U.S. patent application number 16/425292 was filed with the patent office on 2019-10-17 for methods for thaxtomin production and modified streptomyces with increased thaxtomin production.
The applicant listed for this patent is UNIVERSITE DE LIEGE, University of Florida Research Foundation, Inc.. Invention is credited to ISOLDE MARIA FRANCIS, SAMUEL JOURDAN, ROSEMARY LORIA, SEBASTIEN RIGALI.
Application Number | 20190316160 16/425292 |
Document ID | / |
Family ID | 55533826 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190316160 |
Kind Code |
A1 |
LORIA; ROSEMARY ; et
al. |
October 17, 2019 |
METHODS FOR THAXTOMIN PRODUCTION AND MODIFIED STREPTOMYCES WITH
INCREASED THAXTOMIN PRODUCTION
Abstract
In accordance with the purpose(s) of the present disclosure, as
embodied and broadly described herein, embodiments of the present
disclosure, in some aspects, relate to genetically modified
Streptomyces bacteria capable of increased thaxtomin production,
genetically modified Streptomyces bacteria with reduced activity of
a CebR protein encoded by a cebR gene and/or reduced activity of a
.beta.-glucosidase enzyme encoded by the bglC gene, genetically
modified Streptomyces bacteria including a mutation of a native
cebR gene and/or a native bglC gene, methods of increasing
thaxtomin production in Streptomyces bacteria, methods of
suppressing CebR and/or BglC activity, methods of producing
thaxtomin, and thaxtomin produced by the methods of the present
disclosure.
Inventors: |
LORIA; ROSEMARY;
(GAINESVILLE, FL) ; JOURDAN; SAMUEL; (BASTOGNE,
BE) ; RIGALI; SEBASTIEN; (ESNEUX, BE) ;
FRANCIS; ISOLDE MARIA; (BAKERSFIELD, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Inc.
UNIVERSITE DE LIEGE |
Gainesville
LIEGE |
FL |
US
BE |
|
|
Family ID: |
55533826 |
Appl. No.: |
16/425292 |
Filed: |
May 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15510863 |
Mar 13, 2017 |
10385372 |
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PCT/US2015/050582 |
Sep 17, 2015 |
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16425292 |
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62052681 |
Sep 19, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 403/06 20130101;
C12N 9/2445 20130101; A01N 43/60 20130101; C07K 14/36 20130101;
C12P 17/165 20130101; C12N 15/102 20130101; A01N 63/10
20200101 |
International
Class: |
C12P 17/16 20060101
C12P017/16; C12N 15/10 20060101 C12N015/10; C07D 403/06 20060101
C07D403/06; A01N 43/60 20060101 A01N043/60; C07K 14/36 20060101
C07K014/36; A01N 63/02 20060101 A01N063/02; C12N 9/42 20060101
C12N009/42 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. 2010-65110-20372 awarded by the USDA/NIFA. The government has
certain rights in the invention.
Claims
1. A genetically modified Streptomyces bacterium comprising: a
mutation that reduces activity of a CebR protein encoded by a cebR
gene, such that the genetically modified Streptomyces has increased
production of a thaxtomin compound as compared to a corresponding
wild type Streptomyces bacterium, wherein the genetically modified
Streptomyces bacterium is selected from the group of Streptomyces
species in which the corresponding wild type Streptomyces bacterium
is capable of producing one or more thaxtomin compounds under
standard thaxtomin-inducing conditions.
2. The genetically modified Streptomyces bacterium of claim 1,
wherein the genetically modified Streptomyces bacterium is selected
from the group of Streptomyces species consisting of: Streptomyces
scabies, Streptomyces acidiscabies, and Streptomyces
turgidiscabies.
3. The genetically modified Streptomyces bacterium of claim 1,
wherein the Streptomyces bacterium is a genetically modified
Streptomyces scabies bacterium.
4. The genetically modified Streptomyces bacterium of claim 1,
wherein the mutation comprises a mutation of a native cebR gene,
wherein the mutation reduces production or functionality of a CebR
protein encoded by the cebR gene.
5. The genetically modified Streptomyces bacterium of claim 4,
wherein the mutation of cebR is a null mutation.
6. The genetically modified Streptomyces bacterium of claim 1,
wherein the genetically modified bacterium does not produce
functional CebR.
7. The genetically modified Streptomyces bacterium of claim 1,
wherein the mutation comprises an exogenous nucleic acid sequence
introduced into the bacterium, wherein the exogenous nucleic acid
sequence reduces activity of a CebR protein encoded by the cebR
gene.
