtgatgggt gtggactgga
gtgatgcgaa tcttgccggg 960 agtttgattg gacagaaact ggcaataaat
gaatttgtcg cttatctcaa tttctcaccc 1020 tatctgcaaa cggctggcac
tctcgatgct aaaactgtgg cgattatttc cttcgcgttg 1080 tgcggtttcg
ctaactttgg ttctatcggg gtggtggtgg gggcgttttc tgcggttgcg 1140
ccacaccgtg cgccggaaat cgcccagctt ggtttacggg cgctggcggc ggcgacgctt
1200 tccaacttga tgagtgcgac cattgccggg ttctttattg gtttagcttg a 1251
<210> SEQ ID NO 115 <211> LENGTH: 1251 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1251) <223> OTHER INFORMATION: yeiM - Escherichia coli
K-12 W3110 (AAC75225; JW2151) <400> SEQUENCE: 115 atggatataa
tgagaagtgt tgtggggatg gtggtgttac tggcaatagc atttctgttg 60
tcagtgaata aaaagagcat cagtttgcgc acggttggag ccgcactgct gctgcaaatc
120 gctattggtg gcatcatgct ctacttccca ccgggaaaat gggcagtaga
acaggcggca 180 ttaggcgttc ataaagtgat gtcttacagt gatgccggta
gcgccttcat ttttggttcg 240 ctggttgggc cgaaaatgga tgtcctgttt
gacggtgcgg gttttatctt cgcctttcgc 300 gtacttccgg cgattatttt
cgttactgcg ctcatcagtc tgctgtacta cattggcgtg 360 atggggctgc
tgattcgcat ccttggcagc attttccaga aagccctcaa catcagcaaa 420
atcgaatctt ttgttgcggt tactactatt ttcctcgggc aaaatgagat cccggcgatc
480 gttaaaccgt ttatcgatcg catgaatcgc aacgagttgt ttaccgcaat
ttgtagcggg 540 atggcgtcca ttgctggttc gatgatgatt ggttatgccg
gaatgggcgt accaattgac 600 tacctgttag cggcatcgct gatggcgatc
cctggcggga ttttgtttgc acgtattctt 660 agcccggcaa ccgagccttc
gcaggtcaca tttgaaaatc tgtcgttcag cgaaacgccg 720 ccaaaaagct
ttatcgaagc ggcggcgagc ggtgcgatga ccgggctaaa aatcgccgct 780
ggtgtggcga cggtggtaat ggcgtttgtc gcaattattg cgctgatcaa cggcattatc
840 ggcggaattg gcggctggtt tggtttcgcc aatgcctctc tggaaagtat
ttttggctat 900 gtgctggcac cgctggcgtg gatcatgggt gtggactgga
gtgatgccaa tcttgcgggt 960 agcctgattg ggcagaaact ggcgattaac
gaattcgtcg cttacctgag tttctcccca 1020 tacctgcaaa cgggcggcac
gctggaagtg aaaaccattg cgattatctc ctttgcgctt 1080 tgtggttttg
ctaactttgg ttctatcggt gttgtcgttg gcgcattttc ggctatttcg 1140
ccaaaacgcg cgccggaaat cgcccagctt ggtttacggg cgctggcagc agcaacgctt
1200 tccaacctga tgagtgcgac tattgccggg ttctttattg gtctggcgta a 1251
<210> SEQ ID NO 116 <211> LENGTH: 1254 <212>
TYPE: DNA <213> ORGANISM: Haemophilus influenzae <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1254) <223> OTHER INFORMATION: HI0519 - Haemophilus
influenzae Rd KW20 serotype d(AAC22177) <400> SEQUENCE: 116
atgagtgtgt taagcagcat tttgggaatg gtcgtattaa tcgctattgc cgtgttactt
60 tctaataatc gtaaagcgat tagtattcga accgtagtag gggcgttagc
aatccaagta 120 ggatttgccg cccttatttt atatgtgcca gcaggtaaac
aagcgttggg tgccgctgcg 180 gatatggtat ccaatgttat tgcctatggt
aatgacggga ttaatttcgt tttcggcgga 240 ttggcagatc caagtaaacc
atccggtttc atttttgcag tgaaagtatt accgattatc 300 gtgttcttct
ctggcttaat ttctgtgctt tactatctcg gcattatgca agtcgtgatt 360
aaagtattag gtggcgcatt acaaaaagca ttgggtacgt caaaagcgga atcaatgtca
420 gcggcggcga atatcttcgt cggtcaaact gaagcaccat tagttgttcg
cccttacatt 480 aaaaatatga cccaatctga attatttgcc attatggtgg
gtggtacagc gtctatcgcg 540 ggttcagtaa tggcaggtta tgctggaatg
ggcgtgccat tgacatactt aatcgctgcg 600 tcatttatgg cggcaccagc
aggtttatta tttgcgaaat taatgttccc acaaaccgaa 660 caattcacag
ataaacaacc agaagacaat gattcagaaa aaccaactaa cgtacttgaa 720
gcaatggcgg gcggtgcgag tgcaggtatg caacttgcgt taaacgtagg tgcaatgtta
780 atcgcattcg ttggtttaat tgcattaatt aatggtattt taagtggcgt
aggcggatgg 840 ttcggctatg gcgacttaac cttacaatct atctttggtt
taatttttaa accattagca 900 tacttaatcg gtgtaactga tggtgctgaa
gcaggtattg caggacaaat gatcgggatg 960 aaattagcgg ttaatgaatt
tgtgggttat cttgaatttg caaaatattt acaaccagat 1020 tctgcaattg
tattaactga aaaaaccaaa gcgattatta ctttcgcact ttgtggtttt 1080
gctaacttca gctcaattgc aatcttaatt ggtggtttag gtggtatggc accaagccgt
1140 cgtagtgatg ttgctcgttt aggtatcaaa gccgttatcg ctggtactct
cgctaactta 1200 atgagtgcaa ctattgctgg tttatttatc ggcttaggtg
ctgcagcact ttaa 1254 <210> SEQ ID NO 117 <211> LENGTH:
1257 <212> TYPE: DNA <213> ORGANISM: Helicobacter
pylori 26695 <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1257) <223> OTHER
INFORMATION: nupC (HP1180) - Helicobacter pylori 26695 (AAD08224)
<400> SEQUENCE: 117 atgattttta gctctctttt tagtgttgta
gggatggcgg tgctttttct tattgcttgg 60 gtgttttcta gcaataaaag
ggctattaat tatcgcacga ttgtcagtgc ctttgtgatt 120 caagtggctt
taggggcgtt ggctttatat gtgcctttgg gtagggaaat gctgcaaggc 180
ttagccagcg gcatacaaag cgtgatttct tacggctatg agggggtgcg ttttttattt
240 ggcaatctcg ctccaaacgc taagggcgat caagggatag gggggtttgt
ctttgcgatc 300 aatgttttag cgatcattat cttttttgct agcttgattt
cacttctata ttatttaaaa 360 atcatgcctt tatttatcaa tctcatcggt
ggggcgttgc aaaaatgctt aggcacttct 420 agagcagaaa gcatgagtgc
agcggctaat atttttgtag cgcacaccga agcgccctta 480 gtcattaaac
cttatttgaa aagcatgagc gattcagaga tttttgcggt catgtgcgtg 540
ggcatggcta gcgttgcggg gcctgtgtta gccgggtatg cgagcatggg cattcctttg
600
ccttatttga tcgccgcttc gtttatgtcc gctcctgggg ggttgttgtt cgctaaaatc
660 atttacccac aaaacgaaac catttctagc catgcagatg tttctataga
aaagcatgtc 720 aatgccatag aagctatcgc taatggggca agcacagggc
taaatttagc cttgcatgtg 780 ggagcgatgc ttttagcctt tgtggggatg
ctcgcgctca ttaacgggct tttaggggtt 840 gtagggggtt ttttaggcat
ggagcatttg tctttagggt tgattttagg cacgctctta 900 aaacccttag
cctttatgtt aggcattcct tggagccagg ccgggattgc cggagaaatc 960
ataggcatta aaatcgcgct caatgaattt gtgggctata tgcagttatt gccttatttg
1020 ggcgataacc ctcctttaat cttgagcgag aaaactaaag cgatcatcac
ttttgcgttg 1080 tgcgggtttg ctaatttaag ctcagtcgct atgctcattg
gagggcttgg cagtttagtg 1140 cctaaaaaga aggatctcat tgtaaggctt
gctttaaaag cggtgcttgt aggcacgctt 1200 tctaatttca tgagcgcgac
tatcgccggg ttattcatag ggctaaacgc tcattaa 1257 <210> SEQ ID NO
118 <211> LENGTH: 1230 <212> TYPE: DNA <213>
ORGANISM: Staphylococcus aureus <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1230)
<223> OTHER INFORMATION: nupC (SA0600) - Staphylococcus
aureus subsp. aureus N315 (BAB41833) <400> SEQUENCE: 118
atgtttttat taatcaacat tattggtcta attgtatttc ttggtattgc ggtattattt
60 tcaagagatc gcaaaaatat ccaatggcaa tcaattggga tcttagttgt
tttaaacctg 120 tttttagcat ggttctttat ttattttgat tggggtcaaa
aagcagtaag aggagcagcc 180 aatggtatcg cttgggtagt tcagtcagcg
catgctggta caggttttgc atttgcaagt 240 ttgacaaatg ttaaaatgat
ggatatggct gttgcagcct tattcccaat attattaata 300 gtgccattat
ttgatatctt aatgtacttt aatattttac cgaaaattat tggaggtatt 360
ggttggttac tagctaaagt aacaagacaa cctaaattcg agtcattctt tgggatagaa
420 atgatgttct taggaaatac tgaagcatta gccgtatcaa gtgagcaact
aaaacgtatg 480 aatgaaatgc gtgtattaac aatcgcaatg atgtcaatga
gctctgtatc cggagctatt 540 gtaggtgcgt atgtacaaat ggtaccagga
gaactggtac taacggcaat tccactaaat 600 atcgttaacg cgattattgt
gtcatgcttg ttgaatccag taagtgttga agagaaagaa 660 gatattattt
acagtcttaa aaacaatgaa gttgaacgtc aaccattctt ctcattcctt 720
ggagattctg tattagcagc aggtaaatta gtattaatca tcatcgcatt tgttattagt
780 tttgtagcgt tagctgatct atttgatcgt tttatcaatt tgattacagg
attgatagca 840 ggatggatag gcataaaagg tagtttcggt ttaaaccaaa
ttttaggtgt gtttatgtat 900 ccatttgcgc tattactcgg tttaccttat
gatgaagcgt ggttggtagc acaacaaatg 960 gctaagaaaa ttgttacaaa
tgaatttgtt gttatgggtg aaatttctaa agatattgca 1020 tcttatacac
cacaccatcg tgcggttatt acaacattct taatttcatt tgcaaacttc 1080
tcaacgattg gtatgattat cggtacattg aaaggcattg ttgataaaaa gacatcagac
1140 tttgtatcta aatatgtacc tatgatgcta ttatcaggta tcctagtttc
attattaaca 1200 gcagctttcg ttggtttatt tgcatggtaa 1230 <210>
SEQ ID NO 119 <211> LENGTH: 1230 <212> TYPE: DNA
<213> ORGANISM: Staphylococcus aureus <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1230) <223> OTHER INFORMATION: nupC (SAV0645)
-Staphylococcus aureus subsp. aureus Mu50 (BAB56807) <400>
SEQUENCE: 119 atgtttttat taatcaacat tattggtcta attgtatttc
ttggtattgc ggtattattt 60 tcaagagatc gcaaaaatat ccaatggcaa
tcaattggga tcttagttgt tttaaacctg 120 tttttagcat ggttctttat
ttattttgat tggggtcaaa aagcagtaag aggagcagcc 180 aatggtatcg
cttgggtagt tcagtcagcg catgctggta caggttttgc atttgcaagt 240
ttgacaaatg ttaaaatgat ggatatggct gttgcagcct tattcccaat attattaata
300 gtgccattat ttgatatctt aatgtacttt aatattttac cgaaaattat
tggaggtatt 360 ggttggttac tagctaaagt aacaagacaa cctaaattcg
agtcattctt tgggatagaa 420 atgatgttct taggaaatac tgaagcatta
gccgtatcaa gtgagcaact aaaacgtatg 480 aatgaaatgc gtgtattaac
aatcgcaatg atgtcaatga gctctgtatc cggagctatt 540 gtaggtgcgt
atgtacaaat ggtaccagga gaactggtac taacggcaat tccactaaat 600
atcgttaacg cgattattgt gtcatgcttg ttgaatccag taagtgttga agagaaagaa
660 gatattattt acagtcttaa aaacaatgaa gttgaacgtc aaccattctt
ctcattcctt 720 ggagattctg tattagcagc aggtaaatta gtattaatca
tcatcgcatt tgttattagt 780 tttgtagcgt tagctgatct atttgatcgt
tttatcaatt tgattacagg attgatagca 840 ggatggatag gcataaaagg
tagtttcggt ttaaaccaaa ttttaggtgt gtttatgtat 900 ccatttgcgc
tattactcgg tttaccttat gatgaagcgt ggttggtagc acaacaaatg 960
gctaagaaaa ttgttacaaa tgaatttgtt gttatgggtg aaatttctaa agatattgca
1020 tcttatacac cacaccatcg tgcggttatt acaacattct taatttcatt
tgcaaacttc 1080 tcaacgattg gtatgattat cggtacattg aaaggcattg
ttgataaaaa gacatcagac 1140 tttgtatcta aatatgtacc tatgatgcta
ttatcaggta tcctagtttc attattaaca 1200 gcagctttcg ttggtttatt
tgcatggtaa 1230 <210> SEQ ID NO 120 <211> LENGTH: 1203
<212> TYPE: DNA <213> ORGANISM: Streptococcus pyogenes
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1203) <223> OTHER INFORMATION: nupC (SpNupC)
- Streptococcus pyogenes SF370 serotype M1 (AAK34582) <400>
SEQUENCE: 120 atgcaattta tttatagtat tattggtatt ttattggtat
taggaattgt gtatgcaatt 60 tctttcaatc gtaagagtgt ttctctaagt
ttaattggaa aagctcttat cgttcaattc 120 attattgcgc taatcttagt
acgtatccca ctaggccaac aaattgttag tgttgtttca 180 actggagtta
ctagcgtaat caactgtggt caagctggtt taaattttgt gtttgggtca 240
ttagcagata gtggcgcaaa aactggtttt attttcgcta ttcaaacgct tggtaatatt
300 gttttcttat ctgccctagt tagtctactt tattatgtag gaatccttgg
atttgtagta 360 aaatggatag gtaagggcgt tggtaaaatt atgaaatcct
cagaggttga gagttttgtt 420 gctgtagcta atatgtttct tggtcaaaca
gacagtccaa tcttggttag caaataccta 480 ggtcgtatga ctgatagtga
gataatggtt gtgttggtat caggtatggg aagtatgtca 540 gtttctattc
ttggtggcta tattgcatta ggcattccaa tggaatatct cttgattgct 600
tcaacaatgg ttcctattgg cagtattctc attgctaaaa tcttattgcc tcaaacagaa
660 cctgttcaaa aaattgatga cattaagatg gataataaag gtaataacgc
caatgtgatt 720 gatgcaatcg ctgagggtgc aagcacaggt gcacaaatgg
ctttctcaat tggtgctagt 780 ttgattgcct ttgttggttt agtttctttg
attaatatga tgttaagtgg attgggaatc 840 cgcttagaac aaatcttttc
atatgttttt gctccatttg gttttcttat gggatttgac 900 cacaaaaaca
ttcttctaga aggaaacctt cttggaagta agttgatttt aaatgagttt 960
gtttcgttcc aacaattggg tcacctaatc aaatctttag attatcgtac agcattggta
1020 gcaactattt cactctgtgg ttttgctaat ttatcaagtt taggtatttg
tgtttcaggt 1080 attgctgttc tttgcccgga gaaacgtagc accctagctc
gacttgtttt ccgtgcaatg 1140 attggtggta ttgctgtaag tatgcttagc
gcctttatcg tcggtattgt aactctattc 1200 taa 1203 <210> SEQ ID
NO 121 <211> LENGTH: 1257 <212> TYPE: DNA <213>
ORGANISM: Vibrio cholerae O1 biovar El Tor N16961 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1257) <223> OTHER INFORMATION: nupC (VC2352) - Vibrio
cholerae O1 biovar El Tor N16961 (AAF95495) <400> SEQUENCE:
121 atgagcctgt ttatgagcct catcggcatg gcagttctgc taggaatcgc
agttctactg 60 tcaagtaacc gtaaagctat caatctaaga actgtgggtg
gcgcttttgc tatccaattt 120 tcactgggtg catttattct gtatgtgcct
tggggccaag agctacttcg tggcttttcg 180 gatgccgtat cgaatgttat
taactacggt aacgatggta cttcattcct cttcggtgga 240 ctggtatcag
gcaaaatgtt tgaagtgttt ggcggcggcg gtttcatttt cgcattccgc 300
gtactaccaa cactgatctt cttctcagca ctgatttctg tactgtacta cttgggtgtt
360 atgcaatggg ttatccgcat tcttggcggt ggtctgcaaa aagcactggg
tacatcacgc 420 gcggaatcta tgtctgcggc tgcaaacatt ttcgtgggtc
aaactgaagc accattagtt 480 gttcgtccat tcgttccaaa aatgactcaa
tctgagctgt ttgcggtaat gtgtggtggc 540 ttggcttcta tcgcaggtgg
tgtacttgcg ggttacgctt caatgggcgt taagatcgaa 600 tacttggtag
cggcgtcatt catggcggca ccgggtggtc tgctgttcgc aaaactgatg 660
atgcctgaaa ctgaaaaacc acaagacaat gaagacatta ctcttgatgg tggtgacgac
720 aaaccggcta acgttatcga tgcggctgct ggcggtgctt ctgctggtct
gcaacttgct 780 ctgaacgttg gtgcaatgtt gattgccttt atcggtttga
ttgctctgat caacggtatg 840 ttgggtggca tcggtggttg gttcggtatg
cctgaactga aactggaaat gctactgggc 900 tggttgtttg cgcctctggc
tttcctgatc ggtgttcctt ggaacgaagc aactgttgcg 960 ggtgagttca
tcggtctaaa aaccgttgct aacgaattcg ttgcttactc tcagtttgcg 1020
ccttacctga ctgaagcggc accagtggtt ctgtctgaga aaaccaaagc gatcatctct