8. The genetically modified Streptomyces bacterium of claim 1,
wherein the genetically modified Streptomyces bacterium produces a
thaxtomin compound in the absence of at least one carbohydrate that
reduces CebR DNA-binding activity.
9. The genetically modified Streptomyces bacterium of claim 8,
wherein the carbohydrate is cellobiose
10. The genetically modified Streptomyces bacterium of claim 1,
wherein the thaxtomin compound is thaxtomin A.
11. The genetically modified Streptomyces bacterium of claim 1,
wherein the cebR gene has a nucleotide sequence of SEQ ID NO: 1 or
a nucleotide sequence having about 60% or more sequence identity
with SEQ ID NO: 1.
12. A method of increasing production of a thaxtomin compound in a
Streptomyces bacterium, the method comprising: providing a
Streptomyces bacterium from a species capable of producing one or
more thaxtomin compounds under standard thaxtomin-inducing
conditions; genetically modifying the Streptomyces bacterium by
creating a mutation in the bacterium, wherein the mutation reduces
activity of a CebR protein encoded by a cebR gene, such that the
genetically modified Streptomyces has increased production of a
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacterium.
13. The method of claim 12, wherein the mutation is a mutation of a
native cebR gene, wherein the mutation reduces production or
functionality of a CebR protein encoded by the cebR gene.
14. The method of claim 13, wherein the mutation of cebR is a null
mutation created by inserting a deletion cassette into the genome
to remove the cebR gene, thereby inhibiting production of CebR.
15. The method of claim 12, wherein the mutation comprises an
exogenous nucleic acid sequence introduced into the bacterium,
wherein the exogenous nucleic acid sequence reduces activity of a
CebR protein encoded by the cebR gene.
16. A method of increasing production of a thaxtomin compound in a
Streptomyces bacterium capable of producing one or more thaxtomin
compounds under standard thaxtomin-inducing conditions, the method
comprising suppressing the activity of a CebR protein encoded by a
cebR gene.
17. The method of claim 16, wherein suppressing the activity of the
CebR protein comprises genetically modifying the Streptomyces
bacterium to inhibit expression of the cebR gene encoding the CebR
protein.
18. The method of claim 16, wherein suppressing the activity of the
CebR protein comprises genetically modifying the Streptomyces
bacterium to introduce an exogenous nucleic acid sequence, wherein
the exogenous nucleic acid sequence reduces activity of the CebR
protein encoded by the cebR gene.
19. The method of claim 16, wherein the gene encoding the CebR
protein has a nucleotide sequence of SEQ ID NO: 1 or a sequence
having about 60% or more sequence identity with SEQ ID NO: 1.
20. A method of producing a thaxtomin compound, the method
comprising: culturing genetically modified Streptomyces bacteria,
wherein the genetically modified Streptomyces bacteria is selected
from the group of Streptomyces species capable of producing one or
more thaxtomin compounds under standard thaxtomin-inducing
conditions and wherein the genetically modified Streptomyces
bacteria comprise a mutation of a native cebR gene, wherein the
mutation reduces production or functionality of a CebR repressor
encoded by the cebR gene, such that the modified Streptomyces
bacteria produce a greater amount of the thaxtomin compound as
compared to a corresponding wild type Streptomyces bacteria.
21. The method of claim 20, further comprising extracting the
thaxtomin compound from the culture media.
22. The method of claim 20, further comprising culturing the
genetically modified Streptomyces bacteria in a culture medium that
does not contain cellobiose, wherein the genetically modified
Streptomyces bacteria produce the thaxtomin compound in the absence
of cellobiose.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application a divisional application of U.S. Ser. No.
15/510,863, entitled METHODS FOR THAXTOMIN PRODUCTION AND MODIFIED
STREPTOMYCES WITH INCREASED THAXTOMIN PRODUCTION, filed on Mar. 13,
2017, which application is the 35 U.S.C. .sctn. 371 national stage
application of PCT Application No. PCT/US2015/050582, filed Sep.
17, 2015 and entitled METHODS FOR THAXTOMIN PRODUCTION AND MODIFIED
STREPTOMYCES WITH INCREASED THAXTOMIN PRODUCTION, where the PCT
claims the benefit of and priority to U.S. Provisional Application
Ser. No. 62/052,681, having the title "METHODS FOR THAXTOMIN
PRODUCTION AND MODIFIED STREPTOMYCES WITH INCREASED THAXTOMIN
PRODUCTION", filed on Sep. 19, 2014, both of which are herein
incorporated by reference in their entireties.