1080 ttcgctctgt gtggttttgc gaacctttct tctatcgcaa ttctgcttgg
tggtttgggt 1140 agcttggcac ctaagcgtcg tggcgacatc gctcgtatgg
gggtcaaagc ggttatcgca 1200 ggtactctat ctaacctgat ggcagcgacc
atcgctggct tcttcctctc tttctaa 1257 <210> SEQ ID NO 122
<211> LENGTH: 1218 <212> TYPE: DNA
<213> ORGANISM: Vibrio cholerae O1 biovar El Tor N16961
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1218) <223> OTHER INFORMATION: nupC (VC1953)
- Vibrio cholerae O1 biovar El Tor N16961 (AAF95101) <400>
SEQUENCE: 122 ttgggcggcg ttatgtcatc actcctcggt atgggcgcaa
ttttgctggt tgcgtggcta 60 ttttctacca atagaaaaaa tatcaacttg
cgtacagttt ctttagcgtt actgctgcaa 120 atcttcttcg ccttactggt
gctgtatgta cctgcgggta aagaggcact caatcgtgtg 180 acgggcgcgg
tgtcacaact gatcaactat gggcaagatg gtatcggttt tgtgtttggt 240
ggcctcgcca atggcagcgt aggttttgtg tttgcgatta atgtccttgg catcatcatt
300 ttcttctctg cactgatttc tggcctttac catttaggca tcatgccgaa
agtgattaac 360 ctcatcggtg gtggtttaca gaaattgctt ggcacaggcc
gtgcagaatc cctttctgct 420 accgcaaaca ttttcgtggg tatgattgaa
gcgccgctgg tggtgaaacc ttatcttcat 480 aaaatgaccg attcgcaatt
ctttgcagtg atgacgggcg gcttagcgtc ggttgctggc 540 ggtactttgg
ttggttatgc ctctttaggt gtggaattga actatctgat cgcggcggct 600
ttcatgtctg cccctgcggg tcttttgatg gcaaaaatca tgttgccaga aaccgaacac
660 gtcgatgccg cgattgcgca agatgagttg gatctgccga aatccactaa
cgtcgtcgaa 720 gcgattgcgg atggcgcgat gtcgggtgtg aaaattgctg
ttgcggtagg ggcgactttg 780 ctcgctttcg tgagtgtgat tgctctgtta
aacggcttgc tcggttggtt tggtggctgg 840 tttggcatcg agctaagctt
tgaactgatc atggggtatg ttttcgctcc ggtagcttgg 900 ctgattggta
ttccatggca tgaggcgatc acggcaggct cgctgattgg taacaaagtg 960
gtggtgaacg agtttgtcgc tttcattcaa ctgattgaag tgaaagagca attgagtgcg
1020 cattcacaag cgatcgtgac tttcgcgctg tgcggttttg cgaatatttc
taccatggcg 1080 attttgattg gtggtttggg tagccttgta cctgaacgtc
gctcttttat ctcccaatac 1140 ggcttccgtg cgattggcgc aggcgtatta
gctaacctaa tgagtgcatc gatcgctgga 1200 gtgattttgt ctttgtga 1218
<210> SEQ ID NO 123 <211> LENGTH: 1209 <212>
TYPE: DNA <213> ORGANISM: Vibrio cholerae O1 biovar El Tor
N16961 <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(1209) <223> OTHER INFORMATION:
nupC (VCA0179) - Vibrio cholerae O1 biovar El Tor N16961 (AAF96092)
<400> SEQUENCE: 123 atggcgattt tgtttggaat catcggtgtt
acggtactga tcttatgcgc gtatctgctc 60 tctgaaagcc gcagtgcgat
taattggaaa accatttccc gagccttgtt gttgcaaatt 120 ggttttgcgg
ctcttgtgct ttatttccca ttggggcaaa ccgcgctaag cagcttgagt 180
aatggggttt ctggtttgct tggttttgcc gatgtcggca ttcgctttct gtttggtgat
240 cttgccgata cgggctttat ttttgctgtt cgtgtattac ctatcatcat
cttcttcagt 300 gcgctgattt ctgcccttta ttaccttggt gtgatgcaaa
aagtgatcgc cctgatcggc 360 ggtggcattc aacgcttctt aggcaccagt
aaggcggaat cactggtcgc gacaggcaat 420 attttcctat cacaaggcga
atcgccactt ttggtgcgcc ccttccttgc caatatgaca 480 cgctctgaac
tgtttgcggt catggcgggc ggtatggcat cggtagcagg ctctgtgctg 540
ggtggttacg caggtttagg ggttgagctg aaatacctga ttgcagcgag tttcatggcg
600 gcgccgggca gtttaatgat ggcgaaaatc atcgttcctg agcgtggtgt
gccaatcgat 660 caaagccaag tcgagttaga taaagcgcaa gacagcaact
tgattgatgc tctcgctagc 720 ggtgcgatga atggtatgaa agtcgccgtt
gcagtgggca ctatgttgat tgcgttcgtc 780 agcgtgatcg ctatggtcaa
cactggcctt gaaaatctgg gcgatctggt tgggtttagc 840 ggcattacct
tacaagccat gttcggttat ctgtttgctc ctctggcatg ggtgattggc 900
attccaagtc acgaagtgct ggcggcaggt tcctacatcg gtcagaaagt ggtgatgaac
960 gaatttgtgg ctttcattga ctttgttgag cataaagcgc tgctttctga
gcatagccaa 1020 gtcatcatca cgtttgcatt gtgtggcttt gccaacattg
gctctatcgc gatccaatta 1080 ggctccattg gcgtgatagc ccctgagcgc
cgctcggaag tggcgaacct aggcataaaa 1140 gcggtcattg ctggcacttt
agccaaccta atgagcgctt gcttagcggg gattttcatc 1200 tcgctataa 1209
<210> SEQ ID NO 124 <211> LENGTH: 1278 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1278) <223> OTHER INFORMATION: yegT - Escherichia coli
K-12 W3110 (P76417; JW2085) <400> SEQUENCE: 124 atgaaaacaa
cagcaaagct gtcgttcatg atgtttgttg aatggtttat ctggggcgcg 60
tggtttgtgc cattgtggtt gtggttaagt aaaagcggtt ttagtgccgg agaaattggc
120 tggtcgtatg cctgtaccgc cattgcggcg atcctgtcgc caattctggt
tggctccatc 180 actgaccgct ttttctcggc gcaaaaagtg ctggcggtat
tgatgttcgc aggcgcgctg 240 ctgatgtatt tcgctgcgca acagaccact
tttgccgggt tcttcccgtt actgctggcc 300 tactcgctaa cctatatgcc
gaccattgcg ctgactaaca gcatcgcttt tgccaacgtg 360 ccggatgttg
agcgtgattt cccgcgcatt cgtgtgatgg gcactatcgg ctggattgcc 420
tccggtctgg catgtggttt cttgccgcaa atactggggt atgccgatat ctcaccgact
480 aacatcccgc tgctgattac cgccggaagt tctgctctgc tcggtgtgtt
tgcgtttttc 540 ctgcccgaca cgccaccaaa aagcaccggc aaaatggata
ttaaagtcat gctcggcctg 600 gatgcgctga tcctgctgcg cgataaaaac
ttcctcgtct ttttcttctg ttcattcctg 660 tttgcgatgc cactagcgtt
ctattacatc tttgccaacg gttatctgac cgaagttggc 720 atgaaaaacg
ccaccggctg gatgacgctc ggccagttct ctgaaatctt ctttatgctg 780
gcattgccgt ttttcactaa acgctttggt atcaaaaagg tattattgct tggtctggtc
840 accgctgcga tccgctatgg cttctttatt tacggtagtg cggatgaata
tttcacctac 900 gcgttactgt tcctcggtat tttgcttcac ggcgtaagtt
acgattttta ctacgttacc 960 gcttacatct atgtcgataa aaaagccccc
gtgcatatgc gtaccgctgc gcaggggctg 1020 atcacgctct gctgccaggg
cttcggcagt ttgctcggct atcgtcttgg cggtgtgatg 1080 atggaaaaga
tgttcgctta tcaggaaccg gtaaacggac tgactttcaa ctggtccggg 1140
atgtggactt tcggcgcggt gatgattgcc attatcgccg tgctgttcat gatttttttc
1200 cgcgaatccg acaacgaaat tacggctatc aaggtcgatg atcgcgatat
tgcgttgaca 1260 caaggggaag ttaaatga 1278 <210> SEQ ID NO 125
<211> LENGTH: 1257 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1257)
<223> OTHER INFORMATION: nupG - Escherichia coli K-12 W3110
(P09452; JW2932) <400> SEQUENCE: 125 atgaatctta agctgcagct
gaaaatcctc tcttttctgc agttctgtct gtggggaagt 60 tggctgacga
ccctcggctc ctatatgttt gttaccctga agtttgacgg tgcttctatt 120
ggcgcagttt atagctcact gggtatcgca gcggtcttta tgcctgcgct gctggggatt
180 gtggccgaca aatggttaag tgcgaaatgg gtatatgcca tttgccacac
cattggcgct 240 atcacgctgt tcatggcggc acaggtcacg acaccggaag
cgatgttcct tgtgatattg 300 attaactcgt ttgcttatat gccaacgctt
gggttaatca acaccatctc ttactatcgc 360 ctgcaaaatg ccgggatgga
tatcgttact gacttcccgc caatccgtat ctggggcacc 420 atcggcttta
tcatggcaat gtgggtggtg agcctgtctg gcttcgaatt aagccacatg 480
cagctgtata ttggcgcagc actttccgcc attctggttc tgtttaccct gactctgccg
540 catattccgg ttgctaaaca gcaagcgaat cagagctgga caaccctgct
gggcctcgat 600 gcattcgcgc tgtttaaaaa caagcgtatg gcaatcttct
ttatcttctc aatgctgctg 660 ggcgcggaac tgcagattac caacatgttc
ggtaatacct tcctgcacag cttcgacaaa 720 gatccgatgt ttgccagcag
ctttattgtg cagcatgcgt caatcatcat gtcgatttcg 780 cagatctctg
aaaccctgtt cattctgacc atcccgttct tcttaagccg ctacggtatt 840
aagaacgtaa tgatgatcag tattgtggcg tggatcctgc gttttgcgct gtttgcttac
900 ggcgacccga ctccgttcgg tactgtactg ctggtactgt cgatgatcgt
ttacggttgc 960 gcattcgact tcttcaacat ctctggttcg gtgtttgtcg
aaaaagaagt tagcccggca 1020 attcgcgcca gtgcacaagg gatgttcctg
atgatgacta acggcttcgg ctgtatcctc 1080 ggcggcatcg tgagcggtaa
agttgttgag atgtacaccc aaaacggcat taccgactgg 1140 cagaccgtat
ggttgatttt cgctggttac tccgtggttc tggccttcgc gttcatggcg 1200
atgttcaaat ataaacacgt tcgtgtcccg acaggcacac agacggttag ccactaa 1257
<210> SEQ ID NO 126 <211> LENGTH: 1257 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1257) <223> OTHER INFORMATION: xapB - Escherichia coli
K-12 W3110 (P45562; JW2397) <400> SEQUENCE: 126 atgagcatcg
cgatgcgctt aaaggtaatg tcctttttgc aatattttat ctgggggagc 60
tggctggtta ccctcggctc ttacatgatt aatactcttc atttcaccgg cgctaatgtt
120 ggcatggttt acagttccaa agggatcgcc gcgattatta tgcctggtat
aatggggatc 180 atcgcagaca aatggctgcg cgcagaacgt gcatacatgc
tgtgtcacct ggtgtgtgcg 240 ggcgtacttt tttatgcggc atccgtaact
gatccggata tgatgttttg ggtgatgtta 300 gtcaatgcga tggcgtttat
gccgactatt gcgttatcga acagcgtctc ttattcctgt 360 cttgcccagg
cagggcttga cccggtgacc gctttcccgc ccattcgcgt ttttggtacg 420
gtggggttca ttgtcgcgat gtgggcagta agcctgctgc atctggaatt gagtagtctg
480
cagctgtata tcgcgtccgg tgcgtcattg ctgctgtcgg cttatgcgct gactttgccg
540 aagattccgg ttgcggagaa aaaagcgacc acatcgcttg ccagcaagct
gggtctggat 600 gccttcgtgc tgtttaaaaa tccacgcatg gccatctttt
tcctctttgc catgatgctg 660 ggtgcggtac tgcaaattac caacgttttt
ggtaatccgt tcctacatga tttcgcccgt 720 aacccggagt ttgctgacag
ttttgtggtg aaatatccct ccattttact gtcagtttca 780 cagatggcag
aagtgggctt tatactgact atcccattct ttttaaagcg atttggcatt 840
aaaaccgtca tgctgatgag tatggtggcc tggacgctgc gctttggctt cttcgcctat
900 ggcgatccgt caacaaccgg atttattttg ctgctgctgt cgatgattgt
ttatggctgt 960 gcattcgatt tcttcaatat ttctggttcg gtatttgtcg
aacaggaagt tgattccagc 1020 attcgtgcca gcgcgcaggg gctctttatg
accatggtaa atggtgtcgg cgcatgggtt 1080 ggctcgattc tgagtggcat
ggcagtagat tacttttcgg tggatggcgt aaaagactgg 1140 caaactatct
ggctggtgtt tgcaggatat gctctttttc tcgcagtgat atttttcttt 1200
gggtttaaat ataatcatga ccctgaaaag ataaagcatc gagcggtgac tcattaa 1257
<210> SEQ ID NO 127 <211> LENGTH: 1242 <212>
TYPE: DNA <213> ORGANISM: Caulobacter crescentus CB15
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1242) <223> OTHER INFORMATION: CC1628 -
Caulobacter crescentus CB15 (AAK23606) <400> SEQUENCE: 127
atggggacga gtttccgtct gttcgtgatg atggtgctgc agctggcgat ctggggcgcc
60 tgggcgccca agatcttccc ctacatgggc atgctgggct tcgcgccctg
gcagcagtcg 120 ctggtcggca gcgcctgggg cgtggcggcg ctggtgggca
tcttcttctc gaatcagttc 180 gccgaccgga acttctcggc cgagcggttc
ctggcggtca gccacctgat cggcggcgtg 240 gcgctgctgg gcacggcctt
ctcgacggag ttctggccgt tctttgcctg ttacctcgtt 300 ttcagcctgg
tctatgtgcc gacgctgtcg gtcaccaact cgatcgcctt cgccaatctg 360
cgcgatccgg cggccggctt cggcggggtg cggatgggcg gaaccgtcgg ctgggtgctg
420 gtcagctggc ccttcgtgtt cctgctgggc gcccaagcga cggtggagca
ggtccgctgg 480 atcttcctgg tggcggcgat cgtctccttc gttttcgccg
gttacgctct gaccctgccg 540 cacacgccgc cgcgcaaggc cgatgacgct
gtcgacaagc tggcctggcg acgggcgttc 600 aagctactgg gcgcgccctt
cgtgtttgtc ctctttgtcg tgaccttcat cgattccgtg 660 atccacaacg
gctacttcgt gatggccgac gccttcctga ccaaccgggt cgggatcgcg 720
ggcaatctca gcatggtcgt gctgagcctg ggccaggtgg ccgaaatcat caccatgctg
780 ctgttgggcc gcgtgctggc caagctgggc tggaaggtca ccatgatcgt
cggcgtgctg 840 ggccacgccg cgcgctttgc ggtcttcgcc tacttcgccg
acagcgtgcc ggtcatcgtg 900 gcggtgcagc tgctgcacgg cgtctgctac
gccttcttct tcgccacggt ttacatcttc 960 gtcgacgccg tcttcccgaa
agatgtccgc tccagcgcgc agggtctgtt caacttgctg 1020 atcctgggcg
tcggcaatgt ggccgccagc ttcatcttcc ccgcgctgat cggtcgcctg 1080
accaccgatg ggtccgtcga ctacacgacg ctgttcctcg tgccgaccgc catggctttg
1140 gcggcggtct gcctgctggc gctgttcttc cggccgccca cgcggggacc
tgtttcggag 1200 gcggattccg cttcatccgc cgccagttcg gcccaagcct ag 1242
<210> SEQ ID NO 128 <211> LENGTH: 1260 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1260) <223> OTHER INFORMATION: codB - Escherichia coli
K-12 W3110 (P25525; JW0327) <400> SEQUENCE: 128 gtgtcgcaag
ataacaactt tagccagggg ccagtcccgc agtcggcgcg gaaaggggta 60
ttggcattga cgttcgtcat gctgggatta accttctttt ccgccagtat gtggaccggc
120 ggcactctcg gaaccggtct tagctatcat gatttcttcc tcgcagttct
catcggtaat 180 cttctcctcg gtatttacac ttcatttctc ggttacattg
gcgcaaaaac cggcctgacc 240 actcatcttc ttgctcgctt ctcgtttggt
gttaaaggct catggctgcc ttcactgcta 300 ctgggcggaa ctcaggttgg
ctggtttggc gtcggtgtgg cgatgtttgc cattccggtg 360 ggtaaggcaa
ccgggctgga tattaatttg ctgattgccg tttccggttt actgatgacc 420
gtcaccgtct tttttggcat ttcggcgctg acggttcttt cggtgattgc ggttccggct
480 atcgcctgcc tgggcggtta ttccgtgtgg ctggctgtta acggcatggg
cggcctggac 540 gcattaaaag cggtcgttcc cgcacaaccg ttagatttca
atgtcgcgct ggcgctggtt 600 gtggggtcat ttatcagtgc gggtacgctc
accgctgact ttgtccggtt tggtcgcaat 660 gccaaactgg cggtgctggt
ggcgatggtg gcctttttcc tcggcaactc gttgatgttt 720 attttcggtg
cagcgggcgc tgcggcactg ggcatggcgg atatctctga tgtgatgatt 780
gctcagggcc tgctgctgcc tgcgattgtg gtgctggggc tgaatatctg gaccaccaac
840 gataacgcac tctatgcgtc gggtttaggt ttcgccaaca ttaccgggat
gtcgagcaaa 900 accctttcgg taatcaacgg tattatcggt acggtctgcg
cattatggct gtataacaat 960 tttgtcggct ggttgacctt cctttcggca
gctattcctc cagtgggtgg cgtgatcatc 1020 gccgactatc tgatgaaccg
tcgccgctat gagcactttg cgaccacgcg tatgatgagt 1080 gtcaattggg
tggcgattct ggcggtcgcc ttggggattg ctgcaggcca ctggttaccg 1140
ggaattgttc cggtcaacgc ggtattaggt ggcgcgctga gctatctgat ccttaacccg
1200 attttgaatc gtaaaacgac agcagcaatg acgcatgtgg aggctaacag
tgtcgaataa 1260 <210> SEQ ID NO 129 <211> LENGTH: 1656
<212> TYPE: DNA <213> ORGANISM: Corynebacterium sp.