SEQUENCE LISTING
[0003] This application contains a sequence listing filed in
electronic form as an ASCII.txt file entitled 02276058.K created on
Sep. 17, 2015, and having a size of 19900 bytes. The content of the
sequence listing is incorporated herein in its entirety.
BACKGROUND
[0004] The thaxtomins are a group of phytotoxins generated by some
species of Streptomyces bacteria, such as Streptomyces scabies (the
main causal organism of potato common scab). The thaxtomins can
cause plant cell necrosis of various plant species.
[0005] Five toxins, including thaxtomin A and thaxtomin B, that
induce the formation of scabs on potato tubers have been isolated
from S. scabies. They are cyclic dipeptides classed as
2,5-Diketopiperazines, with thaxtomin A, the most abundant, having
the chemical formula C.sub.22H.sub.22N.sub.4O.sub.6. Individual
thaxtomins appear to differ only in the presence or absence of
N-methyl and hydroxyl groups and their respective substitution
sites. For instance, thaxtomin A and thaxtomin B differ only in the
presence of a hydrogen at C.sub.20 in thaxtomin B, rather than a
hydroxyl group.
[0006] The pathogenicity of Streptomyces strains is believed to be
related to the amount of thaxtomin A they produce, as thaxtomin A
is believed to be the most physiologically active. It is
synthesized by peptide synthetases encoded by the txtA and txtB
genes. The genes involved in thaxtomin biosynthesis are located on
a part of the genome called the pathogenicity island, also present
in S. acidiscabies and S. turgidiscabies.
[0007] Cellobiose and cellotriose, subunits of cellulose, activate
thaxtomin production in some strains. The target of the thaxtomins
is unknown, but evidence suggests that they inhibit the growth of
plant cell walls, having an apparent ability to inhibit cellulose
synthesis in developing plant cells. They are not organ- or
plant-specific and cause death in various plant species.
[0008] Although studies have indicated that the thaxtomins have
many of the biological properties desirable in a commercial
herbicide, production on an industrial scale presents problems,
such as slow production, low yields, and the need for certain
inducers, such as cellobiose.
SUMMARY
[0009] In accordance with the purpose(s) of the present disclosure,
as embodied and broadly described herein, embodiments of the
present disclosure relate to genetically modified Streptomyces
bacteria capable of increased thaxtomin production, genetically
modified Streptomyces bacteria with reduced activity of a CebR
protein encoded by a cebR gene and/or reduced activity of a
.beta.-glucosidase enzyme encoded by the bglC gene, genetically
modified Streptomyces bacteria including a mutation of a native
cebR gene and/or a native bglC gene, methods of increasing
thaxtomin production in Streptomyces bacteria, methods of
suppressing CebR and/or BglC activity, methods of producing
thaxtomin, and thaxtomin produced by the methods of the present
disclosure.
[0010] An embodiment of the present disclosure includes a
genetically modified Streptomyces bacterium comprising a mutation
that reduces activity of a CebR protein encoded by a cebR gene,
such that the genetically modified Streptomyces has increased
production of a thaxtomin compound as compared to a corresponding
wild type Streptomyces bacterium.
[0011] An embodiment of the present disclosure also includes a
genetically modified Streptomyces bacterium comprising a mutation
that reduces activity of a .beta.-glucosidase enzyme encoded by a
bglC gene, such that the genetically modified Streptomyces has
increased production of a thaxtomin compound as compared to a
corresponding wild type Streptomyces bacterium.
[0012] An embodiment of the present disclosure includes a method of
increasing production of a thaxtomin compound in a Streptomyces
bacterium, the method comprising providing a Streptomyces bacterium
from a species capable of producing one or more thaxtomin compounds
under standard thaxtomin-inducing conditions and genetically
modifying the Streptomyces bacterium by creating a mutation in the
bacterium, wherein the mutation reduces activity of a CebR protein
encoded by a cebR gene, such that the genetically modified
Streptomyces has increased production of a thaxtomin compound as
compared to a corresponding wild type Streptomyces bacterium.
[0013] An embodiment of the present disclosure includes a method of
increasing production of a thaxtomin compound in a Streptomyces
bacterium, the method comprising providing a Streptomyces bacterium
from a species capable of producing one or more thaxtomin compounds
under standard thaxtomin-inducing conditions, and genetically
modifying the Streptomyces bacterium by creating a mutation in the
bacterium, wherein the mutation reduces activity of a
.beta.-glucosidase enzyme encoded by a bglC gene, such that the
genetically modified Streptomyces has increased production of a
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacterium.