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1656) <223> OTHER INFORMATION: mctC
Corynebacterium <400> SEQUENCE: 129 atgaattcca ctattctcct
tgcacaagac gctgtttctg agggcgtcgg taatccgatt 60 cttaacatca
gtgtcttcgt cgtcttcatt attgtgacga tgaccgtggt gcttcgcgtg 120
ggcaagagca ccagcgaatc caccgacttc tacaccggtg gtgcttcctt ctccggaacc
180 cagaacggtc tggctatcgc aggtgactac ctgtctgcag cgtccttcct
cggaatcgtt 240 ggtgcaattt cactcaacgg ttacgacgga ttcctttact
ccatcggctt cttcgtcgca 300 tggcttgttg cactgctgct cgtggcagag
ccacttcgta acgtgggccg cttcaccatg 360 gctgacgtgc tgtccttccg
actgcgtcag aaaccagtcc gcgtcgctgc ggcctgcggt 420 accctcgcgg
ttaccctctt ttacttgatc gctcagatgg ctggtgcagg ttcgcttgtg 480
tccgttctgc tggacatcca cgagttcaag tggcaggcag ttgttgtcgg tatcgttggc
540 attgtcatga tcgcctacgt tcttcttggc ggtatgaagg gcaccacata
cgttcagatg 600 attaaggcag ttctgctggt cggtggcgtt gccattatga
ccgttctgac cttcgtcaag 660 gtgtctggtg gcctgaccac ccttttaaat
gacgctgttg agaagcacgc cgcttcagat 720 tacgctgcca ccaaggggta
cgatccaacc cagatcctgg agcctggtct gcagtacggt 780 gcaactctga
ccactcagct ggacttcatt tccttggctc tcgctctgtg tcttggaacc 840
gctggtctgc cacacgttct gatgcgcttc tacaccgttc ctaccgccaa ggaagcacgt
900 aagtctgtga cctgggctat cgtcctcatt ggtgcgttct acctgatgac
cctggtcctt 960 ggttacggcg ctgcggcact ggtcggtcca gaccgcgtca
ttgccgcacc aggtgctgct 1020 aatgctgctg ctcctctgct ggccttcgag
cttggtggtt ccatcttcat ggcgctgatt 1080 tccgcagttg cgttcgctac
cgttctcgcc gtggtcgcag gtcttgcaat taccgcatcc 1140 gctgctgttg
gtcacgacat ctacaacgct gttatccgca acggtcagtc caccgaagcg 1200
gagcaggtcc gagtatcccg catcaccgtt gtcgtcattg gcctgatttc cattgtcctg
1260 ggaattcttg caatgaccca gaacgttgcg ttcctcgtgg ccctggcctt
cgcagttgca 1320 gcatccgcta acctgccaac catcctgtac tccctgtact
ggaagaagtt caacaccacc 1380 ggcgctgtgg ccgctatcta caccggtctc
atctccgcgc tgctgctgat cttcctgtcc 1440 ccagcagtct ccggtaatga
cagcgcaatg gttccaggtg cagactgggc aatcttccca 1500 ctgaagaacc
caggcctcgt ctccatccca ctggcattca tcgctggttg gatcggcact 1560
ttggttggca agccagacaa catggatgat cttgctgccg aaatggaagt tcgttccctc
1620 accggtgtcg gtgttgaaaa ggctgttgat cactaa 1656 <210> SEQ
ID NO 130 <211> LENGTH: 1521 <212> TYPE: DNA
<213> ORGANISM: Virgibacillus sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1521) <223> OTHER INFORMATION: putP_6 - Virgibacillus
sp. <400> SEQUENCE: 130 atggatctta cgacattaat aacttttata
gtatatctac tagggatgtt ggcgattggc 60 ctcatcatgt attatcgaac
caataattta tcagattatg ttcttggtgg acgtgatctt 120 ggtccaggcg
tagctgcatt gagtgctggt gcatcggata tgagtggttg gctgttatta 180
ggtttgcctg gagcgattta tgcatctggt atgtctgaag cttggatggg gatcgggtta
240 gctgtaggtg cttatttaaa ttggcaattt gtagctaagc gattacgcgt
ttataccgag 300 gtatcaaata attccattac gatcccagat tattttgaaa
atcggtttaa agataactca 360 catattcttc gtgttatatc tgctatcgta
attttgttat tcttcacttt ttatacatct 420 tcaggaatgg ttgcaggagc
aaaattattt gaggcttcat tcggtctcca atacgaaact 480 gctctgtgga
ttggtgcggt tgtagttgta tcttatacgt tacttggagg atttctagcg 540
gttgcatgga cagactttat tcaaggtatt cttatgttcc ttgcactaat tgttgttcca
600 atcgtcgcat tagatcaaat gggtggctgg aatcaagcgg tacaagctgt
tggtgaaatt 660 aatccttccc acctcaatat ggttgaaggt gttggaataa
tggcaattat ttcatcactt 720 gcttggggct taggttattt tggacagcca
catattattg ttcgttttat ggcattacgt 780 tcggcgaaag atgttccgaa
agcgaaattt attggaacag cttggatgat tttaggactt 840 tatggagcaa
tctttactgg ttttgtagga ctagcattta tcagtacaca agaagtaccg 900
attctgtctg aattcgggat tcaagtagtt aatgagaatg gtttacaaat gttagccgat
960 cctgaaaaga tatttattgc tttctcccaa atactattcc atccagtagt
tgccggtatc 1020
ttactagcgg caatcttgtc tgcaattatg agtaccgttg attcacagtt acttgtatca
1080 tcttcagcgg ttgcagaaga tttctataaa gctattttcc gtaaaaaagc
tactggtaaa 1140 gagcttgttt gggttggacg tattgctaca gtgataattg
cgattgttgc tttaattatt 1200 gcaatgaacc cagatagctc tgtattggat
ctagttagtt atgcatgggc tggatttggt 1260 gcagcatttg gaccaattat
catcttgtca ttattctgga agagaatcac aagaaatggt 1320 gcactagcgg
gtatcattgt aggtgccatt acggtaattg tatggggaga ctttctatct 1380
ggaggtatct ttgacctcta cgaaattgtt ccaggcttta tcttaaatat gattgtcacc
1440 gttattgtga gtcttatcga taaaccgaat ccagatttag aagctgactt
tgatgaaacc 1500 gtagaaaaaa tgaaagaata a 1521 <210> SEQ ID NO
131 <211> LENGTH: 1323 <212> TYPE: DNA <213>
ORGANISM: Lactobacillus johnsonii <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1323)
<223> OTHER INFORMATION: cbsT1 - Lactobacillus johnsonii
<400> SEQUENCE: 131 atgtcgacca caccgacaca gccatcatca
cgaaaacagg ctgtttaccc gtacttgatc 60 gtgctgtcgg gcatcgtctt
cacggccatc ccggtatcgc tggtctgcag ttgcgcaggt 120 atcttcttca
cgcctgtcag cagctacttc catgttccca aggccgcatt caccggatat 180
ttcagcatat tcagcatcac catggtcgcc ttcctgccgg tggccggatg gctgatgcac
240 cgctacgatc tgcgcatcgt actgaccgca agcaccgtcc tggctggact
gggctgcctg 300 ggtatgtccc gatcatccgc catgtggcag ttctatctat
gcggagtggt tctgggaatc 360 ggcatgccgg ccgtcctcta tctgtcagtg
ccaacactca tcaacgcctg gttccgcaag 420 cgggtcgggt tcttcatcgg
cctgtgcatg gccttcaccg gcataggcgg cgtgatcttc 480 aaccagatag
gcaccatgat catcagatcc gcccctgatg gatggaggcg gggatatctg 540
gttttcgcta ttctcatcct ggtgatcacc ctgcccttca ccattttcgt cattcgcagc
600 acacccgaac agatgggtct gcatccctac ggcgccgacc aggagcctga
tgcagctgag 660 acggccacca atagtgcagg caccgggagc aaagaccaaa
agagtcctga gcctgcagcg 720 tcaaccgtag gcatgactgc ctcccaggcc
ttgcgctccc ctgccttctg ggcgctggcg 780 ctcttctgcg gtctgatcac
catgaatcag accatttacc agttcctgcc ctcctacgcg 840 gcatccctgc
catccatggc agcctacacg ggactgatcg cctcctcctg catggccggc 900
caggccatcg gcaagatcat cctgggcatg gtcaacgacg gcagcatcgt aggcggtctc
960 tgtctgggca tcggcggcgg cattctcggc gtctgcctca tggtcgcctt
ccccggattg 1020 cccgtgctcc tcctgctggg agcctttgcc ttcggccttg
tctacgcctg cactactgtg 1080 cagacaccaa tcctggttac agcggtcttc
ggctcgcgcg actacaccaa catctatgca 1140 cgtatccaga tggttgggtc
cctagcctcg gccttcgcag ctctcttctg gggcgccatc 1200 gctgaccagc
cccacggcta catcatcatg ttcggtctga gcatcctgat catggttgtg 1260
gccttgttcc taggcattat ccctctgaaa ggtacgcgca agttgaccga tcagatcgcc
1320 tga 1323 <210> SEQ ID NO 132 <211> LENGTH: 1356
<212> TYPE: DNA <213> ORGANISM: Lactobacillus johnsonii
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1356) <223> OTHER INFORMATION: cbsT2 -
Lactobacillus johnsonii <400> SEQUENCE: 132 atgtctactg
atgccgctac taaagataaa gtagtaagca agggctataa atacttcatg 60
gttttccttt gtatgttaac ccaagctatt ccttatggaa ttgctcaaaa cattcagcct
120 ttgtttatcc accctttagt taatactttc cactttacct tagcatcgta
cacattaatt 180 tttacgtttg gtgcggtttt tgcttcagtt gcttctccat
ttattggtaa ggcattagaa 240 aaagttaact tccgactaat gtatttaatt
ggtattggtc tttctgctat tgcctacgta 300 atttttggaa ttagtacaaa
actacccggt ttctatattg ccgctatcat ttgtatggtt 360 ggttcaacct
tttactccgg ccaaggtgtt ccctgggtta ttaaccactg gttcccagca 420
aagggacgtg gggctgcctt aggaattgcc ttctgcggtg gttctattgg taatatcttt
480 ttacaaccag caacccaagc tattttaaaa cactacatga caggtaatac
taagaccggt 540 catttaacct ctatggcacc attctttatc tttgccgtag
ctttattagt aatcggtgta 600 attatcgcct gcttcattag aacccctaag
aaagacgaaa ttgttgtttc tgatgcagaa 660 ctagctgaaa gcaagaaagc
tgaagccgca gccaaagcta aagagtttaa aggctggact 720 agtaaacaag
tgttacaaat gaaatggttc tggattttca gccttggttt cttaatcatt 780
ggtttaggct tagcttcttt aaatgaagac tatgccgcct tccttgatac taagctttct
840 ttaaccgatg ttggtttagt tgggtcaatg tacggtgttg gttgtttaat
cggaaatatt 900 tctggtggtt tcttatttga taaatttggt acagcaaaat
caatgaccta tgctggttgt 960 atgtatattt tatctattct gatgatgatc
tttattagct tccagccata tggttcatct 1020 attagtaagg ctgctggcat
tggctatgct atcttttgcg gcttagctgt atttagttac 1080 atgtcaggcc
cagccttcat ggcaaaagac ctctttggtt caagagatca aggtgtcatg 1140
cttggatacg ttggtttagc ttatgcaatt ggctatgcca ttggtgctcc actatttggg
1200 attattaagg gagcggcaag ctttacagtt gcttggtact ttatgattgc
ctttgttgca 1260 attggtttta tcattttagt atttgccgtt atccaaatta
agagatacca aaagaaatac 1320 attgcagagc aagcagcaaa agctaatgct aaataa
1356 <210> SEQ ID NO 133 <211> LENGTH: 1287 <212>
TYPE: DNA <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1287) <223> OTHER INFORMATION: amtB - Escherichia coli
K-12 MG1655 (B0451; 945084) <400> SEQUENCE: 133 atgaagatag
cgacgataaa aactgggctt gcttcactgg cgatgcttcc gggactggta 60
atggctgcac ctgcggtggc cgataaagcc gacaatgcgt ttatgatgat ttgtactgcg
120 ctggtgctgt ttatgactat tccggggatt gccctgtttt acggtgggtt
gattcgcggc 180 aaaaacgtgc tgtcgatgct gacgcaggtg acggtgacat
ttgcactggt ctgtattctc 240 tgggtggttt acggttactc gctggcgttt
ggtgagggca acaacttctt cggcaacatt 300 aactggttga tgctgaaaaa
catcgaactg acggcggtga tgggcagcat ttatcagtat 360 atccacgtgg
cgtttcaggg atcgtttgcc tgcattaccg tcggcttgat agttggggcg 420
ctggcggaac gaatccgctt ctcagctgtg ttgattttcg tggtggtatg gctgacgctc
480 tcttacattc cgattgcgca tatggtgtgg ggcggtggtt tgctggcttc
tcacggtgcg 540 ctggatttcg cgggtggcac cgtggtgcac attaacgccg
caatcgccgg tctggtgggc 600 gcgtatctga taggaaaacg cgtgggcttc
ggtaaagagg cgtttaaacc gcacaacctg 660 ccgatggtct tcaccgggac
tgccattctc tatatcggtt ggtttggctt taacgccggg 720 tcagcgggca
cggcgaatga aatcgcggca ctggcatttg tgaatactgt ggtcgcaacg 780
gcggcggcaa ttcttggctg gatcttcggt gaatgggcgc tgcgtggtaa gccttcactg
840 ctgggggcgt gttctggcgc gattgccggt ctggtcggcg tgacgccagc
ctgcggctac 900 attggggttg gcggcgcgtt gattatcggc gtggtagctg
gtctggcggg cttgtggggc 960 gttaccatgc tcaaacgctt gctgcgggtg
gatgatccct gcgatgtctt cggtgtgcac 1020 ggcgtttgtg gcattgtcgg
ctgtatcatg accgggattt ttgccgccag ctcgctgggc 1080 ggcgtgggct
tcgctgaagg tgtgacgatg ggccatcagt tgctggtaca gctggaaagc 1140
atcgccatta cgatcgtctg gtccggtgtt gtggcattta tcggctacaa attggcggat
1200 ctgacggttg gtctgcgtgt accggaagag caggagcgag aagggctgga
tgtcaacagc 1260 cacggcgaga atgcctataa cgcgtaa 1287 <210> SEQ
ID NO 134 <211> LENGTH: 1401 <212> TYPE: DNA
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1401) <223> OTHER INFORMATION: GABA permease GabP
Escherichia coli <400> SEQUENCE: 134 atggggcaat catcgcaacc
acatgagtta ggcggcgggc tgaagtcacg ccacgtcacc 60 atgttgtcta
ttgccggtgt tatcggcgca agtctgtttg tcggttccag cgtcgccatc 120
gccgaagcgg gcccggcggt attactggcc tatctgttcg ccggattact ggtggttatg
180 attatgcgga tgttggcgga aatggcagtt gccacgcccg ataccggttc
gttttccacc 240 tatgccgata aagccattgg ccgctgggcg ggttatacca
tcggctggct gtactggtgg 300 ttttgggtac tggttatccc gctggaagcc
aacatcgccg ctatgatcct gcactcgtgg 360 gttccaggca ttcccatctg
gttattttcc ctcgtcatta ccctcgcctt aactggcagt 420 aatttattaa
gcgttaaaaa ctacggcgaa tttgagttct ggctggcgct gtgcaaagtc 480
atcgctatcc tggcctttat tttccttggt gcagtcgcaa ttagcggttt ttacccttat
540 gccgaagtga gcgggatctc aagattgtgg gatagcggcg gctttatgcc
caacggtttc 600 ggtgcggtat taagcgcgat gttgatcacc atgttctcgt
ttatgggcgc agaaattgtc 660 accattgccg ccgcggaatc cgacacgccg
gaaaaacata ttgtccgcgc cactaactcg 720 gttatctggc gtatttctat
cttctatttg tgctctattt ttgtcgtagt ggcgttaata 780 ccgtggaata
tgccggggct gaaagccgtt ggttcttatc gctcggttct ggaattgctc 840
aatattcccc atgcgaaatt aatcatggac tgcgtgatat tactttccgt aaccagctgt
900 ctgaactcgg cgctgtatac cgcgtcaagg atgctctact ccttaagccg
tcgcggtgat 960 gcgcccgcgg taatgggcaa aatcaaccgc agtaaaaccc
cgtatgtggc ggtgttactc 1020 tccaccggag cggcattttt aacggtggtg
gtgaactatt acgcacctgc gaaagtgttt 1080 aaattcctga tagacagctc
cggtgctatc gccctgctgg tttatttagt catcgccgtt 1140 tcacagttgc
ggatgcgtaa aattctgcga gcagaaggaa gcgaaattcg cttgcgcatg 1200
tggctttacc cgtggctcac ctggctggta ataggcttta ttacctttgt gttggtagtg
1260 atgctattcc gcccggcgca acagttagaa gtgatctcta ccggcttatt
agcgataggg 1320 attatctgta ccgtgccgat tatggcgcgc tggaaaaagc
tggtattgtg gcaaaaaaca 1380
cccgttcata atacgcgctg a 1401 <210> SEQ ID NO 135 <211>
LENGTH: 1239 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(1239) <223> OTHER
INFORMATION: mtnH Escherichia coli <400> SEQUENCE: 135
atgacgaact atcgcgttga gagtagcagc ggacgggcgg cgcgcaagat gaggctcgca
60 ttaatgggac ctgcgttcat tgcggcgatt ggttatatcg atcccggtaa
ctttgcgacc 120 aatattcagg cgggtgccag cttcggctat cagctactgt
gggttgtcgt ttgggccaac 180 ctgatggcga tgctgattca gatcctctct
gccaaactag ggattgccac cggtaaaaat 240 ctggcggagc agattcgcga
tcactatccg cgtcccgtag tgtggttcta ttgggttcag 300 gcagaaatta
ttgcgatggc aaccgacctg gcggaattta ttggtgcggc gatcggtttt 360
aaactcattc ttggtgtctc gttgttgcag ggcgcggtgc tgacggggat cgcgactttc
420 ctgattttaa tgctgcaacg tcgcgggcaa aaaccgctgg agaaagtgat
tggcgggtta 480 ctgttgtttg ttgccgcggc ttacattgtc gagttgattt
tctcccagcc taacctggcg 540 cagctgggta aaggaatggt gatcccgagt
ttacctactt cggaggcggt cttcctggca 600 gcaggcgtgt taggggcgac
gattatgccg catgtgattt atttgcactc ctcgctcact 660 cagcatttac
atggcggttc gcgtcaacaa cgttattccg ccaccaaatg ggatgtggct 720
atcgccatga cgattgccgg ttttgtcaat ctggcgatga tggctacagc tgcggcggcg
780 ttccactttt ctggtcatac tggtgttgcc gatcttgatg aggcttatct
gacgctgcaa 840 ccgctgttaa gccatgctgc ggcaacggtc tttgggttaa
gtctggttgc tgccggactg 900 tcctcaacgg tggtggggac actggcgggg
caggtggtga tgcagggatt cattcgcttc 960 catatcccgc tgtgggtgcg
tcgtacagtc accatgttgc cgtcatttat tgtcattctg 1020 atgggattag
atccgacacg gattctggtt atgagtcagg tgctgttaag ttttggtatc 1080
gccctggcgc tggttccact gctgattttc accagtgaca gcaagttgat gggcgatctg
1140 gtgaacagca aacgcgtaaa acagacaggc tgggtgattg tagtgctggt
cgtggcgctg 1200 aatatctggt tgttggtggg gacggcgctg ggattgtag 1239
<210> SEQ ID NO 136 <211> LENGTH: 287 <212> TYPE:
PRT <213> ORGANISM: Salmonella enterica <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(287)
<223> OTHER INFORMATION: Tsx - Salmonella enterica subsp.
enterica serovar Typhimurium LT2 (STM0413) <400> SEQUENCE:
136 Met Lys Lys Thr Leu Leu Ala Val Ser Ala Ala Leu Ala Leu Thr Ser
1 5 10 15 Ser Phe Thr Ala Asn Ala Ala Glu Asn Asp Gln Pro Gln Tyr
Leu Ser 20 25 30 Asp Trp Trp His Gln Ser Val Asn Val Val Gly Ser
Tyr His Thr Arg 35 40 45 Phe Ser Pro Lys Leu Asn Asn Asp Val Tyr
Leu Glu Tyr Glu Ala Phe 50 55 60 Ala Lys Lys Asp Trp Phe Asp Phe
Tyr Gly Tyr Ile Asp Ile Pro Lys 65 70 75 80 Thr Phe Asp Trp Gly Asn
Gly Asn Asp Lys Gly Ile Trp Ser Asp Gly 85 90 95 Ser Pro Leu Phe
Met Glu Ile Glu Pro Arg Phe Ser Ile Asp Lys Leu 100 105 110 Thr Gly
Ala Asp Leu Ser Phe Gly Pro Phe Lys Glu Trp Tyr Phe Ala 115 120 125
Asn Asn Tyr Ile Tyr Asp Met Gly Asp Asn Lys Ala Ser Arg Gln Ser 130
135 140 Thr Trp Tyr Met Gly Leu Gly Thr Asp Ile Asp Thr Gly Leu Pro
Met 145 150 155 160 Gly Leu Ser Leu Asn Val Tyr Ala Lys Tyr Gln Trp
Gln Asn Tyr Gly 165 170 175 Ala Ser Asn Glu Asn Glu Trp Asp Gly Tyr
Arg Phe Lys Val Lys Tyr 180 185 190 Phe Val Pro Ile Thr Asp Leu Trp
Gly Gly Lys Leu Ser Tyr Ile Gly 195 200 205 Phe Thr Asn Phe Asp Trp
Gly Ser Asp Leu Gly Asp Asp Pro Asn Arg 210 215 220 Thr Ser Asn Ser
Ile Ala Ser Ser His Ile Leu Ala Leu Asn Tyr Asp 225 230 235 240 His
Trp His Tyr Ser Val Val Ala Arg Tyr Phe His Asn Gly Gly Gln 245 250
255 Trp Gln Asn Gly Ala Lys Leu Asn Trp Gly Asp Gly Asp Phe Ser Ala
260 265 270 Lys Ser Thr Gly Trp Gly Gly Tyr Leu Val Val Gly Tyr Asn
Phe 275 280 285 <210> SEQ ID NO 137 <211> LENGTH: 406
<212> TYPE: PRT <213> ORGANISM: Bacillus halodurans
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(406) <223> OTHER INFORMATION: BH1446 -
Bacillus halodurans (BAB05165) <400> SEQUENCE: 137 Met Asn
Ile Leu Trp Gly Leu Leu Gly Ile Val Val Val Phe Leu Ile 1 5 10 15
Ala Phe Ala Phe Ser Thr Asn Arg Arg Ala Ile Lys Pro Arg Thr Ile 20
25 30 Leu Gly Gly Leu Ala Ile Gln Leu Leu Phe Ala Ile Ile Val Leu
Lys 35 40 45 Ile Pro Ala Gly Gln Ala Leu Leu Glu Ser Leu Thr Asn
Val Val Leu 50 55 60 Asn Ile Ile Ser Tyr Ala Asn Glu Gly Ile Asp
Phe Val Phe Gly Gly 65 70 75 80 Phe Phe Glu Glu Gly Ser Gly Val Gly
Phe Val Phe Ala Ile Asn Val 85 90 95 Leu Ser Val Val Ile Phe Phe
Ser Ala Leu Ile Ser Ile Leu Tyr Tyr 100 105 110 Leu Gly Ile Met Gln
Phe Val Ile Lys Ile Ile Gly Gly Ala Leu Ser 115 120 125 Trp Leu Leu
Gly Thr Ser Lys Ala Glu Ser Met Ser Ala Ala Ala Asn 130 135 140 Ile
Phe Val Gly Gln Thr Glu Ala Pro Leu Val Val Lys Pro Tyr Leu 145 150
155 160 Pro Lys Met Thr Gln Ser Glu Leu Phe Ala Val Met Thr Gly Gly
Leu 165 170 175 Ala Ser Val Ala Gly Ser Val Leu Ile Gly Tyr Ser Leu
Leu Gly Val 180 185 190 Pro Leu Gln Tyr Leu Leu Ala Ala Ser Phe Met
Ala Ala Pro Ala Gly 195 200 205 Leu Ile Met Ala Lys Met Ile Met Pro
Glu Thr Glu Lys Thr Thr Asp 210 215 220 Ala Glu Asp Asp Phe Lys Leu
Ala Lys Asp Glu Glu Ser Thr Asn Leu 225 230 235 240 Ile Asp Ala Ala
Ala Asn Gly Ala Ser Thr Gly Leu Met Leu Val Leu 245 250 255 Asn Ile
Ala Ala Met Leu Leu Ala Phe Val Ala Leu Ile Ala Leu Ile 260 265 270
Asn Gly Ile Leu Gly Trp Ile Gly Gly Leu Phe Gly Ala Ser Gln Leu 275
280 285 Ser Leu Glu Leu Ile Leu Gly Tyr Val Phe Ala Pro Leu Ala Phe
Val 290 295 300 Ile Gly Ile Pro Trp Ala Glu Ala Leu Gln Ala Gly Ser
Tyr Ile Gly 305 310 315 320 Gln Lys Leu Val Val Asn Glu Phe Val Ala
Tyr Leu Ser Phe Ala Pro 325 330 335 Glu Ile Glu Asn Leu Ser Asp Lys
Ala Val Met Val Ile Ser Phe Ala 340 345 350 Leu Cys Gly Phe Ala Asn
Phe Ser Ser Leu Gly Ile Leu Leu Gly Gly 355 360 365 Leu Gly Lys Leu
Ala Pro Ser Arg Arg Pro Asp Ile Ala Arg Leu Gly 370 375 380 Leu Arg
Ala Ile Leu Ala Gly Thr Leu Ala Ser Leu Leu Ser Ala Ser 385 390 395
400 Ile Ala Gly Met Leu Phe 405 <210> SEQ ID NO 138
<211> LENGTH: 393 <212> TYPE: PRT <213> ORGANISM:
Bacillus subtilis <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(393) <223> OTHER
INFORMATION: nupC Bacillus subtilis subsp. subtilis 168 (BSU39410;
CAA57663) <400> SEQUENCE: 138 Met Lys Tyr Leu Ile Gly Ile Ile
Gly Leu Ile Val Phe Leu Gly Leu 1 5 10 15 Ala Trp Ile Ala Ser Ser
Gly Lys Lys Arg Ile Lys Ile Arg Pro Ile 20 25 30 Val Val Met Leu
Ile Leu Gln Phe Ile Leu Gly Tyr Ile Leu Leu Asn 35 40 45 Thr Gly
Ile Gly Asn Phe Leu Val Gly Gly Phe Ala Lys Gly Phe Gly 50 55 60
Tyr Leu Leu Glu Tyr Ala Ala Glu Gly Ile Asn Phe Val Phe Gly Gly 65
70 75 80 Leu Val Asn Ala Asp Gln Thr Thr Phe Phe Met Asn Val Leu
Leu Pro 85 90 95 Ile Val Phe Ile Ser Ala Leu Ile Gly Ile Leu Gln
Lys Trp Lys Val 100 105 110 Leu Pro Phe Ile Ile Arg Tyr Ile Gly Leu
Ala Leu Ser Lys Val Asn 115 120 125 Gly Met Gly Arg Leu Glu Ser Tyr
Asn Ala Val Ala Ser Ala Ile Leu
130 135 140 Gly Gln Ser Glu Val Phe Ile Ser Leu Lys Lys Glu Leu Gly
Leu Leu 145 150 155 160 Asn Gln Gln Arg Leu Tyr Thr Leu Cys Ala Ser
Ala Met Ser Thr Val 165 170 175 Ser Met Ser Ile Val Gly Ala Tyr Met
Thr Met Leu Lys Pro Glu Tyr 180 185 190 Val Val Thr Ala Leu Val Leu
Asn Leu Phe Gly Gly Phe Ile Ile Ala 195 200 205 Ser Ile Ile Asn Pro
Tyr Glu Val Ala Lys Glu Glu Asp Met Leu Arg 210 215 220 Val Glu Glu
Glu Glu Lys Gln Ser Phe Phe Glu Val Leu Gly Glu Tyr 225 230 235 240
Ile Leu Asp Gly Phe Lys Val Ala Val Val Val Ala Ala Met Leu Ile 245
250 255 Gly Phe Val Ala Ile Ile Ala Leu Ile Asn Gly Ile Phe Asn Ala
Val 260 265 270 Phe Gly Ile Ser Phe Gln Gly Ile Leu Gly Tyr Val Phe
Ala Pro Phe 275 280 285 Ala Phe Leu Val Gly Ile Pro Trp Asn Glu Ala
Val Asn Ala Gly Ser 290 295 300 Ile Met Ala Thr Lys Met Val Ser Asn
Glu Phe Val Ala Met Thr Ser 305 310 315 320 Leu Thr Gln Asn Gly Phe
His Phe Ser Gly Arg Thr Thr Ala Ile Val 325 330 335 Ser Val Phe Leu
Val Ser Phe Ala Asn Phe Ser Ser Ile Gly Ile Ile 340 345 350 Ala Gly
Ala Val Lys Gly Leu Asn Glu Lys Gln Gly Asn Val Val Ala 355 360 365
Arg Phe Gly Leu Lys Leu Leu Tyr Gly Ala Thr Leu Val Ser Phe Leu 370
375 380 Ser Ala Ala Ile Val Gly Leu Ile Tyr 385 390 <210> SEQ
ID NO 139 <211> LENGTH: 404 <212> TYPE: PRT <213>
ORGANISM: Bacillus subtilis <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(404) <223>
OTHER INFORMATION: yutK - Bacillus subtilis subsp. subtilis 168:
BSU32180 <400> SEQUENCE: 139 Met Asn Val Leu Trp Gly Leu Leu
Gly Ala Val Ala Ile Ile Ala Ile 1 5 10 15 Ala Phe Leu Phe Ser Glu
Lys Lys Ser Asn Ile Lys Ile Arg Thr Val 20 25 30 Ile Val Gly Leu
Cys Thr Gln Val Ala Phe Gly Tyr Ile Val Leu Lys 35 40 45 Trp Glu
Ala Gly Arg Ala Val Phe Leu Trp Phe Ser Ser Arg Val Gln 50 55 60
Leu Leu Ile Asp Tyr Ala Asn Glu Gly Ile Ser Phe Ile Phe Gly Pro 65
70 75 80 Leu Leu Lys Val Gly Asp Ser Pro Ala Phe Ala Leu Ser Val
Leu Pro 85 90 95 Val Ile Ile Phe Phe Ser Ala Leu Ile Ala Val Leu
Tyr His Leu Lys 100 105 110 Ile Met Gln Leu Val Phe Arg Val Ile Gly
Gly Gly Leu Ser Lys Leu 115 120 125 Leu Gly Thr Ser Lys Thr Glu Ser
Leu Ala Ala Ala Ala Asn Ile Phe 130 135 140 Val Gly Gln Ser Glu Ser
Pro Leu Val Ile Lys Pro Leu Ile Ala Gly 145 150 155 160 Leu Thr Arg
Ser Glu Leu Phe Thr Ile Met Thr Ser Gly Leu Ser Ala 165 170 175 Val
Ala Gly Ser Thr Leu Phe Gly Tyr Ala Leu Leu Gly Ile Pro Ile 180 185
190 Glu Tyr Leu Leu Ala Ala Ser Phe Met Ala Ala Pro Ala Gly Leu Val
195 200 205 Phe Gly Lys Leu Ile Ile Pro Glu Thr Glu Lys Thr Gln Thr
Val Lys 210 215 220 Ser Asp Phe Lys Met Asp Glu Gly Glu Gly Ala Ala
Asn Val Ile Asp 225 230 235 240 Ala Ala Ala Lys Gly Ala Ser Thr Gly
Leu Gln Ile Ala Leu Asn Val 245 250 255 Gly Ala Met Leu Leu Ala Phe
Val Ala Leu Ile Ala Val Val Asn Gly 260 265 270 Ile Leu Gly Gly Ala
Phe Gly Leu Phe Gly Leu Lys Gly Val Thr Leu 275 280 285 Glu Ser Ile
Leu Gly Tyr Val Phe Ser Pro Ile Ala Phe Leu Ile Gly 290 295 300 Val
Pro Trp His Glu Ala Leu Gln Ala Gly Ser Tyr Ile Gly Gln Lys 305 310
315 320 Leu Val Leu Asn Glu Phe Val Ala Tyr Ser Asn Phe Gly Ser His
Ile 325 330 335 Gly Glu Phe Ser Lys Lys Thr Ala Thr Ile Ile Ser Phe
Ala Leu Cys 340 345 350 Gly Phe Ala Asn Phe Ser Ser Ile Ala Ile Met
Leu Gly Thr Leu Gly 355 360 365 Gly Leu Ala Pro Ser Arg Arg Ser Asp
Ile Ala Arg Leu Gly Leu Lys 370 375 380 Ala Val Leu Ala Gly Thr Leu
Ala Asn Leu Leu Ser Ala Ala Ile Ala 385 390 395 400 Gly Met Phe Ile
<210> SEQ ID NO 140 <211> LENGTH: 397 <212> TYPE:
PRT <213> ORGANISM: Bacillus subtilis <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(397)
<223> OTHER INFORMATION: yxjA - Bacillus subtilis subsp.
spizizenii W23 (BSUW23_19355) <400> SEQUENCE: 140 Met Tyr Phe
Leu Leu Asn Leu Val Gly Leu Ile Val Ile Met Ala Val 1 5 10 15 Val
Phe Leu Cys Ser Pro Gln Lys Lys Lys Ile Gln Trp Arg Pro Ile 20 25
30 Ile Thr Leu Ile Val Leu Glu Leu Leu Ile Thr Trp Phe Met Leu Gly
35 40 45 Thr Lys Val Gly Ser Trp Ala Ile Gly Lys Ile Gly Asp Phe
Phe Thr 50 55 60 Trp Leu Ile Ala Cys Ala Ser Asp Gly Ile Ala Phe
Ala Phe Pro Ser 65 70 75 80 Val Met Ala Asn Glu Thr Val Asp Phe Phe
Phe Ser Ala Leu Leu Pro 85 90 95 Ile Ile Phe Ile Val Thr Phe Phe
Asp Ile Leu Thr Tyr Phe Gly Ile 100 105 110 Leu Pro Trp Leu Ile Asp
Lys Ile Gly Trp Val Ile Ser Lys Ala Ser 115 120 125 Arg Leu Pro Lys
Leu Glu Ser Phe Phe Ser Ile Gln Met Met Phe Leu 130 135 140 Gly Asn
Thr Glu Ala Leu Ala Val Ile Arg Gln Gln Leu Thr Val Leu 145 150 155
160 Asn Asn Asn Arg Leu Leu Thr Phe Gly Leu Met Ser Met Ser Ser Ile
165 170 175 Ser Gly Ser Ile Ile Gly Ser Tyr Leu Ser Met Val Pro Ala
Thr Tyr 180 185 190 Val Phe Thr Ala Ile Pro Leu Asn Cys Leu Asn Ala
Leu Ile Ile Ala 195 200 205 Asn Leu Leu Asn Pro Val His Val Pro Glu
Asp Glu Asp Ile Ile Tyr 210 215 220 Thr Pro Pro Lys Glu Glu Lys Lys
Asp Phe Phe Ser Thr Ile Ser Asn 225 230 235 240 Ser Met Leu Val Gly
Met Asn Met Val Ile Val Ile Leu Ala Met Val 245 250 255 Ile Gly Tyr
Val Ala Leu Thr Ser Ala Val Asn Gly Ile Leu Gly Val 260 265 270 Phe
Val His Gly Leu Thr Ile Gln Thr Ile Phe Ala Tyr Leu Phe Ser 275 280
285 Pro Phe Ala Phe Leu Leu Gly Leu Pro Val His Asp Ala Met Tyr Val
290 295 300 Ala Gln Leu Met Gly Met Lys Leu Ala Thr Asn Glu Phe Val
Ala Met 305 310 315 320 Leu Asp Leu Lys Asn Asn Leu Thr Thr Leu Pro
Pro His Thr Val Ala 325 330 335 Val Ala Thr Thr Phe Leu Thr Ser Phe
Ala Asn Phe Ser Thr Val Gly 340 345 350 Met Ile Tyr Gly Thr Tyr Asn
Ser Ile Leu Asp Gly Glu Lys Ser Thr 355 360 365 Val Ile Gly Lys Asn
Val Trp Lys Leu Leu Val Ser Gly Ile Ala Val 370 375 380 Ser Leu Leu
Ser Ala Ala Ile Val Gly Leu Phe Val Trp 385 390 395 <210> SEQ
ID NO 141 <211> LENGTH: 426 <212> TYPE: PRT <213>
ORGANISM: Caulobacter crescentus CB15 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(426)
<223> OTHER INFORMATION: ccCNT (CC2089) - Caulobacter
crescentus CB15 (AAK24060) <400> SEQUENCE: 141 Met Phe Arg
Pro Glu Asn Val Gln Ala Leu Ala Gly Leu Ala Leu Thr 1 5 10 15 Leu
Gly Leu Cys Trp Leu Val Ser Glu Asn Arg Lys Arg Phe Pro Trp 20 25
30 Gly Leu Ala Ile Gly Ala Val Val Ile Gln Val Leu Leu Val Leu
Val
35 40 45 Leu Phe Gly Leu Pro Gln Ala Gln Gln Met Leu Arg Gly Val
Asn Gly 50 55 60 Ala Val Glu Gly Leu Ala Ala Ser Thr Gln Ala Gly
Thr Ala Phe Val 65 70 75 80 Phe Gly Phe Leu Ala Gly Gly Asp Gln Pro
Tyr Pro Val Ser Asn Pro 85 90 95 Gly Ala Gly Phe Ile Phe Ala Phe
Arg Val Leu Pro Val Ile Leu Val 100 105 110 Val Cys Ala Leu Ser Ala
Leu Leu Trp His Trp Lys Ile Leu Lys Trp 115 120 125 Leu Ala Gln Gly
Phe Gly Phe Val Phe Gln Lys Thr Leu Gly Leu Arg 130 135 140 Gly Pro
Pro Ala Leu Ala Thr Ala Ala Thr Ile Phe Met Gly Gln Val 145 150 155
160 Glu Gly Pro Ile Phe Ile Arg Ala Tyr Leu Asp Lys Leu Ser Arg Ser
165 170 175 Glu Leu Phe Met Leu Ile Ala Val Gly Met Ala Cys Val Ser
Gly Ser 180 185 190 Thr Met Val Ala Tyr Ala Thr Ile Leu Ala Asp Val
Leu Pro Asn Ala 195 200 205 Ala Ala His Val Leu Thr Ala Ser Ile Ile
Ser Ala Pro Ala Gly Val 210 215 220 Leu Leu Ala Arg Ile Ile Val Pro
Ser Asp Pro Met Glu Lys Ser Ala 225 230 235 240 Asp Leu Asp Leu Ser
Thr Glu Asp Lys Thr Tyr Gly Ser Ser Ile Asp 245 250 255 Ala Val Met
Lys Gly Thr Thr Asp Gly Leu Gln Ile Ala Leu Asn Val 260 265 270 Gly
Ala Thr Leu Ile Val Phe Val Ala Leu Ala Thr Met Val Asp Lys 275 280
285 Val Leu Gly Ala Phe Pro Pro Val Gly Gly Glu Pro Leu Ser Ile Ala
290 295 300 Arg Gly Leu Gly Val Val Phe Ala Pro Leu Ala Trp Ser Met
Gly Ile 305 310 315 320 Pro Trp Lys Glu Ala Gly Thr Ala Gly Gly Leu
Leu Gly Val Lys Leu 325 330 335 Ile Leu Thr Glu Phe Thr Ala Phe Ile
Gln Leu Ser Lys Val Gly Glu 340 345 350 Ala Leu Leu Asp Glu Arg Thr
Arg Met Ile Met Thr Tyr Ala Leu Cys 355 360 365 Gly Phe Ala Asn Ile
Gly Ser Val Gly Met Asn Val Ala Gly Phe Ser 370 375 380 Val Leu Val
Pro Gln Arg Arg Gln Glu Val Leu Gly Leu Val Trp Lys 385 390 395 400
Ala Met Met Ala Gly Phe Leu Ala Thr Cys Leu Thr Ala Ser Leu Val 405
410 415 Gly Leu Met Pro Arg Ser Leu Phe Gly Leu 420 425 <210>
SEQ ID NO 142 <211> LENGTH: 416 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: yeiJ - Escherichia coli K-12 W3110
(AAC75222; JW2148) <400> SEQUENCE: 142 Met Asp Val Met Arg
Ser Val Leu Gly Met Val Val Leu Leu Thr Ile 1 5 10 15 Ala Phe Leu
Leu Ser Val Asn Lys Lys Lys Ile Ser Leu Arg Thr Val 20 25 30 Gly
Ala Ala Leu Val Leu Gln Val Val Ile Gly Gly Ile Met Leu Trp 35 40
45 Leu Pro Pro Gly Arg Trp Val Ala Glu Lys Val Ala Phe Gly Val His
50 55 60 Lys Val Met Ala Tyr Ser Asp Ala Gly Ser Ala Phe Ile Phe
Gly Ser 65 70 75 80 Leu Val Gly Pro Lys Met Asp Thr Leu Phe Asp Gly
Ala Gly Phe Ile 85 90 95 Phe Gly Phe Arg Val Leu Pro Ala Ile Ile
Phe Val Thr Ala Leu Val 100 105 110 Ser Ile Leu Tyr Tyr Ile Gly Val
Met Gly Ile Leu Ile Arg Ile Leu 115 120 125 Gly Gly Ile Phe Gln Lys
Ala Leu Asn Ile Ser Lys Ile Glu Ser Phe 130 135 140 Val Ala Val Thr
Thr Ile Phe Leu Gly Gln Asn Glu Ile Pro Ala Ile 145 150 155 160 Val
Lys Pro Phe Ile Asp Arg Leu Asn Arg Asn Glu Leu Phe Thr Ala 165 170
175 Ile Cys Ser Gly Met Ala Ser Ile Ala Gly Ser Thr Met Ile Gly Tyr
180 185 190 Ala Ala Leu Gly Val Pro Val Glu Tyr Leu Leu Ala Ala Ser
Leu Met 195 200 205 Ala Ile Pro Gly Gly Ile Leu Phe Ala Arg Leu Leu
Ser Pro Ala Thr 210 215 220 Glu Ser Ser Gln Val Ser Phe Asn Asn Leu
Ser Phe Thr Glu Thr Pro 225 230 235 240 Pro Lys Ser Ile Ile Glu Ala
Ala Ala Thr Gly Ala Met Thr Gly Leu 245 250 255 Lys Ile Ala Ala Gly
Val Ala Thr Val Val Met Ala Phe Val Ala Ile 260 265 270 Ile Ala Leu
Ile Asn Gly Ile Ile Gly Gly Val Gly Gly Trp Phe Gly 275 280 285 Phe
Glu His Ala Ser Leu Glu Ser Ile Leu Gly Tyr Leu Leu Ala Pro 290 295
300 Leu Ala Trp Val Met Gly Val Asp Trp Ser Asp Ala Asn Leu Ala Gly
305 310 315 320 Ser Leu Ile Gly Gln Lys Leu Ala Ile Asn Glu Phe Val
Ala Tyr Leu 325 330 335 Asn Phe Ser Pro Tyr Leu Gln Thr Ala Gly Thr