[0014] An embodiment of the present disclosure includes a method of
increasing production of a thaxtomin compound in a Streptomyces
bacterium, the method comprising suppressing the activity of one or
more of the group selected from a CebR protein encoded by a cebR
gene or a .beta.-glucosidase enzyme encoded by a bglC gene.
[0015] An embodiment of the present disclosure includes a method of
producing a thaxtomin compound, the method comprising culturing
genetically modified Streptomyces bacteria, wherein the genetically
modified Streptomyces bacteria have a mutation selected from at
least one of a mutation of a native cebR gene and a mutation of a
native bglC gene, wherein the mutation reduces production or
functionality of at least one of a CebR repressor encoded by the
cebR gene and a .beta.-glucosidase enzyme encoded by the bglC gene,
such that the modified Streptomyces bacteria have increased
production of a thaxtomin compound as compared to a corresponding
wild type Streptomyces bacteria.
[0016] Other genetically modified strains of bacteria, methods,
features, and advantages will be or become apparent to one with
skill in the art upon examination of the following drawings and
detailed description. It is intended that all such additional
genetically modified strains of bacteria, methods, features and
advantages be included within this description, be within the scope
of the present disclosure, and be protected by the accompanying
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Further aspects of the present disclosure will be more
readily appreciated upon review of the detailed description of its
various embodiments, described below, when taken in conjunction
with the accompanying drawings.
[0018] FIGS. 1A-C illustrate Streptomyces scabies and thaxtomin A.
FIG. 1A is an image of Streptomyces scabies, FIG. 1B shows potato
scab lesions caused by Streptomyces scabies, and FIG. 10 shows the
structure of thaxtomin A.
[0019] FIG. 2 shows the CebR-binding sites associated with the
cellobiose uptake system and the thaxtomin biosynthetic genes.
Numbers below cbs are scores obtained by the predicted CebR target
sequences using the PREDetctor software and the position weight
matrix. Numbers associated with genes/orfs are SCAB numbers from
the annotated genome of S. scabies (strain 87-22).
[0020] FIG. 3 shows an electromobility gel shift assay (EMSA)
demonstrating CebR binding to DNA motifs identified within the
txtRA and cebRE intergenic region, upstream of bglC and within
txtB. Numbers 1 to 14 refer to increasing concentrations of pure
CebR-His6, i.e., 0 (free probe, 30 nM), 80, 160, 240, 320, 400,
480, 560, 640, 720, 960, 1200, 1600, 3200 nM, respectively.
[0021] FIG. 4 shows EMSAs demonstrating cellobiose as the best
allosteric effector of CebR. Numbers 1 and 2 refer to EMSAs with
free probes (6 nM) and with probes incubated with CebR-His.sub.6,
respectively. Numbers 3 to 7 refer to EMSAs with CebR-His.sub.6
pre-incubated with oligosaccharides, i.e. cellobiose (3),
cellotriose (4), cellotetraose (5), cellopentaose (6), and
cellohexaose (7). Note that cellobiose (3) is the best
oligosaccharide for preventing CebR interaction with all
CebR-binding sites.
[0022] FIG. 5 illustrates a model of cellobiose-dependent
production of thaxtomin in S. scabies.
[0023] FIG. 6 shows the effect of cebR deletion in S. scabies on
the transcription levels of the thaxtomin biosynthetic and
regulatory genes. Quantitative real-time RT-PCR analysis of gene
expression levels in S. scabies 87-22 and in the .DELTA.cebR
strain. Data were normalized using the gyrA, murX and the hrdB gene
as internal controls. Mean normalized expression levels
(.+-.standard deviation) from three biological replicates analyzed
in triplicate are shown.
[0024] FIGS. 7A-7B show effects of the cebR deletion on thaxtomin
production in S. scabies. FIG. 7A illustrates an analysis of growth
conditions showing that deletion of cebR in S. scabies resulted in
higher thaxtomin production (determined by intensity of
pigmentation, as graphed in FIG. 7B) on OBA and even in production
under conditions which do not trigger thaxtomin production in the
wild type strain 87-22. FIG. 7B is a graphic representation of HPLC
analysis of thaxtomin A extracted from the plates shown in FIG.
7A.