Leu Asp Ala Lys Thr 340 345 350 Val Ala Ile Ile Ser Phe Ala Leu Cys
Gly Phe Ala Asn Phe Gly Ser 355 360 365 Ile Gly Val Val Val Gly Ala
Phe Ser Ala Val Ala Pro His Arg Ala 370 375 380 Pro Glu Ile Ala Gln
Leu Gly Leu Arg Ala Leu Ala Ala Ala Thr Leu 385 390 395 400 Ser Asn
Leu Met Ser Ala Thr Ile Ala Gly Phe Phe Ile Gly Leu Ala 405 410 415
<210> SEQ ID NO 143 <211> LENGTH: 416 <212> TYPE:
PRT <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: yeiM - Escherichia coli K-12 W3110
(AAC75225; JW2151) <400> SEQUENCE: 143 Met Asp Ile Met Arg
Ser Val Val Gly Met Val Val Leu Leu Ala Ile 1 5 10 15 Ala Phe Leu
Leu Ser Val Asn Lys Lys Ser Ile Ser Leu Arg Thr Val 20 25 30 Gly
Ala Ala Leu Leu Leu Gln Ile Ala Ile Gly Gly Ile Met Leu Tyr 35 40
45 Phe Pro Pro Gly Lys Trp Ala Val Glu Gln Ala Ala Leu Gly Val His
50 55 60 Lys Val Met Ser Tyr Ser Asp Ala Gly Ser Ala Phe Ile Phe
Gly Ser 65 70 75 80 Leu Val Gly Pro Lys Met Asp Val Leu Phe Asp Gly
Ala Gly Phe Ile 85 90 95 Phe Ala Phe Arg Val Leu Pro Ala Ile Ile
Phe Val Thr Ala Leu Ile 100 105 110 Ser Leu Leu Tyr Tyr Ile Gly Val
Met Gly Leu Leu Ile Arg Ile Leu 115 120 125 Gly Ser Ile Phe Gln Lys
Ala Leu Asn Ile Ser Lys Ile Glu Ser Phe 130 135 140 Val Ala Val Thr
Thr Ile Phe Leu Gly Gln Asn Glu Ile Pro Ala Ile 145 150 155 160 Val
Lys Pro Phe Ile Asp Arg Met Asn Arg Asn Glu Leu Phe Thr Ala 165 170
175 Ile Cys Ser Gly Met Ala Ser Ile Ala Gly Ser Met Met Ile Gly Tyr
180 185 190 Ala Gly Met Gly Val Pro Ile Asp Tyr Leu Leu Ala Ala Ser
Leu Met 195 200 205 Ala Ile Pro Gly Gly Ile Leu Phe Ala Arg Ile Leu
Ser Pro Ala Thr 210 215 220 Glu Pro Ser Gln Val Thr Phe Glu Asn Leu
Ser Phe Ser Glu Thr Pro 225 230 235 240 Pro Lys Ser Phe Ile Glu Ala
Ala Ala Ser Gly Ala Met Thr Gly Leu 245 250 255 Lys Ile Ala Ala Gly
Val Ala Thr Val Val Met Ala Phe Val Ala Ile 260 265 270 Ile Ala Leu
Ile Asn Gly Ile Ile Gly Gly Ile Gly Gly Trp Phe Gly 275 280 285 Phe
Ala Asn Ala Ser Leu Glu Ser Ile Phe Gly Tyr Val Leu Ala Pro 290 295
300 Leu Ala Trp Ile Met Gly Val Asp Trp Ser Asp Ala Asn Leu Ala Gly
305 310 315 320 Ser Leu Ile Gly Gln Lys Leu Ala Ile Asn Glu Phe Val
Ala Tyr Leu 325 330 335 Ser Phe Ser Pro Tyr Leu Gln Thr Gly Gly Thr
Leu Glu Val Lys Thr 340 345 350 Ile Ala Ile Ile Ser Phe Ala Leu Cys
Gly Phe Ala Asn Phe Gly Ser 355 360 365 Ile Gly Val Val Val Gly Ala
Phe Ser Ala Ile Ser Pro Lys Arg Ala 370 375 380
Pro Glu Ile Ala Gln Leu Gly Leu Arg Ala Leu Ala Ala Ala Thr Leu 385
390 395 400 Ser Asn Leu Met Ser Ala Thr Ile Ala Gly Phe Phe Ile Gly
Leu Ala 405 410 415 <210> SEQ ID NO 144 <211> LENGTH:
417 <212> TYPE: PRT <213> ORGANISM: Haemophilus
influenzae <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (1)..(417) <223> OTHER INFORMATION:
HI0519 - Haemophilus influenzae Rd KW20 serotype d(AAC22177)
<400> SEQUENCE: 144 Met Ser Val Leu Ser Ser Ile Leu Gly Met
Val Val Leu Ile Ala Ile 1 5 10 15 Ala Val Leu Leu Ser Asn Asn Arg
Lys Ala Ile Ser Ile Arg Thr Val 20 25 30 Val Gly Ala Leu Ala Ile
Gln Val Gly Phe Ala Ala Leu Ile Leu Tyr 35 40 45 Val Pro Ala Gly
Lys Gln Ala Leu Gly Ala Ala Ala Asp Met Val Ser 50 55 60 Asn Val
Ile Ala Tyr Gly Asn Asp Gly Ile Asn Phe Val Phe Gly Gly 65 70 75 80
Leu Ala Asp Pro Ser Lys Pro Ser Gly Phe Ile Phe Ala Val Lys Val 85
90 95 Leu Pro Ile Ile Val Phe Phe Ser Gly Leu Ile Ser Val Leu Tyr
Tyr 100 105 110 Leu Gly Ile Met Gln Val Val Ile Lys Val Leu Gly Gly
Ala Leu Gln 115 120 125 Lys Ala Leu Gly Thr Ser Lys Ala Glu Ser Met
Ser Ala Ala Ala Asn 130 135 140 Ile Phe Val Gly Gln Thr Glu Ala Pro
Leu Val Val Arg Pro Tyr Ile 145 150 155 160 Lys Asn Met Thr Gln Ser
Glu Leu Phe Ala Ile Met Val Gly Gly Thr 165 170 175 Ala Ser Ile Ala
Gly Ser Val Met Ala Gly Tyr Ala Gly Met Gly Val 180 185 190 Pro Leu
Thr Tyr Leu Ile Ala Ala Ser Phe Met Ala Ala Pro Ala Gly 195 200 205
Leu Leu Phe Ala Lys Leu Met Phe Pro Gln Thr Glu Gln Phe Thr Asp 210
215 220 Lys Gln Pro Glu Asp Asn Asp Ser Glu Lys Pro Thr Asn Val Leu
Glu 225 230 235 240 Ala Met Ala Gly Gly Ala Ser Ala Gly Met Gln Leu
Ala Leu Asn Val 245 250 255 Gly Ala Met Leu Ile Ala Phe Val Gly Leu
Ile Ala Leu Ile Asn Gly 260 265 270 Ile Leu Ser Gly Val Gly Gly Trp
Phe Gly Tyr Gly Asp Leu Thr Leu 275 280 285 Gln Ser Ile Phe Gly Leu
Ile Phe Lys Pro Leu Ala Tyr Leu Ile Gly 290 295 300 Val Thr Asp Gly
Ala Glu Ala Gly Ile Ala Gly Gln Met Ile Gly Met 305 310 315 320 Lys
Leu Ala Val Asn Glu Phe Val Gly Tyr Leu Glu Phe Ala Lys Tyr 325 330
335 Leu Gln Pro Asp Ser Ala Ile Val Leu Thr Glu Lys Thr Lys Ala Ile
340 345 350 Ile Thr Phe Ala Leu Cys Gly Phe Ala Asn Phe Ser Ser Ile
Ala Ile 355 360 365 Leu Ile Gly Gly Leu Gly Gly Met Ala Pro Ser Arg
Arg Ser Asp Val 370 375 380 Ala Arg Leu Gly Ile Lys Ala Val Ile Ala
Gly Thr Leu Ala Asn Leu 385 390 395 400 Met Ser Ala Thr Ile Ala Gly
Leu Phe Ile Gly Leu Gly Ala Ala Ala 405 410 415 Leu <210> SEQ
ID NO 145 <211> LENGTH: 418 <212> TYPE: PRT <213>
ORGANISM: Helicobacter pylori 26695 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(418)
<223> OTHER INFORMATION: nupC (HP1180) - Helicobacter pylori
26695 (AAD08224) <400> SEQUENCE: 145 Met Ile Phe Ser Ser Leu
Phe Ser Val Val Gly Met Ala Val Leu Phe 1 5 10 15 Leu Ile Ala Trp
Val Phe Ser Ser Asn Lys Arg Ala Ile Asn Tyr Arg 20 25 30 Thr Ile
Val Ser Ala Phe Val Ile Gln Val Ala Leu Gly Ala Leu Ala 35 40 45
Leu Tyr Val Pro Leu Gly Arg Glu Met Leu Gln Gly Leu Ala Ser Gly 50
55 60 Ile Gln Ser Val Ile Ser Tyr Gly Tyr Glu Gly Val Arg Phe Leu
Phe 65 70 75 80 Gly Asn Leu Ala Pro Asn Ala Lys Gly Asp Gln Gly Ile
Gly Gly Phe 85 90 95 Val Phe Ala Ile Asn Val Leu Ala Ile Ile Ile
Phe Phe Ala Ser Leu 100 105 110 Ile Ser Leu Leu Tyr Tyr Leu Lys Ile
Met Pro Leu Phe Ile Asn Leu 115 120 125 Ile Gly Gly Ala Leu Gln Lys
Cys Leu Gly Thr Ser Arg Ala Glu Ser 130 135 140 Met Ser Ala Ala Ala
Asn Ile Phe Val Ala His Thr Glu Ala Pro Leu 145 150 155 160 Val Ile
Lys Pro Tyr Leu Lys Ser Met Ser Asp Ser Glu Ile Phe Ala 165 170 175
Val Met Cys Val Gly Met Ala Ser Val Ala Gly Pro Val Leu Ala Gly 180
185 190 Tyr Ala Ser Met Gly Ile Pro Leu Pro Tyr Leu Ile Ala Ala Ser
Phe 195 200 205 Met Ser Ala Pro Gly Gly Leu Leu Phe Ala Lys Ile Ile
Tyr Pro Gln 210 215 220 Asn Glu Thr Ile Ser Ser His Ala Asp Val Ser
Ile Glu Lys His Val 225 230 235 240 Asn Ala Ile Glu Ala Ile Ala Asn
Gly Ala Ser Thr Gly Leu Asn Leu 245 250 255 Ala Leu His Val Gly Ala
Met Leu Leu Ala Phe Val Gly Met Leu Ala 260 265 270 Leu Ile Asn Gly
Leu Leu Gly Val Val Gly Gly Phe Leu Gly Met Glu 275 280 285 His Leu
Ser Leu Gly Leu Ile Leu Gly Thr Leu Leu Lys Pro Leu Ala 290 295 300
Phe Met Leu Gly Ile Pro Trp Ser Gln Ala Gly Ile Ala Gly Glu Ile 305
310 315 320 Ile Gly Ile Lys Ile Ala Leu Asn Glu Phe Val Gly Tyr Met
Gln Leu 325 330 335 Leu Pro Tyr Leu Gly Asp Asn Pro Pro Leu Ile Leu
Ser Glu Lys Thr 340 345 350 Lys Ala Ile Ile Thr Phe Ala Leu Cys Gly
Phe Ala Asn Leu Ser Ser 355 360 365 Val Ala Met Leu Ile Gly Gly Leu
Gly Ser Leu Val Pro Lys Lys Lys 370 375 380 Asp Leu Ile Val Arg Leu
Ala Leu Lys Ala Val Leu Val Gly Thr Leu 385 390 395 400 Ser Asn Phe
Met Ser Ala Thr Ile Ala Gly Leu Phe Ile Gly Leu Asn 405 410 415 Ala
His <210> SEQ ID NO 146 <211> LENGTH: 409 <212>
TYPE: PRT <213> ORGANISM: Staphylococcus aureus <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(409) <223> OTHER INFORMATION: nupC (SA0600) -
Staphylococcus aureus subsp. aureus N315 (BAB41833) <400>
SEQUENCE: 146 Met Phe Leu Leu Ile Asn Ile Ile Gly Leu Ile Val Phe
Leu Gly Ile 1 5 10 15 Ala Val Leu Phe Ser Arg Asp Arg Lys Asn Ile
Gln Trp Gln Ser Ile 20 25 30 Gly Ile Leu Val Val Leu Asn Leu Phe
Leu Ala Trp Phe Phe Ile Tyr 35 40 45 Phe Asp Trp Gly Gln Lys Ala
Val Arg Gly Ala Ala Asn Gly Ile Ala 50 55 60 Trp Val Val Gln Ser
Ala His Ala Gly Thr Gly Phe Ala Phe Ala Ser 65 70 75 80 Leu Thr Asn
Val Lys Met Met Asp Met Ala Val Ala Ala Leu Phe Pro 85 90 95 Ile
Leu Leu Ile Val Pro Leu Phe Asp Ile Leu Met Tyr Phe Asn Ile 100 105
110 Leu Pro Lys Ile Ile Gly Gly Ile Gly Trp Leu Leu Ala Lys Val Thr
115 120 125 Arg Gln Pro Lys Phe Glu Ser Phe Phe Gly Ile Glu Met Met
Phe Leu 130 135 140 Gly Asn Thr Glu Ala Leu Ala Val Ser Ser Glu Gln
Leu Lys Arg Met 145 150 155 160 Asn Glu Met Arg Val Leu Thr Ile Ala
Met Met Ser Met Ser Ser Val 165 170 175 Ser Gly Ala Ile Val Gly Ala
Tyr Val Gln Met Val Pro Gly Glu Leu 180 185 190 Val Leu Thr Ala Ile
Pro Leu Asn Ile Val Asn Ala Ile Ile Val Ser 195 200 205 Cys Leu Leu
Asn Pro Val Ser Val Glu Glu Lys Glu Asp Ile Ile Tyr 210 215 220 Ser
Leu Lys Asn Asn Glu Val Glu Arg Gln Pro Phe Phe Ser Phe Leu 225 230
235 240
Gly Asp Ser Val Leu Ala Ala Gly Lys Leu Val Leu Ile Ile Ile Ala 245
250 255 Phe Val Ile Ser Phe Val Ala Leu Ala Asp Leu Phe Asp Arg Phe
Ile 260 265 270 Asn Leu Ile Thr Gly Leu Ile Ala Gly Trp Ile Gly Ile
Lys Gly Ser 275 280 285 Phe Gly Leu Asn Gln Ile Leu Gly Val Phe Met
Tyr Pro Phe Ala Leu 290 295 300 Leu Leu Gly Leu Pro Tyr Asp Glu Ala
Trp Leu Val Ala Gln Gln Met 305 310 315 320 Ala Lys Lys Ile Val Thr
Asn Glu Phe Val Val Met Gly Glu Ile Ser 325 330 335 Lys Asp Ile Ala
Ser Tyr Thr Pro His His Arg Ala Val Ile Thr Thr 340 345 350 Phe Leu
Ile Ser Phe Ala Asn Phe Ser Thr Ile Gly Met Ile Ile Gly 355 360 365
Thr Leu Lys Gly Ile Val Asp Lys Lys Thr Ser Asp Phe Val Ser Lys 370
375 380 Tyr Val Pro Met Met Leu Leu Ser Gly Ile Leu Val Ser Leu Leu
Thr 385 390 395 400 Ala Ala Phe Val Gly Leu Phe Ala Trp 405
<210> SEQ ID NO 147 <211> LENGTH: 409 <212> TYPE:
PRT <213> ORGANISM: Staphylococcus aureus <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(409) <223> OTHER INFORMATION: nupC (SAV0645)
-Staphylococcus aureus subsp. aureus Mu50 (BAB56807) <400>
SEQUENCE: 147 Met Phe Leu Leu Ile Asn Ile Ile Gly Leu Ile Val Phe
Leu Gly Ile 1 5 10 15 Ala Val Leu Phe Ser Arg Asp Arg Lys Asn Ile
Gln Trp Gln Ser Ile 20 25 30 Gly Ile Leu Val Val Leu Asn Leu Phe
Leu Ala Trp Phe Phe Ile Tyr 35 40 45 Phe Asp Trp Gly Gln Lys Ala
Val Arg Gly Ala Ala Asn Gly Ile Ala 50 55 60 Trp Val Val Gln Ser
Ala His Ala Gly Thr Gly Phe Ala Phe Ala Ser 65 70 75 80 Leu Thr Asn
Val Lys Met Met Asp Met Ala Val Ala Ala Leu Phe Pro 85 90 95 Ile
Leu Leu Ile Val Pro Leu Phe Asp Ile Leu Met Tyr Phe Asn Ile 100 105
110 Leu Pro Lys Ile Ile Gly Gly Ile Gly Trp Leu Leu Ala Lys Val Thr
115 120 125 Arg Gln Pro Lys Phe Glu Ser Phe Phe Gly Ile Glu Met Met
Phe Leu 130 135 140 Gly Asn Thr Glu Ala Leu Ala Val Ser Ser Glu Gln
Leu Lys Arg Met 145 150 155 160 Asn Glu Met Arg Val Leu Thr Ile Ala
Met Met Ser Met Ser Ser Val 165 170 175 Ser Gly Ala Ile Val Gly Ala
Tyr Val Gln Met Val Pro Gly Glu Leu 180 185 190 Val Leu Thr Ala Ile
Pro Leu Asn Ile Val Asn Ala Ile Ile Val Ser 195 200 205 Cys Leu Leu
Asn Pro Val Ser Val Glu Glu Lys Glu Asp Ile Ile Tyr 210 215 220 Ser
Leu Lys Asn Asn Glu Val Glu Arg Gln Pro Phe Phe Ser Phe Leu 225 230
235 240 Gly Asp Ser Val Leu Ala Ala Gly Lys Leu Val Leu Ile Ile Ile
Ala 245 250 255 Phe Val Ile Ser Phe Val Ala Leu Ala Asp Leu Phe Asp
Arg Phe Ile 260 265 270 Asn Leu Ile Thr Gly Leu Ile Ala Gly Trp Ile
Gly Ile Lys Gly Ser 275 280 285 Phe Gly Leu Asn Gln Ile Leu Gly Val
Phe Met Tyr Pro Phe Ala Leu 290 295 300 Leu Leu Gly Leu Pro Tyr Asp
Glu Ala Trp Leu Val Ala Gln Gln Met 305 310 315 320 Ala Lys Lys Ile
Val Thr Asn Glu Phe Val Val Met Gly Glu Ile Ser 325 330 335 Lys Asp
Ile Ala Ser Tyr Thr Pro His His Arg Ala Val Ile Thr Thr 340 345 350
Phe Leu Ile Ser Phe Ala Asn Phe Ser Thr Ile Gly Met Ile Ile Gly 355
360 365 Thr Leu Lys Gly Ile Val Asp Lys Lys Thr Ser Asp Phe Val Ser
Lys 370 375 380 Tyr Val Pro Met Met Leu Leu Ser Gly Ile Leu Val Ser
Leu Leu Thr 385 390 395 400 Ala Ala Phe Val Gly Leu Phe Ala Trp 405
<210> SEQ ID NO 148 <211> LENGTH: 400 <212> TYPE:
PRT <213> ORGANISM: Streptococcus pyogenes SF370 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(400) <223> OTHER INFORMATION: nupC (SpNupC) -
Streptococcus pyogenes SF370 serotype M1 (AAK34582) <400>
SEQUENCE: 148 Met Gln Phe Ile Tyr Ser Ile Ile Gly Ile Leu Leu Val
Leu Gly Ile 1 5 10 15 Val Tyr Ala Ile Ser Phe Asn Arg Lys Ser Val
Ser Leu Ser Leu Ile 20 25 30 Gly Lys Ala Leu Ile Val Gln Phe Ile
Ile Ala Leu Ile Leu Val Arg 35 40 45 Ile Pro Leu Gly Gln Gln Ile
Val Ser Val Val Ser Thr Gly Val Thr 50 55 60 Ser Val Ile Asn Cys
Gly Gln Ala Gly Leu Asn Phe Val Phe Gly Ser 65 70 75 80 Leu Ala Asp
Ser Gly Ala Lys Thr Gly Phe Ile Phe Ala Ile Gln Thr 85 90 95 Leu
Gly Asn Ile Val Phe Leu Ser Ala Leu Val Ser Leu Leu Tyr Tyr 100 105
110 Val Gly Ile Leu Gly Phe Val Val Lys Trp Ile Gly Lys Gly Val Gly
115 120 125 Lys Ile Met Lys Ser Ser Glu Val Glu Ser Phe Val Ala Val
Ala Asn 130 135 140 Met Phe Leu Gly Gln Thr Asp Ser Pro Ile Leu Val
Ser Lys Tyr Leu 145 150 155 160 Gly Arg Met Thr Asp Ser Glu Ile Met
Val Val Leu Val Ser Gly Met 165 170 175 Gly Ser Met Ser Val Ser Ile
Leu Gly Gly Tyr Ile Ala Leu Gly Ile 180 185 190 Pro Met Glu Tyr Leu
Leu Ile Ala Ser Thr Met Val Pro Ile Gly Ser 195 200 205 Ile Leu Ile
Ala Lys Ile Leu Leu Pro Gln Thr Glu Pro Val Gln Lys 210 215 220 Ile
Asp Asp Ile Lys Met Asp Asn Lys Gly Asn Asn Ala Asn Val Ile 225 230
235 240 Asp Ala Ile Ala Glu Gly Ala Ser Thr Gly Ala Gln Met Ala Phe
Ser 245 250 255 Ile Gly Ala Ser Leu Ile Ala Phe Val Gly Leu Val Ser
Leu Ile Asn 260 265 270 Met Met Leu Ser Gly Leu Gly Ile Arg Leu Glu
Gln Ile Phe Ser Tyr 275 280 285 Val Phe Ala Pro Phe Gly Phe Leu Met
Gly Phe Asp His Lys Asn Ile 290 295 300 Leu Leu Glu Gly Asn Leu Leu
Gly Ser Lys Leu Ile Leu Asn Glu Phe 305 310 315 320 Val Ser Phe Gln
Gln Leu Gly His Leu Ile Lys Ser Leu Asp Tyr Arg 325 330 335 Thr Ala
Leu Val Ala Thr Ile Ser Leu Cys Gly Phe Ala Asn Leu Ser 340 345 350
Ser Leu Gly Ile Cys Val Ser Gly Ile Ala Val Leu Cys Pro Glu Lys 355
360 365 Arg Ser Thr Leu Ala Arg Leu Val Phe Arg Ala Met Ile Gly Gly
Ile 370 375 380 Ala Val Ser Met Leu Ser Ala Phe Ile Val Gly Ile Val
Thr Leu Phe 385 390 395 400 <210> SEQ ID NO 149 <211>
LENGTH: 418 <212> TYPE: PRT <213> ORGANISM: Vibrio
cholerae O1 biovar El Tor N16961 <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(418) <223>
OTHER INFORMATION: nupC (VC2352) - Vibrio cholerae O1 biovar El Tor
N16961 (AAF95495) <400> SEQUENCE: 149 Met Ser Leu Phe Met Ser
Leu Ile Gly Met Ala Val Leu Leu Gly Ile 1 5 10 15 Ala Val Leu Leu
Ser Ser Asn Arg Lys Ala Ile Asn Leu Arg Thr Val 20 25 30 Gly Gly
Ala Phe Ala Ile Gln Phe Ser Leu Gly Ala Phe Ile Leu Tyr 35 40 45
Val Pro Trp Gly Gln Glu Leu Leu Arg Gly Phe Ser Asp Ala Val Ser 50
55 60 Asn Val Ile Asn Tyr Gly Asn Asp Gly Thr Ser Phe Leu Phe Gly
Gly 65 70 75 80 Leu Val Ser Gly Lys Met Phe Glu Val Phe Gly Gly Gly
Gly Phe Ile 85 90 95 Phe Ala Phe Arg Val Leu Pro Thr Leu Ile Phe
Phe Ser Ala Leu Ile 100 105 110 Ser Val Leu Tyr Tyr Leu Gly Val Met
Gln Trp Val Ile Arg Ile Leu
115 120 125 Gly Gly Gly Leu Gln Lys Ala Leu Gly Thr Ser Arg Ala Glu
Ser Met 130 135 140 Ser Ala Ala Ala Asn Ile Phe Val Gly Gln Thr Glu
Ala Pro Leu Val 145 150 155 160 Val Arg Pro Phe Val Pro Lys Met Thr
Gln Ser Glu Leu Phe Ala Val 165 170 175 Met Cys Gly Gly Leu Ala Ser
Ile Ala Gly Gly Val Leu Ala Gly Tyr 180 185 190 Ala Ser Met Gly Val
Lys Ile Glu Tyr Leu Val Ala Ala Ser Phe Met 195 200 205 Ala Ala Pro
Gly Gly Leu Leu Phe Ala Lys Leu Met Met Pro Glu Thr 210 215 220 Glu
Lys Pro Gln Asp Asn Glu Asp Ile Thr Leu Asp Gly Gly Asp Asp 225 230
235 240 Lys Pro Ala Asn Val Ile Asp Ala Ala Ala Gly Gly Ala Ser Ala
Gly 245 250 255 Leu Gln Leu Ala Leu Asn Val Gly Ala Met Leu Ile Ala
Phe Ile Gly 260 265 270 Leu Ile Ala Leu Ile Asn Gly Met Leu Gly Gly
Ile Gly Gly Trp Phe 275 280 285 Gly Met Pro Glu Leu Lys Leu Glu Met
Leu Leu Gly Trp Leu Phe Ala 290 295 300 Pro Leu Ala Phe Leu Ile Gly
Val Pro Trp Asn Glu Ala Thr Val Ala 305 310 315 320 Gly Glu Phe Ile
Gly Leu Lys Thr Val Ala Asn Glu Phe Val Ala Tyr 325 330 335 Ser Gln
Phe Ala Pro Tyr Leu Thr Glu Ala Ala Pro Val Val Leu Ser 340 345 350
Glu Lys Thr Lys Ala Ile Ile Ser Phe Ala Leu Cys Gly Phe Ala Asn 355
360 365 Leu Ser Ser Ile Ala Ile Leu Leu Gly Gly Leu Gly Ser Leu Ala
Pro 370 375 380 Lys Arg Arg Gly Asp Ile Ala Arg Met Gly Val Lys Ala
Val Ile Ala 385 390 395 400 Gly Thr Leu Ser Asn Leu Met Ala Ala Thr
Ile Ala Gly Phe Phe Leu 405 410 415 Ser Phe <210> SEQ ID NO
150 <211> LENGTH: 405 <212> TYPE: PRT <213>
ORGANISM: Vibrio cholerae O1 biovar El Tor N16961 <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(405) <223> OTHER INFORMATION: nupC (VC1953) - Vibrio
cholerae O1 biovar El Tor N16961 (AAF95101) <400> SEQUENCE:
150 Met Gly Gly Val Met Ser Ser Leu Leu Gly Met Gly Ala Ile Leu Leu
1 5 10 15 Val Ala Trp Leu Phe Ser Thr Asn Arg Lys Asn Ile Asn Leu
Arg Thr 20 25 30 Val Ser Leu Ala Leu Leu Leu Gln Ile Phe Phe Ala
Leu Leu Val Leu 35 40 45 Tyr Val Pro Ala Gly Lys Glu Ala Leu Asn
Arg Val Thr Gly Ala Val 50 55 60 Ser Gln Leu Ile Asn Tyr Gly Gln
Asp Gly Ile Gly Phe Val Phe Gly 65 70 75 80 Gly Leu Ala Asn Gly Ser
Val Gly Phe Val Phe Ala Ile Asn Val Leu 85 90 95 Gly Ile Ile Ile
Phe Phe Ser Ala Leu Ile Ser Gly Leu Tyr His Leu 100 105 110 Gly Ile
Met Pro Lys Val Ile Asn Leu Ile Gly Gly Gly Leu Gln Lys 115 120 125
Leu Leu Gly Thr Gly Arg Ala Glu Ser Leu Ser Ala Thr Ala Asn Ile 130
135 140 Phe Val Gly Met Ile Glu Ala Pro Leu Val Val Lys Pro Tyr Leu
His 145 150 155 160 Lys Met Thr Asp Ser Gln Phe Phe Ala Val Met Thr
Gly Gly Leu Ala 165 170 175 Ser Val Ala Gly Gly Thr Leu Val Gly Tyr
Ala Ser Leu Gly Val Glu 180 185 190 Leu Asn Tyr Leu Ile Ala Ala Ala
Phe Met Ser Ala Pro Ala Gly Leu 195 200 205 Leu Met Ala Lys Ile Met
Leu Pro Glu Thr Glu His Val Asp Ala Ala 210 215 220 Ile Ala Gln Asp
Glu Leu Asp Leu Pro Lys Ser Thr Asn Val Val Glu 225 230 235 240 Ala
Ile Ala Asp Gly Ala Met Ser Gly Val Lys Ile Ala Val Ala Val 245 250
255 Gly Ala Thr Leu Leu Ala Phe Val Ser Val Ile Ala Leu Leu Asn Gly
260 265 270 Leu Leu Gly Trp Phe Gly Gly Trp Phe Gly Ile Glu Leu Ser
Phe Glu 275 280 285 Leu Ile Met Gly Tyr Val Phe Ala Pro Val Ala Trp
Leu Ile Gly Ile 290 295 300 Pro Trp His Glu Ala Ile Thr Ala Gly Ser
Leu Ile Gly Asn Lys Val 305 310 315 320 Val Val Asn Glu Phe Val Ala
Phe Ile Gln Leu Ile Glu Val Lys Glu 325 330 335 Gln Leu Ser Ala His
Ser Gln Ala Ile Val Thr Phe Ala Leu Cys Gly 340 345 350 Phe Ala Asn
Ile Ser Thr Met Ala Ile Leu Ile Gly Gly Leu Gly Ser 355 360 365 Leu
Val Pro Glu Arg Arg Ser Phe Ile Ser Gln Tyr Gly Phe Arg Ala 370 375
380 Ile Gly Ala Gly Val Leu Ala Asn Leu Met Ser Ala Ser Ile Ala Gly
385 390 395 400 Val Ile Leu Ser Leu 405 <210> SEQ ID NO 151
<211> LENGTH: 425 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(425) <223> OTHER
INFORMATION: yegT - Escherichia coli K-12 W3110 (P76417; JW2085)
<400> SEQUENCE: 151 Met Lys Thr Thr Ala Lys Leu Ser Phe Met
Met Phe Val Glu Trp Phe 1 5 10 15 Ile Trp Gly Ala Trp Phe Val Pro
Leu Trp Leu Trp Leu Ser Lys Ser 20 25 30 Gly Phe Ser Ala Gly Glu
Ile Gly Trp Ser Tyr Ala Cys Thr Ala Ile 35 40 45 Ala Ala Ile Leu
Ser Pro Ile Leu Val Gly Ser Ile Thr Asp Arg Phe 50 55 60 Phe Ser
Ala Gln Lys Val Leu Ala Val Leu Met Phe Ala Gly Ala Leu 65 70 75 80
Leu Met Tyr Phe Ala Ala Gln Gln Thr Thr Phe Ala Gly Phe Phe Pro 85
90 95 Leu Leu Leu Ala Tyr Ser Leu Thr Tyr Met Pro Thr Ile Ala Leu
Thr 100 105 110 Asn Ser Ile Ala Phe Ala Asn Val Pro Asp Val Glu Arg
Asp Phe Pro 115 120 125 Arg Ile Arg Val Met Gly Thr Ile Gly Trp Ile
Ala Ser Gly Leu Ala 130 135 140 Cys Gly Phe Leu Pro Gln Ile Leu Gly
Tyr Ala Asp Ile Ser Pro Thr 145 150 155 160 Asn Ile Pro Leu Leu Ile
Thr Ala Gly Ser Ser Ala Leu Leu Gly Val 165 170 175 Phe Ala Phe Phe
Leu Pro Asp Thr Pro Pro Lys Ser Thr Gly Lys Met 180 185 190 Asp Ile
Lys Val Met Leu Gly Leu Asp Ala Leu Ile Leu Leu Arg Asp 195 200 205
Lys Asn Phe Leu Val Phe Phe Phe Cys Ser Phe Leu Phe Ala Met Pro 210
215 220 Leu Ala Phe Tyr Tyr Ile Phe Ala Asn Gly Tyr Leu Thr Glu Val
Gly 225 230 235 240 Met Lys Asn Ala Thr Gly Trp Met Thr Leu Gly Gln
Phe Ser Glu Ile 245 250 255 Phe Phe Met Leu Ala Leu Pro Phe Phe Thr
Lys Arg Phe Gly Ile Lys 260 265 270 Lys Val Leu Leu Leu Gly Leu Val
Thr Ala Ala Ile Arg Tyr Gly Phe 275 280 285 Phe Ile Tyr Gly Ser Ala
Asp Glu Tyr Phe Thr Tyr Ala Leu Leu Phe 290 295 300 Leu Gly Ile Leu
Leu His Gly Val Ser Tyr Asp Phe Tyr Tyr Val Thr 305 310 315 320 Ala
Tyr Ile Tyr Val Asp Lys Lys Ala Pro Val His Met Arg Thr Ala 325 330
335 Ala Gln Gly Leu Ile Thr Leu Cys Cys Gln Gly Phe Gly Ser Leu Leu
340 345 350 Gly Tyr Arg Leu Gly Gly Val Met Met Glu Lys Met Phe Ala
Tyr Gln 355 360 365 Glu Pro Val Asn Gly Leu Thr Phe Asn Trp Ser Gly
Met Trp Thr Phe 370 375 380 Gly Ala Val Met Ile Ala Ile Ile Ala Val
Leu Phe Met Ile Phe Phe 385 390 395 400 Arg Glu Ser Asp Asn Glu Ile
Thr Ala Ile Lys Val Asp Asp Arg Asp 405 410 415 Ile Ala Leu Thr Gln
Gly Glu Val Lys 420 425 <210> SEQ ID NO 152 <211>
LENGTH: 418 <212> TYPE: PRT <213> ORGANISM: Escherichia
coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(418)
<223> OTHER INFORMATION: nupG - Escherichia coli K-12 W3110
(P09452; JW2932 <400> SEQUENCE: 152 Met Asn Leu Lys Leu Gln
Leu Lys Ile Leu Ser Phe Leu Gln Phe Cys 1 5 10 15 Leu Trp Gly Ser
Trp Leu Thr Thr Leu Gly Ser Tyr Met Phe Val Thr 20 25 30 Leu Lys
Phe Asp Gly Ala Ser Ile Gly Ala Val Tyr Ser Ser Leu Gly 35 40 45
Ile Ala Ala Val Phe Met Pro Ala Leu Leu Gly Ile Val Ala Asp Lys 50
55 60 Trp Leu Ser Ala Lys Trp Val Tyr Ala Ile Cys His Thr Ile Gly
Ala 65 70 75 80 Ile Thr Leu Phe Met Ala Ala Gln Val Thr Thr Pro Glu
Ala Met Phe 85 90 95 Leu Val Ile Leu Ile Asn Ser Phe Ala Tyr Met
Pro Thr Leu Gly Leu 100 105 110 Ile Asn Thr Ile Ser Tyr Tyr Arg Leu
Gln Asn Ala Gly Met Asp Ile 115 120 125 Val Thr Asp Phe Pro Pro Ile
Arg Ile Trp Gly Thr Ile Gly Phe Ile 130 135 140 Met Ala Met Trp Val
Val Ser Leu Ser Gly Phe Glu Leu Ser His Met 145 150 155 160 Gln Leu
Tyr Ile Gly Ala Ala Leu Ser Ala Ile Leu Val Leu Phe Thr 165 170 175
Leu Thr Leu Pro His Ile Pro Val Ala Lys Gln Gln Ala Asn Gln Ser 180
185 190 Trp Thr Thr Leu Leu Gly Leu Asp Ala Phe Ala Leu Phe Lys Asn
Lys 195 200 205 Arg Met Ala Ile Phe Phe Ile Phe Ser Met Leu Leu Gly
Ala Glu Leu 210 215 220 Gln Ile Thr Asn Met Phe Gly Asn Thr Phe Leu
His Ser Phe Asp Lys 225 230 235 240 Asp Pro Met Phe Ala Ser Ser Phe
Ile Val Gln His Ala Ser Ile Ile 245 250 255 Met Ser Ile Ser Gln Ile
Ser Glu Thr Leu Phe Ile Leu Thr Ile Pro 260 265 270 Phe Phe Leu Ser
Arg Tyr Gly Ile Lys Asn Val Met Met Ile Ser Ile 275 280 285 Val Ala
Trp Ile Leu Arg Phe Ala Leu Phe Ala Tyr Gly Asp Pro Thr 290 295 300
Pro Phe Gly Thr Val Leu Leu Val Leu Ser Met Ile Val Tyr Gly Cys 305
310 315 320 Ala Phe Asp Phe Phe Asn Ile Ser Gly Ser Val Phe Val Glu
Lys Glu 325 330 335 Val Ser Pro Ala Ile Arg Ala Ser Ala Gln Gly Met
Phe Leu Met Met 340 345 350 Thr Asn Gly Phe Gly Cys Ile Leu Gly Gly
Ile Val Ser Gly Lys Val 355 360 365 Val Glu Met Tyr Thr Gln Asn Gly
Ile Thr Asp Trp Gln Thr Val Trp 370 375 380 Leu Ile Phe Ala Gly Tyr
Ser Val Val Leu Ala Phe Ala Phe Met Ala 385 390 395 400 Met Phe Lys
Tyr Lys His Val Arg Val Pro Thr Gly Thr Gln Thr Val 405 410 415 Ser
His <210> SEQ ID NO 153 <211> LENGTH: 418 <212>
TYPE: PRT <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(418) <223> OTHER INFORMATION: xapB - Escherichia coli
K-12 W3110 (P45562; JW2397) <400> SEQUENCE: 153 Met Ser Ile
Ala Met Arg Leu Lys Val Met Ser Phe Leu Gln Tyr Phe 1 5 10 15 Ile
Trp Gly Ser Trp Leu Val Thr Leu Gly Ser Tyr Met Ile Asn Thr 20 25
30 Leu His Phe Thr Gly Ala Asn Val Gly Met Val Tyr Ser Ser Lys Gly
35 40 45 Ile Ala Ala Ile Ile Met Pro Gly Ile Met Gly Ile Ile Ala
Asp Lys 50 55 60 Trp Leu Arg Ala Glu Arg Ala Tyr Met Leu Cys His
Leu Val Cys Ala 65 70 75 80 Gly Val Leu Phe Tyr Ala Ala Ser Val Thr
Asp Pro Asp Met Met Phe 85 90 95 Trp Val Met Leu Val Asn Ala Met
Ala Phe Met Pro Thr Ile Ala Leu 100 105 110 Ser Asn Ser Val Ser Tyr
Ser Cys Leu Ala Gln Ala Gly Leu Asp Pro 115 120 125 Val Thr Ala Phe
Pro Pro Ile Arg Val Phe Gly Thr Val Gly Phe Ile 130 135 140 Val Ala
Met Trp Ala Val Ser Leu Leu His Leu Glu Leu Ser Ser Leu 145 150 155
160 Gln Leu Tyr Ile Ala Ser Gly Ala Ser Leu Leu Leu Ser Ala Tyr Ala
165 170 175 Leu Thr Leu Pro Lys Ile Pro Val Ala Glu Lys Lys Ala Thr
Thr Ser 180 185 190 Leu Ala Ser Lys Leu Gly Leu Asp Ala Phe Val Leu
Phe Lys Asn Pro 195 200 205 Arg Met Ala Ile Phe Phe Leu Phe Ala Met
Met Leu Gly Ala Val Leu 210 215 220 Gln Ile Thr Asn Val Phe Gly Asn
Pro Phe Leu His Asp Phe Ala Arg 225 230 235 240 Asn Pro Glu Phe Ala
Asp Ser Phe Val Val Lys Tyr Pro Ser Ile Leu 245 250 255 Leu Ser Val
Ser Gln Met Ala Glu Val Gly Phe Ile Leu Thr Ile Pro 260 265 270 Phe
Phe Leu Lys Arg Phe Gly Ile Lys Thr Val Met Leu Met Ser Met 275 280
285 Val Ala Trp Thr Leu Arg Phe Gly Phe Phe Ala Tyr Gly Asp Pro Ser
290 295 300 Thr Thr Gly Phe Ile Leu Leu Leu Leu Ser Met Ile Val Tyr
Gly Cys 305 310 315 320 Ala Phe Asp Phe Phe Asn Ile Ser Gly Ser Val
Phe Val Glu Gln Glu 325 330 335 Val Asp Ser Ser Ile Arg Ala Ser Ala
Gln Gly Leu Phe Met Thr Met 340 345 350 Val Asn Gly Val Gly Ala Trp
Val Gly Ser Ile Leu Ser Gly Met Ala 355 360 365 Val Asp Tyr Phe Ser
Val Asp Gly Val Lys Asp Trp Gln Thr Ile Trp 370 375 380 Leu Val Phe
Ala Gly Tyr Ala Leu Phe Leu Ala Val Ile Phe Phe Phe 385 390 395 400
Gly Phe Lys Tyr Asn His Asp Pro Glu Lys Ile Lys His Arg Ala Val 405
410 415 Thr His <210> SEQ ID NO 154 <211> LENGTH: 413
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(413) <223> OTHER INFORMATION: CC1628 -
Caulobacter crescentus CB15 (AAK23606) <400> SEQUENCE: 154
Met Gly Thr Ser Phe Arg Leu Phe Val Met Met Val Leu Gln Leu Ala 1 5
10 15 Ile Trp Gly Ala Trp Ala Pro Lys Ile Phe Pro Tyr Met Gly Met
Leu 20 25 30 Gly Phe Ala Pro Trp Gln Gln Ser Leu Val Gly Ser Ala
Trp Gly Val 35 40 45 Ala Ala Leu Val Gly Ile Phe Phe Ser Asn Gln
Phe Ala Asp Arg Asn 50 55 60 Phe Ser Ala Glu Arg Phe Leu Ala Val
Ser His Leu Ile Gly Gly Val 65 70 75 80 Ala Leu Leu Gly Thr Ala Phe
Ser Thr Glu Phe Trp Pro Phe Phe Ala 85 90 95 Cys Tyr Leu Val Phe
Ser Leu Val Tyr Val Pro Thr Leu Ser Val Thr 100 105 110 Asn Ser Ile
Ala Phe Ala Asn Leu Arg Asp Pro Ala Ala Gly Phe Gly 115 120 125 Gly
Val Arg Met Gly Gly Thr Val Gly Trp Val Leu Val Ser Trp Pro 130 135
140 Phe Val Phe Leu Leu Gly Ala Gln Ala Thr Val Glu Gln Val Arg Trp
145 150 155 160 Ile Phe Leu Val Ala Ala Ile Val Ser Phe Val Phe Ala
Gly Tyr Ala 165 170 175 Leu Thr Leu Pro His Thr Pro Pro Arg Lys Ala
Asp Asp Ala Val Asp 180 185 190 Lys Leu Ala Trp Arg Arg Ala Phe Lys
Leu Leu Gly Ala Pro Phe Val 195 200 205 Phe Val Leu Phe Val Val Thr
Phe Ile Asp Ser Val Ile His Asn Gly 210 215 220 Tyr Phe Val Met Ala
Asp Ala Phe Leu Thr Asn Arg Val Gly Ile Ala 225 230 235 240 Gly Asn
Leu Ser Met Val Val Leu Ser Leu Gly Gln Val Ala Glu Ile 245 250 255
Ile Thr Met Leu Leu Leu Gly Arg Val Leu Ala Lys Leu Gly Trp Lys 260
265 270 Val Thr Met Ile Val Gly Val Leu Gly His Ala Ala Arg Phe Ala
Val 275 280 285 Phe Ala Tyr Phe Ala Asp Ser Val Pro Val Ile Val Ala
Val Gln Leu 290 295 300
Leu His Gly Val Cys Tyr Ala Phe Phe Phe Ala Thr Val Tyr Ile Phe 305
310 315 320 Val Asp Ala Val Phe Pro Lys Asp Val Arg Ser Ser Ala Gln
Gly Leu 325 330 335 Phe Asn Leu Leu Ile Leu Gly Val Gly Asn Val Ala
Ala Ser Phe Ile 340 345 350 Phe Pro Ala Leu Ile Gly Arg Leu Thr Thr
Asp Gly Ser Val Asp Tyr 355 360 365 Thr Thr Leu Phe Leu Val Pro Thr
Ala Met Ala Leu Ala Ala Val Cys 370 375 380 Leu Leu Ala Leu Phe Phe
Arg Pro Pro Thr Arg Gly Pro Val Ser Glu 385 390 395 400 Ala Asp Ser
Ala Ser Ser Ala Ala Ser Ser Ala Gln Ala 405 410 <210> SEQ ID
NO 155 <211> LENGTH: 419 <212> TYPE: PRT <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(419) <223>
OTHER INFORMATION: codB - Escherichia coli K-12 W3110 (P25525;
JW0327) <400> SEQUENCE: 155 Met Ser Gln Asp Asn Asn Phe Ser
Gln Gly Pro Val Pro Gln Ser Ala 1 5 10 15 Arg Lys Gly Val Leu Ala
Leu Thr Phe Val Met Leu Gly Leu Thr Phe 20 25 30 Phe Ser Ala Ser
Met Trp Thr Gly Gly Thr Leu Gly Thr Gly Leu Ser 35 40 45 Tyr His
Asp Phe Phe Leu Ala Val Leu Ile Gly Asn Leu Leu Leu Gly 50 55 60
Ile Tyr Thr Ser Phe Leu Gly Tyr Ile Gly Ala Lys Thr Gly Leu Thr 65
70 75 80 Thr His Leu Leu Ala Arg Phe Ser Phe Gly Val Lys Gly Ser
Trp Leu 85 90 95 Pro Ser Leu Leu Leu Gly Gly Thr Gln Val Gly Trp
Phe Gly Val Gly 100 105 110 Val Ala Met Phe Ala Ile Pro Val Gly Lys
Ala Thr Gly Leu Asp Ile 115 120 125 Asn Leu Leu Ile Ala Val Ser Gly
Leu Leu Met Thr Val Thr Val Phe 130 135 140 Phe Gly Ile Ser Ala Leu
Thr Val Leu Ser Val Ile Ala Val Pro Ala 145 150 155 160 Ile Ala Cys
Leu Gly Gly Tyr Ser Val Trp Leu Ala Val Asn Gly Met 165 170 175 Gly
Gly Leu Asp Ala Leu Lys Ala Val Val Pro Ala Gln Pro Leu Asp 180 185
190 Phe Asn Val Ala Leu Ala Leu Val Val Gly Ser Phe Ile Ser Ala Gly
195 200 205 Thr Leu Thr Ala Asp Phe Val Arg Phe Gly Arg Asn Ala Lys
Leu Ala 210 215 220 Val Leu Val Ala Met Val Ala Phe Phe Leu Gly Asn
Ser Leu Met Phe 225 230 235 240 Ile Phe Gly Ala Ala Gly Ala Ala Ala
Leu Gly Met Ala Asp Ile Ser 245 250 255 Asp Val Met Ile Ala Gln Gly
Leu Leu Leu Pro Ala Ile Val Val Leu 260 265 270 Gly Leu Asn Ile Trp
Thr Thr Asn Asp Asn Ala Leu Tyr Ala Ser Gly 275 280 285 Leu Gly Phe
Ala Asn Ile Thr Gly Met Ser Ser Lys Thr Leu Ser Val 290 295 300 Ile
Asn Gly Ile Ile Gly Thr Val Cys Ala Leu Trp Leu Tyr Asn Asn 305 310
315 320 Phe Val Gly Trp Leu Thr Phe Leu Ser Ala Ala Ile Pro Pro Val
Gly 325 330 335 Gly Val Ile Ile Ala Asp Tyr Leu Met Asn Arg Arg Arg
Tyr Glu His 340 345 350 Phe Ala Thr Thr Arg Met Met Ser Val Asn Trp
Val Ala Ile Leu Ala 355 360 365 Val Ala Leu Gly Ile Ala Ala Gly His
Trp Leu Pro Gly Ile Val Pro 370 375 380 Val Asn Ala Val Leu Gly Gly
Ala Leu Ser Tyr Leu Ile Leu Asn Pro 385 390 395 400 Ile Leu Asn Arg
Lys Thr Thr Ala Ala Met Thr His Val Glu Ala Asn 405 410 415 Ser Val
Glu <210> SEQ ID NO 156 <211> LENGTH: 428 <212>
TYPE: PRT <213> ORGANISM: Escherichia coli <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(428) <223> OTHER INFORMATION: amtB - Escherichia coli
K-12 MG1655 (B0451; 945084) <400> SEQUENCE: 156 Met Lys Ile
Ala Thr Ile Lys Thr Gly Leu Ala Ser Leu Ala Met Leu 1 5 10 15 Pro
Gly Leu Val Met Ala Ala Pro Ala Val Ala Asp Lys Ala Asp Asn 20 25
30 Ala Phe Met Met Ile Cys Thr Ala Leu Val Leu Phe Met Thr Ile Pro
35 40 45 Gly Ile Ala Leu Phe Tyr Gly Gly Leu Ile Arg Gly Lys Asn
Val Leu 50 55 60 Ser Met Leu Thr Gln Val Thr Val Thr Phe Ala Leu
Val Cys Ile Leu 65 70 75 80 Trp Val Val Tyr Gly Tyr Ser Leu Ala Phe
Gly Glu Gly Asn Asn Phe 85 90 95 Phe Gly Asn Ile Asn Trp Leu Met
Leu Lys Asn Ile Glu Leu Thr Ala 100 105 110 Val Met Gly Ser Ile Tyr
Gln Tyr Ile His Val Ala Phe Gln Gly Ser 115 120 125 Phe Ala Cys Ile
Thr Val Gly Leu Ile Val Gly Ala Leu Ala Glu Arg 130 135 140 Ile Arg
Phe Ser Ala Val Leu Ile Phe Val Val Val Trp Leu Thr Leu 145 150 155
160 Ser Tyr Ile Pro Ile Ala His Met Val Trp Gly Gly Gly Leu Leu Ala
165 170 175 Ser His Gly Ala Leu Asp Phe Ala Gly Gly Thr Val Val His
Ile Asn 180 185 190 Ala Ala Ile Ala Gly Leu Val Gly Ala Tyr Leu Ile
Gly Lys Arg Val 195 200 205 Gly Phe Gly Lys Glu Ala Phe Lys Pro His
Asn Leu Pro Met Val Phe 210 215 220 Thr Gly Thr Ala Ile Leu Tyr Ile
Gly Trp Phe Gly Phe Asn Ala Gly 225 230 235 240 Ser Ala Gly Thr Ala
Asn Glu Ile Ala Ala Leu Ala Phe Val Asn Thr 245 250 255 Val Val Ala
Thr Ala Ala Ala Ile Leu Gly Trp Ile Phe Gly Glu Trp 260 265 270 Ala
Leu Arg Gly Lys Pro Ser Leu Leu Gly Ala Cys Ser Gly Ala Ile 275 280
285 Ala Gly Leu Val Gly Val Thr Pro Ala Cys Gly Tyr Ile Gly Val Gly
290 295 300 Gly Ala Leu Ile Ile Gly Val Val Ala Gly Leu Ala Gly Leu
Trp Gly 305 310 315 320 Val Thr Met Leu Lys Arg Leu Leu Arg Val Asp
Asp Pro Cys Asp Val 325 330 335 Phe Gly Val His Gly Val Cys Gly Ile
Val Gly Cys Ile Met Thr Gly 340 345 350 Ile Phe Ala Ala Ser Ser Leu
Gly Gly Val Gly Phe Ala Glu Gly Val 355 360 365 Thr Met Gly His Gln
Leu Leu Val Gln Leu Glu Ser Ile Ala Ile Thr 370 375 380 Ile Val Trp
Ser Gly Val Val Ala Phe Ile Gly Tyr Lys Leu Ala Asp 385 390 395 400
Leu Thr Val Gly Leu Arg Val Pro Glu Glu Gln Glu Arg Glu Gly Leu 405
410 415 Asp Val Asn Ser His Gly Glu Asn Ala Tyr Asn Ala 420 425
<210> SEQ ID NO 157 <211> LENGTH: 466 <212> TYPE:
PRT <213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(466)
<223> OTHER INFORMATION: GABA permease GabP Escherichia coli
<400> SEQUENCE: 157 Met Gly Gln Ser Ser Gln Pro His Glu Leu
Gly Gly Gly Leu Lys Ser 1 5 10 15 Arg His Val Thr Met Leu Ser Ile
Ala Gly Val Ile Gly Ala Ser Leu 20 25 30 Phe Val Gly Ser Ser Val
Ala Ile Ala Glu Ala Gly Pro Ala Val Leu 35 40 45 Leu Ala Tyr Leu
Phe Ala Gly Leu Leu Val Val Met Ile Met Arg Met 50 55 60 Leu Ala
Glu Met Ala Val Ala Thr Pro Asp Thr Gly Ser Phe Ser Thr 65 70 75 80
Tyr Ala Asp Lys Ala Ile Gly Arg Trp Ala Gly Tyr Thr Ile Gly Trp 85
90 95 Leu Tyr Trp Trp Phe Trp Val Leu Val Ile Pro Leu Glu Ala Asn
Ile 100 105 110 Ala Ala Met Ile Leu His Ser Trp Val Pro Gly Ile Pro
Ile Trp Leu 115 120 125 Phe Ser Leu Val Ile Thr Leu Ala Leu Thr Gly
Ser Asn Leu Leu Ser 130 135 140 Val Lys Asn Tyr Gly Glu Phe Glu Phe
Trp Leu Ala Leu Cys Lys Val
145 150 155 160 Ile Ala Ile Leu Ala Phe Ile Phe Leu Gly Ala Val Ala
Ile Ser Gly 165 170 175 Phe Tyr Pro Tyr Ala Glu Val Ser Gly Ile Ser
Arg Leu Trp Asp Ser 180 185 190 Gly Gly Phe Met Pro Asn Gly Phe Gly
Ala Val Leu Ser Ala Met Leu 195 200 205 Ile Thr Met Phe Ser Phe Met
Gly Ala Glu Ile Val Thr Ile Ala Ala 210 215 220 Ala Glu Ser Asp Thr
Pro Glu Lys His Ile Val Arg Ala Thr Asn Ser 225 230 235 240 Val Ile
Trp Arg Ile Ser Ile Phe Tyr Leu Cys Ser Ile Phe Val Val 245 250 255
Val Ala Leu Ile Pro Trp Asn Met Pro Gly Leu Lys Ala Val Gly Ser 260
265 270 Tyr Arg Ser Val Leu Glu Leu Leu Asn Ile Pro His Ala Lys Leu
Ile 275 280 285 Met Asp Cys Val Ile Leu Leu Ser Val Thr Ser Cys Leu
Asn Ser Ala 290 295 300 Leu Tyr Thr Ala Ser Arg Met Leu Tyr Ser Leu
Ser Arg Arg Gly Asp 305 310 315 320 Ala Pro Ala Val Met Gly Lys Ile
Asn Arg Ser Lys Thr Pro Tyr Val 325 330 335 Ala Val Leu Leu Ser Thr
Gly Ala Ala Phe Leu Thr Val Val Val Asn 340 345 350 Tyr Tyr Ala Pro
Ala Lys Val Phe Lys Phe Leu Ile Asp Ser Ser Gly 355 360 365 Ala Ile
Ala Leu Leu Val Tyr Leu Val Ile Ala Val Ser Gln Leu Arg 370 375 380
Met Arg Lys Ile Leu Arg Ala Glu Gly Ser Glu Ile Arg Leu Arg Met 385
390 395 400 Trp Leu Tyr Pro Trp Leu Thr Trp Leu Val Ile Gly Phe Ile
Thr Phe 405 410 415 Val Leu Val Val Met Leu Phe Arg Pro Ala Gln Gln
Leu Glu Val Ile 420 425 430 Ser Thr Gly Leu Leu Ala Ile Gly Ile Ile
Cys Thr Val Pro Ile Met 435 440 445 Ala Arg Trp Lys Lys Leu Val Leu
Trp Gln Lys Thr Pro Val His Asn 450 455 460 Thr Arg 465 <210>
SEQ ID NO 158 <211> LENGTH: 412 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(412)
<223> OTHER INFORMATION: mtnH Escherichia coli <400>
SEQUENCE: 158 Met Thr Asn Tyr Arg Val Glu Ser Ser Ser Gly Arg Ala
Ala Arg Lys 1 5 10 15 Met Arg Leu Ala Leu Met Gly Pro Ala Phe Ile
Ala Ala Ile Gly Tyr 20 25 30 Ile Asp Pro Gly Asn Phe Ala Thr Asn
Ile Gln Ala Gly Ala Ser Phe 35 40 45 Gly Tyr Gln Leu Leu Trp Val
Val Val Trp Ala Asn Leu Met Ala Met 50 55 60 Leu Ile Gln Ile Leu
Ser Ala Lys Leu Gly Ile Ala Thr Gly Lys Asn 65 70 75 80 Leu Ala Glu
Gln Ile Arg Asp His Tyr Pro Arg Pro Val Val Trp Phe 85 90 95 Tyr
Trp Val Gln Ala Glu Ile Ile Ala Met Ala Thr Asp Leu Ala Glu 100 105
110 Phe Ile Gly Ala Ala Ile Gly Phe Lys Leu Ile Leu Gly Val Ser Leu
115 120 125 Leu Gln Gly Ala Val Leu Thr Gly Ile Ala Thr Phe Leu Ile
Leu Met 130 135 140 Leu Gln Arg Arg Gly Gln Lys Pro Leu Glu Lys Val
Ile Gly Gly Leu 145 150 155 160 Leu Leu Phe Val Ala Ala Ala Tyr Ile
Val Glu Leu Ile Phe Ser Gln 165 170 175 Pro Asn Leu Ala Gln Leu Gly
Lys Gly Met Val Ile Pro Ser Leu Pro 180 185 190 Thr Ser Glu Ala Val
Phe Leu Ala Ala Gly Val Leu Gly Ala Thr Ile 195 200 205 Met Pro His
Val Ile Tyr Leu His Ser Ser Leu Thr Gln His Leu His 210 215 220 Gly
Gly Ser Arg Gln Gln Arg Tyr Ser Ala Thr Lys Trp Asp Val Ala 225 230
235 240 Ile Ala Met Thr Ile Ala Gly Phe Val Asn Leu Ala Met Met Ala
Thr 245 250 255 Ala Ala Ala Ala Phe His Phe Ser Gly His Thr Gly Val
Ala Asp Leu 260 265 270 Asp Glu Ala Tyr Leu Thr Leu Gln Pro Leu Leu
Ser His Ala Ala Ala 275 280 285 Thr Val Phe Gly Leu Ser Leu Val Ala
Ala Gly Leu Ser Ser Thr Val 290 295 300 Val Gly Thr Leu Ala Gly Gln
Val Val Met Gln Gly Phe Ile Arg Phe 305 310 315 320 His Ile Pro Leu
Trp Val Arg Arg Thr Val Thr Met Leu Pro Ser Phe 325 330 335 Ile Val
Ile Leu Met Gly Leu Asp Pro Thr Arg Ile Leu Val Met Ser 340 345 350
Gln Val Leu Leu Ser Phe Gly Ile Ala Leu Ala Leu Val Pro Leu Leu 355
360 365 Ile Phe Thr Ser Asp Ser Lys Leu Met Gly Asp Leu Val Asn Ser
Lys 370 375 380 Arg Val Lys Gln Thr Gly Trp Val Ile Val Val Leu Val
Val Ala Leu 385 390 395 400 Asn Ile Trp Leu Leu Val Gly Thr Ala Leu
Gly Leu 405 410 <210> SEQ ID NO 159 <211> LENGTH: 1320
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1320) <223> OTHER INFORMATION: Nucleotide
sequence: BCAA transporter BrnQ from E. coli <400> SEQUENCE:
159 atgacccatc aattaagatc gcgcgatatc atcgctctgg gctttatgac
atttgcgttg 60 ttcgtcggcg caggtaacat tattttccct ccaatggtcg
gcttgcaggc aggcgaacac 120 gtctggactg cggcattcgg cttcctcatt
actgccgttg gcctaccggt attaacggta 180 gtggcgctgg caaaagttgg
cggcggtgtt gacagtctca gcacgccaat tggtaaagtc 240 gctggcgtac
tgctggcaac agtttgttac ctggcggtgg ggccgctttt tgctacgccg 300
cgtacagcta ccgtttcttt tgaagtgggc attgcgccgc tgacgggtga ttccgcgctg
360 ccgctgttta tttacagcct ggtctatttc gctatcgtta ttctggtttc
gctctatccg 420 ggcaagctgc tggataccgt gggcaacttc cttgcgccgc
tgaaaattat cgcgctggtc 480 atcctgtctg ttgccgcaat tatctggccg
gcgggttcta tcagtacggc gactgaggct 540 tatcaaaacg ctgcgttttc
taacggcttc gtcaacggct atctgaccat ggatacgctg 600 ggcgcaatgg
tgtttggtat cgttattgtt aacgcggcgc gttctcgtgg cgttaccgaa 660
gcgcgtctgc tgacccgtta taccgtctgg gctggcctga tggcgggtgt tggtctgact
720 ctgctgtacc tggcgctgtt ccgtctgggt tcagacagcg cgtcgctggt
cgatcagtct 780 gcaaacggtg cggcgatcct gcatgcttac gttcagcata
cctttggcgg cggcggtagc 840 ttcctgctgg cggcgttaat cttcatcgcc
tgcctggtca cggcggttgg cctgacctgt 900 gcttgtgcag aattcttcgc
ccagtacgta ccgctctctt atcgtacgct ggtgtttatc 960 ctcggcggct
tctcgatggt ggtgtctaac ctcggcttga gccagctgat tcagatctct 1020
gtaccggtgc tgaccgccat ttatccgccg tgtatcgcac tggttgtatt aagttttaca
1080 cgctcatggt ggcataattc gtcccgcgtg attgctccgc cgatgtttat
cagcctgctt 1140 tttggtattc tcgacgggat caaggcatct gcattcagcg
atatcttacc gtcctgggcg 1200 cagcgtttac cgctggccga acaaggtctg
gcgtggttaa tgccaacagt ggtgatggtg 1260 gttctggcca ttatctggga
tcgtgcggca ggtcgtcagg tgacctccag cgctcactaa 1320 <210> SEQ ID
NO 160 <211> LENGTH: 439 <212> TYPE: PRT <213>
ORGANISM: Escherichia coli <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(439) <223>
OTHER INFORMATION: AA sequence: BCAA transporter BrnQ from E. coli
<400> SEQUENCE: 160 Met Thr His Gln Leu Arg Ser Arg Asp Ile
Ile Ala Leu Gly Phe Met 1 5 10 15 Thr Phe Ala Leu Phe Val Gly Ala
Gly Asn Ile Ile Phe Pro Pro Met 20 25 30 Val Gly Leu Gln Ala Gly
Glu His Val Trp Thr Ala Ala Phe Gly Phe 35 40 45 Leu Ile Thr Ala
Val Gly Leu Pro Val Leu Thr Val Val Ala Leu Ala 50 55 60 Lys Val
Gly Gly Gly Val Asp Ser Leu Ser Thr Pro Ile Gly Lys Val 65 70 75 80
Ala Gly Val Leu Leu Ala Thr Val Cys Tyr Leu Ala Val Gly Pro Leu 85
90 95 Phe Ala Thr Pro Arg Thr Ala Thr Val Ser Phe Glu Val Gly Ile
Ala 100 105 110 Pro Leu Thr Gly Asp Ser Ala Leu Pro Leu Phe Ile Tyr
Ser Leu Val 115 120 125 Tyr Phe Ala Ile Val Ile Leu Val Ser Leu Tyr
Pro Gly Lys Leu Leu 130 135 140 Asp Thr Val Gly Asn Phe Leu Ala Pro
Leu Lys Ile Ile Ala Leu Val
145 150 155 160 Ile Leu Ser Val Ala Ala Ile Ile Trp Pro Ala Gly Ser
Ile Ser Thr 165 170 175 Ala Thr Glu Ala Tyr Gln Asn Ala Ala Phe Ser
Asn Gly Phe Val Asn 180 185 190 Gly Tyr Leu Thr Met Asp Thr Leu Gly
Ala Met Val Phe Gly Ile Val 195 200 205 Ile Val Asn Ala Ala Arg Ser
Arg Gly Val Thr Glu Ala Arg Leu Leu 210 215 220 Thr Arg Tyr Thr Val
Trp Ala Gly Leu Met Ala Gly Val Gly Leu Thr 225 230 235 240 Leu Leu
Tyr Leu Ala Leu Phe Arg Leu Gly Ser Asp Ser Ala Ser Leu 245 250 255
Val Asp Gln Ser Ala Asn Gly Ala Ala Ile Leu His Ala Tyr Val Gln 260
265 270 His Thr Phe Gly Gly Gly Gly Ser Phe Leu Leu Ala Ala Leu Ile
Phe 275 280 285 Ile Ala Cys Leu Val Thr Ala Val Gly Leu Thr Cys Ala
Cys Ala Glu 290 295 300 Phe Phe Ala Gln Tyr Val Pro Leu Ser Tyr Arg
Thr Leu Val Phe Ile 305 310 315 320 Leu Gly Gly Phe Ser Met Val Val
Ser Asn Leu Gly Leu Ser Gln Leu 325 330 335 Ile Gln Ile Ser Val Pro
Val Leu Thr Ala Ile Tyr Pro Pro Cys Ile 340 345 350 Ala Leu Val Val
Leu Ser Phe Thr Arg Ser Trp Trp His Asn Ser Ser 355 360 365 Arg Val
Ile Ala Pro Pro Met Phe Ile Ser Leu Leu Phe Gly Ile Leu 370 375 380
Asp Gly Ile Lys Ala Ser Ala Phe Ser Asp Ile Leu Pro Ser Trp Ala 385
390 395 400 Gln Arg Leu Pro Leu Ala Glu Gln Gly Leu Ala Trp Leu Met
Pro Thr 405 410 415 Val Val Met Val Val Leu Ala Ile Ile Trp Asp Arg
Ala Ala Gly Arg 420 425 430 Gln Val Thr Ser Ser Ala His 435
<210> SEQ ID NO 161 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: consensus sequence
<400> SEQUENCE: 161 ttgttgayry rtcaacwa 18 <210> SEQ ID
NO 162 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic: consensus sequence <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: n is a, c, g, or t
<400> SEQUENCE: 162 ttataatnat tataa 15 <210> SEQ ID NO
163 <211> LENGTH: 355 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic: katG Regulatory sequence <400>
SEQUENCE: 163 tgtggctttt atgaaaatca cacagtgatc acaaatttta
aacagagcac aaaatgctgc 60 ctcgaaatga gggcgggaaa ataaggttat
cagccttgtt ttctccctca ttacttgaag 120 gatatgaagc taaaaccctt
ttttataaag catttgtccg aattcggaca taatcaaaaa 180 agcttaatta
agatcaattt gatctacatc tctttaacca acaatatgta agatctcaac 240
tatcgcatcc gtggattaat tcaattataa cttctctcta acgctgtgta tcgtaacggt
300 aacactgtag aggggagcac attgatgcga attcattaaa gaggagaaag gtacc
355 <210> SEQ ID NO 164 <211> LENGTH: 228 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic: dps Regulatory
sequence <400> SEQUENCE: 164 ttccgaaaat tcctggcgag cagataaata
agaattgttc ttatcaatat atctaactca 60 ttgaatcttt attagttttg
tttttcacgc ttgttaccac tattagtgtg ataggaacag 120 ccagaatagc
ggaacacata gccggtgcta tacttaatct cgttaattac tgggacataa 180
catcaagagg atatgaaatt cgaattcatt aaagaggaga aaggtacc 228
<210> SEQ ID NO 165 <211> LENGTH: 334 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: ahpC Regulatory sequence
<400> SEQUENCE: 165 gcttagatca ggtgattgcc ctttgtttat
gagggtgttg taatccatgt cgttgttgca 60 tttgtaaggg caacacctca
gcctgcaggc aggcactgaa gataccaaag ggtagttcag 120 attacacggt
cacctggaaa gggggccatt ttacttttta tcgccgctgg cggtgcaaag 180
ttcacaaagt tgtcttacga aggttgtaag gtaaaactta tcgatttgat aatggaaacg
240 cattagccga atcggcaaaa attggttacc ttacatctca tcgaaaacac
ggaggaagta 300 tagatgcgaa ttcattaaag aggagaaagg tacc 334
<210> SEQ ID NO 166 <211> LENGTH: 134 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic: oxyS Regulatory sequence
<400> SEQUENCE: 166 ctcgagttca ttatccatcc tccatcgcca
cgatagttca tggcgatagg tagaatagca 60 atgaacgatt atccctatca
agcattctga ctgataattg ctcacacgaa ttcattaaag 120 aggagaaagg tacc 134
<210> SEQ ID NO 167 <211> LENGTH: 416 <212> TYPE:
PRT <213> ORGANISM: Pseudomonas sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(416)
<223> OTHER INFORMATION: Pseudomonas kynureninase <400>
SEQUENCE: 167 Met Thr Thr Arg Asn Asp Cys Leu Ala Leu Asp Ala Gln
Asp Ser Leu 1 5 10 15 Ala Pro Leu Arg Gln Gln Phe Ala Leu Pro Glu
Gly Val Ile Tyr Leu 20 25 30 Asp Gly Asn Ser Leu Gly Ala Arg Pro
Val Ala Ala Leu Ala Arg Ala 35 40 45 Gln Ala Val Ile Ala Glu Glu
Trp Gly Asn Gly Leu Ile Arg Ser Trp 50 55 60 Asn Ser Ala Gly Trp
Arg Asp Leu Ser Glu Arg Leu Gly Asn Arg Leu 65 70 75 80 Ala Thr Leu
Ile Gly Ala Arg Asp Gly Glu Val Val Val Thr Asp Thr 85 90 95 Thr
Ser Ile Asn Leu Phe Lys Val Leu Ser Ala Ala Leu Arg Val Gln 100 105
110 Ala Thr Arg Ser Pro Glu Arg Arg Val Ile Val Thr Glu Thr Ser Asn
115 120 125 Phe Pro Thr Asp Leu Tyr Ile Ala Glu Gly Leu Ala Asp Met
Leu Gln 130 135 140 Gln Gly Tyr Thr Leu Arg Leu Val Asp Ser Pro Glu
Glu Leu Pro Gln 145 150 155 160 Ala Ile Asp Gln Asp Thr Ala Val Val
Met Leu Thr His Val Asn Tyr 165 170 175 Lys Thr Gly Tyr Met His Asp
Met Gln Ala Leu Thr Ala Leu Ser His 180 185 190 Glu Cys Gly Ala Leu
Ala Ile Trp Asp Leu Ala His Ser Ala Gly Ala 195 200 205 Val Pro Val
Asp Leu His Gln Ala Gly Ala Asp Tyr Ala Ile Gly Cys 210 215 220 Thr
Tyr Lys Tyr Leu Asn Gly Gly Pro Gly Ser Gln Ala Phe Val Trp 225 230
235 240 Val Ser Pro Gln Leu Cys Asp Leu Val Pro Gln Pro Leu Ser Gly
Trp 245 250 255 Phe Gly His Ser Arg Gln Phe Ala Met Glu Pro Arg Tyr
Glu Pro Ser 260 265 270 Asn Gly Ile Ala Arg Tyr Leu Cys Gly Thr Gln
Pro Ile Thr Ser Leu 275 280 285 Ala Met Val Glu Cys Gly Leu Asp Val
Phe Ala Gln Thr Asp Met Ala 290 295 300 Ser Leu Arg Arg Lys Ser Leu
Ala Leu Thr Asp Leu Phe Ile Glu Leu 305 310 315 320 Val Glu Gln Arg
Cys Ala Ala His Glu Leu Thr Leu Val Thr Pro Arg 325 330 335 Glu His
Ala Lys Arg Gly Ser His Val Ser Phe Glu His Pro Glu Gly 340 345 350
Tyr Ala Val Ile Gln Ala Leu Ile Asp Arg Gly Val Ile Gly Asp Tyr 355
360 365 Arg Glu Pro Arg Ile Met Arg Phe Gly Phe Thr Pro Leu Tyr Thr
Thr 370 375 380 Phe Thr Glu Val Trp Asp Ala Val Gln Ile Leu Gly Glu
Ile Leu Asp 385 390 395 400 Arg Lys Thr Trp Ala Gln Ala Gln Phe Gln
Val Arg His Ser Val Thr 405 410 415
<210> SEQ ID NO 168 <211> LENGTH: 465 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(465)
<223> OTHER INFORMATION: Human kynureninase <400>
SEQUENCE: 168 Met Glu Pro Ser Ser Leu Glu Leu Pro Ala Asp Thr Val
Gln Arg Ile 1 5 10 15 Ala Ala Glu Leu Lys Cys His Pro Thr Asp Glu
Arg Val Ala Leu His 20 25 30 Leu Asp Glu Glu Asp Lys Leu Arg His
Phe Arg Glu Cys Phe Tyr Ile 35 40 45 Pro Lys Ile Gln Asp Leu Pro
Pro Val Asp Leu Ser Leu Val Asn Lys 50 55 60 Asp Glu Asn Ala Ile
Tyr Phe Leu Gly Asn Ser Leu Gly Leu Gln Pro 65 70 75 80 Lys Met Val
Lys Thr Tyr Leu Glu Glu Glu Leu Asp Lys Trp Ala Lys 85 90 95 Ile
Ala Ala Tyr Gly His Glu Val Gly Lys Arg Pro Trp Ile Thr Gly 100 105
110 Asp Glu Ser Ile Val Gly Leu Met Lys Asp Ile Val Gly Ala Asn Glu
115 120 125 Lys Glu Ile Ala Leu Met Asn Ala Leu Thr Val Asn Leu His
Leu Leu 130 135 140 Met Leu Ser Phe Phe Lys Pro Thr Pro Lys Arg Tyr
Lys Ile Leu Leu 145 150 155 160 Glu Ala Lys Ala Phe Pro Ser Asp His
Tyr Ala Ile Glu Ser Gln Leu 165 170 175 Gln Leu His Gly Leu Asn Ile
Glu Glu Ser Met Arg Met Ile Lys Pro 180 185 190 Arg Glu Gly Glu Glu
Thr Leu Arg Ile Glu Asp Ile Leu Glu Val Ile 195 200 205 Glu Lys Glu
Gly Asp Ser Ile Ala Val Ile Leu Phe Ser Gly Val His 210 215 220 Phe
Tyr Thr Gly Gln His Phe Asn Ile Pro Ala Ile Thr Lys Ala Gly 225 230
235 240 Gln Ala Lys Gly Cys Tyr Val Gly Phe Asp Leu Ala His Ala Val
Gly 245 250 255 Asn Val Glu Leu Tyr Leu His Asp Trp Gly Val Asp Phe
Ala Cys Trp 260 265 270 Cys Ser Tyr Lys Tyr Leu Asn Ala Gly Ala Gly
Gly Ile Ala Gly Ala 275 280 285 Phe Ile His Glu Lys His Ala His Thr
Ile Lys Pro Ala Leu Val Gly 290 295 300 Trp Phe Gly His Glu Leu Ser
Thr Arg Phe Lys Met Asp Asn Lys Leu 305 310 315 320 Gln Leu Ile Pro
Gly Val Cys Gly Phe Arg Ile Ser Asn Pro Pro Ile 325 330 335 Leu Leu
Val Cys Ser Leu His Ala Ser Leu Glu Ile Phe Lys Gln Ala 340 345 350
Thr Met Lys Ala Leu Arg Lys Lys Ser Val Leu Leu Thr Gly Tyr Leu 355
360 365 Glu Tyr Leu Ile Lys His Asn Tyr Gly Lys Asp Lys Ala Ala Thr
Lys 370 375 380 Lys Pro Val Val Asn Ile Ile Thr Pro Ser His Val Glu
Glu Arg Gly 385 390 395 400 Cys Gln Leu Thr Ile Thr Phe Ser Val Pro
Asn Lys Asp Val Phe Gln 405 410 415 Glu Leu Glu Lys Arg Gly Val Val
Cys Asp Lys Arg Asn Pro Asn Gly 420 425 430 Ile Arg Val Ala Pro Val
Pro Leu Tyr Asn Ser Phe His Asp Val Tyr 435 440 445 Lys Phe Thr Asn
Leu Leu Thr Ser Ile Leu Asp Ser Ala Glu Thr Lys 450 455 460 Asn 465
<210> SEQ ID NO 169 <211> LENGTH: 378 <212> TYPE:
PRT <213> ORGANISM: Shewanella sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(378)
<223> OTHER INFORMATION: Shewanella kynureninase <400>
SEQUENCE: 169 Met Leu Leu Asn Val Lys Gln Asp Phe Cys Leu Ala Gly
Pro Gly Tyr 1 5 10 15 Leu Leu Asn His Ser Val Gly Arg Pro Leu Lys
Ser Thr Glu Gln Ala 20 25 30 Leu Lys Gln Ala Phe Phe Ala Pro Trp
Gln Glu Ser Gly Arg Glu Pro 35 40 45 Trp Gly Gln Trp Leu Gly Val
Ile Asp Asn Phe Thr Ala Ala Leu Ala 50 55 60 Ser Leu Phe Asn Gly
Gln Pro Gln Asp Phe Cys Pro Gln Val Asn Leu 65 70 75 80 Ser Ser Ala
Leu Thr Lys Ile Val Met Ser Leu Asp Arg Leu Thr Arg 85 90 95 Asp
Leu Thr Arg Asn Gly Gly Ala Val Val Leu Met Ser Glu Ile Asp 100 105
110 Phe Pro Ser Met Gly Phe Ala Leu Lys Lys Ala Leu Pro Ala Ser Cys
115 120 125 Glu Leu Arg Phe Ile Pro Lys Ser Leu Asp Val Thr Asp Pro
Asn Val 130 135 140 Trp Asp Ala His Ile Cys Asp Asp Val Asp Leu Val
Phe Val Ser His 145 150 155 160 Ala Tyr Ser Asn Thr Gly Gln Gln Ala
Pro Leu Ala Gln Ile Ile Ser 165 170 175 Leu Ala Arg Glu Arg Gly Cys
Leu Ser Leu Val Asp Val Ala Gln Ser 180 185 190 Ala Gly Ile Leu Pro
Leu Asp Leu Ala Lys Leu Gln Pro Asp Phe Met 195 200 205 Ile Gly Ser
Ser Val Lys Trp Leu Cys Ser Gly Pro Gly Ala Ala Tyr 210 215 220 Leu
Trp Val Asn Pro Ala Ile Leu Pro Glu Cys Gln Pro Gln Asp Val 225 230
235 240 Gly Trp Phe Ser His Glu Asn Pro Phe Glu Phe Asp Ile His Asp
Phe 245 250 255 Arg Tyr His Pro Thr Ala Leu Arg Phe Trp Gly Gly Thr
Pro Ser Ile 260 265 270 Ala Pro Tyr Ala Ile Ala Ala His Ser Ile Glu
Tyr Phe Ala Asn Ile 275 280 285 Gly Ser Gln Val Met Arg Glu His Asn
Leu Gln Leu Met Glu Pro Val 290 295 300 Val Gln Ala Leu Asp Asn Glu
Leu Val Ser Pro Gln Glu Val Asp Lys 305 310 315 320 Arg Ser Gly Thr
Ile Ile Leu Gln Phe Gly Glu Arg Gln Pro Gln Ile 325 330 335 Leu Ala
Ala Leu Ala Ala Ala Asn Ile Ser Val Asp Thr Arg Ser Leu 340 345 350
Gly Ile Arg Val Ser Pro His Ile Tyr Asn Asp Glu Ala Asp Ile Ala 355
360 365 Arg Leu Leu Gly Val Ile Lys Ala Asn Arg 370 375 <210>
SEQ ID NO 170 <211> LENGTH: 1424 <212> TYPE: DNA
<213> ORGANISM: Pseudomonas sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1424) <223> OTHER INFORMATION: Ptet-kynU(Pseudomonas)
<400> SEQUENCE: 170 atctaatcta gacatcatta attcctaatt
tttgttgaca ctctatcatt gatagagtta 60 ttttaccact ccctatcagt
gatagagaaa agtgaattat ataaaagtgg gaggtgcccg 120 aatgacgacc
cgaaatgatt gcctagcgtt ggatgcacag gacagtctgg ctccgctgcg 180
ccaacaattt gcgctgccgg agggtgtgat atacctggat ggcaattcgc tgggcgcacg
240 tccggtagct gcgctggctc gcgcgcaggc tgtgatcgca gaagaatggg
gcaacgggtt 300 gatccgttca tggaactctg cgggctggcg tgatctgtct
gaacgcctgg gtaatcgcct 360 ggctaccctg attggtgcgc gcgatgggga
agtagttgtt actgatacca cctcgattaa 420 tctgtttaaa gtgctgtcag
cggcgctgcg cgtgcaagct acccgtagcc cggagcgccg 480 tgttatcgtg
actgagacct cgaatttccc gaccgacctg tatattgcgg aagggttggc 540
ggatatgctg caacaaggtt acactctgcg tttggtggat tcaccggaag agctgccaca
600 ggctatagat caggacaccg cggtggtgat gctgacgcac gtaaattata
aaaccggtta 660 tatgcacgac atgcaggctc tgaccgcgtt gagccacgag
tgtggggctc tggcgatttg 720 ggatctggcg cactctgctg gcgctgtgcc
ggtggacctg caccaagcgg gcgcggacta 780 tgcgattggc tgcacgtaca
aatacctgaa tggcggcccg ggttcgcaag cgtttgtttg 840 ggtttcgccg
caactgtgcg acctggtacc gcagccgctg tctggttggt tcggccatag 900
tcgccaattc gcgatggagc cgcgctacga accttctaac ggcattgctc gctatctgtg
960 cggcactcag cctattacta gcttggctat ggtggagtgc ggcctggatg
tgtttgcgca 1020 gacggatatg gcttcgctgc gccgtaaaag tctggcgctg
actgatctgt tcatcgagct 1080 ggttgaacaa cgctgcgctg cacacgaact
gaccctggtt actccacgtg aacacgcgaa 1140 acgcggctct cacgtgtctt
ttgaacaccc cgagggttac gctgttattc aagctctgat 1200 tgatcgtggc
gtgatcggcg attaccgtga gccacgtatt atgcgtttcg gtttcactcc 1260
tctgtatact acttttacgg aagtttggga tgcagtacaa atcctgggcg aaatcctgga
1320 tcgtaagact tgggcgcagg ctcagtttca ggtgcgccac tctgttactt
aaaaataaaa 1380 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttg
1424 <210> SEQ ID NO 171 <211> LENGTH: 1571 <212>
TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(1571)
<223> OTHER INFORMATION: Ptet-kynU(Human) <400>
SEQUENCE: 171 atctaatcta gacatcatta attcctaatt tttgttgaca
ctctatcatt gatagagtta 60 ttttaccact ccctatcagt gatagagaaa
agtgaatatc aagacacgag gaggtaagat 120 tatggagcct tcatctttag
aactgccagc ggacacggtg cagcgcatcg cggcggaact 180 gaagtgccat
ccgactgatg agcgtgtggc gctgcatctg gacgaagaag ataaactgcg 240
ccactttcgt gaatgttttt atattcctaa aattcaagac ttgccgccgg tagatttgag
300 tctcgttaac aaagatgaaa acgcgatcta ctttctgggc aactctctgg
gtctgcaacc 360 aaaaatggtt aaaacgtacc tggaggaaga actggataaa
tgggcaaaaa tcgcggctta 420 tggtcacgaa gtgggcaagc gtccttggat
tactggcgac gagtctattg tgggtttgat 480 gaaagatatt gtgggcgcga
atgaaaagga aattgcactg atgaatgctc tgaccgttaa 540 tctgcacctg
ctgatgctgt ctttttttaa accgaccccg aaacgctaca aaatactgct 600
ggaagcgaaa gcgtttccgt cggatcacta tgctatagaa agtcaactgc agttgcatgg
660 tctgaatatc gaggaatcta tgcgcatgat taaaccgcgt gagggtgaag
aaacgctgcg 720 tattgaagac attctggaag ttattgaaaa agaaggtgat
tctatcgcag ttatactgtt 780 ttctggcgtg cacttttata caggtcagca
cttcaatatc ccggcaatca ctaaagcggg 840 gcaggcaaaa ggctgctatg
ttggttttga cctggcgcat gcagtgggga atgttgaact 900 gtatctgcac
gattggggcg ttgatttcgc gtgttggtgt agctacaaat atctgaacgc 960
tggcgcgggt ggcattgctg gcgcttttat tcacgaaaaa cacgcgcaca ccattaaacc
1020 ggctctggtt ggctggttcg gtcatgagct gagtactcgc tttaaaatgg
ataacaaact 1080 gcaattgatt ccgggtgttt gcggcttccg tatcagcaat
ccgccgattc tgctggtttg 1140 cagcctgcac gctagtctgg aaatctttaa
gcaggcgact atgaaagcgc tgcgcaaaaa 1200 atctgtgctg ctgaccggct
atctggagta tctgatcaaa cacaattatg gcaaagataa 1260 agctgcaact
aaaaaaccgg tagtgaacat tatcaccccc tcacacgtgg aggagcgcgg 1320
ttgtcagctg actattactt tcagtgtacc taataaagat gtgttccagg aactggaaaa
1380 acgcggcgtt gtttgtgata aacgtaaccc gaatggtatt cgcgtggctc
ctgtgccgct 1440 gtacaattca ttccacgatg tttataaatt caccaacctg
ctgacttcta ttctcgacag 1500 tgctgagact aaaaattaaa aataaaacga
aaggctcagt cgaaagactg ggcctttcgt 1560 tttatctgtt g 1571 <210>
SEQ ID NO 172 <211> LENGTH: 1310 <212> TYPE: DNA
<213> ORGANISM: Shewanella sp. <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(1310) <223> OTHER INFORMATION: ptet-kynU(Shewanella)
<400> SEQUENCE: 172 atctaatcta gacatcatta attcctaatt
tttgttgaca ctctatcatt gatagagtta 60 ttttaccact ccctatcagt
gatagagaaa agtgaatggt tcaccaccac aaggagggat 120 tatgctgctg
aatgtaaaac aggacttttg cctggcaggc ccgggctacc tgctgaatca 180
ctcggttggc cgtccgctga aatcaactga gcaagcgctg aaacaagcat tttttgctcc
240 gtggcaagag agcggtcgtg aaccgtgggg ccagtggctg ggtgttattg
ataatttcac 300 tgctgcgctg gcatctctgt ttaatggtca accgcaggat
ttttgtccgc aggttaacct 360 gagcagcgcg ctgactaaaa ttgtgatgtc
actggatcgt ctgactcgcg atctgacccg 420 caatggcggt gctgttgtgc
tgatgtctga aatcgatttc ccatctatgg gcttcgcgtt 480 gaaaaaagcg
ctgccagcga gctgcgaact gcgttttatc ccgaaaagtc tggacgtgac 540
tgatccgaac gtatgggatg cacacatctg tgatgatgta gacctggttt ttgtgtctca
600 cgcctatagt aatacgggcc aacaggctcc gctggcgcaa atcatctctc
tggcgcgtga 660 acgtggctgc ctgtcactgg tggatgtagc gcaatcagcg
gggattttgc cgctggatct 720 ggcgaaactg caaccggact tcatgatcgg
cagttcggtt aaatggctgt gctcgggccc 780 tggtgcggca tatctgtggg
ttaatccggc gattctgccg gaatgtcagc cgcaggatgt 840 gggctggttt
tcacatgaga atccctttga attcgacatc cacgatttcc gctaccaccc 900
gactgcactg cgcttttggg gtggtacgcc gtcgatcgcg ccttatgcga tcgcggcgca
960 ctcgatcgaa tattttgcca atatcggctc gcaagtgatg cgtgaacaca
acctgcaact 1020 gatggaaccg gtggttcagg cgctggacaa tgaactggtg
agcccgcagg aagtggataa 1080 acgctcaggc actattattc tgcaattcgg
tgaacgtcaa ccgcaaattc tggcggctct 1140 ggctgcggcg aacatttcgg
tggacactcg ttctttgggg attcgtgtta gtccgcacat 1200 ttataatgat
gaggcggaca ttgcgcgcct gctgggtgtg atcaaagcaa atcgctaaaa 1260
ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 1310
* * * * *