[0025] FIGS. 8A-B illustrate genetic complementation of the cebR
mutant scored by thaxtomin production on ISP-4 plates. FIG. 8A is a
digital image of a visual inspection of ISP-4 plates, where
thaxtomin production is displayed by typical yellow pigmentation.
Although pigmentation is not visible in the black and white photos,
FIG. 8B is a graphic illustration HPLC analysis of thaxtomin
production on the ISP-4 plates from FIG. 8A. The cebR mutant (and
the cebR mutant transformed with the empty pAU3-45 plasmid as a
negative control for the complementation) produce thaxtomin under
conditions that do not induce toxin production in the wild-type
strain. Thaxtomin production is reverted back to wild-type levels
when the mutation is genetically complemented (cebR mutant
transformed with pRLIF8 containing the cebR gene and its promoter
region).
[0026] FIGS. 9A-9B show production of thaxtomin A by wild type S.
scabies (87-22) and by the S. scabies .beta.-glucosidase mutant
(.DELTA.57721) in OBB (FIG. 9A) and on OBA plates and OBA and OBA
with cellobiose (FIG. 9B).
[0027] FIG. 10 shows production of thaxtomin A by wild type S.
scabies (87-22) and by the S. scabies .beta.-glucosidase mutant
(.DELTA.57721) on TDM agar plates with glucose and cellobiose,
respectively, as the only carbon source.
[0028] FIG. 11 is a bar graph illustrating production of thaxtomin
A by wild type S. scabies (87-22) and by the S. scabies
.beta.-glucosidase mutant (.DELTA.57721) on ISP-4 plates and ISP-4
with cellobiose.
DETAILED DESCRIPTION
[0029] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present disclosure
will be limited only by the appended claims.
[0030] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
(unless the context clearly dictates otherwise), between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0032] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0033] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of chemistry, botany, biochemistry,
biology, molecular biology, and the like, which are within the
skill of the art. Such techniques are explained fully in the
literature.
[0034] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is at or near
atmospheric. Standard temperature and pressure are defined as
20.degree. C. and 1 atmosphere.
[0035] Before the embodiments of the present disclosure are
described in detail, it is to be understood that, unless otherwise
indicated, the present disclosure is not limited to particular
materials, reagents, reaction materials, manufacturing processes,
or the like, as such can vary. It is also to be understood that the
terminology used herein is for purposes of describing particular
embodiments only, and is not intended to be limiting. It is also
possible in the present disclosure that steps can be executed in
different sequence where this is logically possible.
[0036] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a cell" includes a plurality of
cells. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
[0037] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise. In this disclosure,
"consisting essentially of" or "consists essentially" or the like,
when applied to methods and compositions encompassed by the present
disclosure refers to compositions like those disclosed herein, but
which may contain additional structural groups, composition
components or method steps (or analogs or derivatives thereof as
discussed above). Such additional structural groups, composition
components or method steps, etc., however, do not materially affect
the basic and novel characteristic(s) of the compositions or
methods, compared to those of the corresponding compositions or
methods disclosed herein. "Consisting essentially of" or "consists
essentially" or the like, when applied to methods and compositions
encompassed by the present disclosure have the meaning ascribed in
U.S. Patent law and the term is open-ended, allowing for the
presence of more than that which is recited so long as basic or
novel characteristics of that which is recited is not changed by
the presence of more than that which is recited, but excludes prior
art embodiments.
[0038] Prior to describing the various embodiments, the following
definitions are provided and should be used unless otherwise
indicated.
Definitions
[0039] In describing and claiming the disclosed subject matter, the
following terminology will be used in accordance with the
definitions set forth below.
[0040] The terms "nucleic acid" and "polynucleotide" are terms that
generally refer to a string of at least two base-sugar-phosphate
combinations. As used herein, the terms include deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA) and generally refer to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. RNA may be in the
form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA
(ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA
interference construct), siRNA (short interfering RNA), or
ribozymes. Thus, for instance, polynucleotides as used herein
refers to, among others, single- and double-stranded DNA, DNA that
is a mixture of single- and double-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or
a mixture of single- and double-stranded regions. The terms
"nucleic acid sequence" and "oligonucleotide" also encompasses a
nucleic acid and polynucleotide as defined above.
[0041] In addition, polynucleotide as used herein refers to
double-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a double-helical region
often is an oligonucleotide.
[0042] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term polynucleotide as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple and complex cells, inter alia. For instance, the
term polynucleotide includes DNAs or RNAs as described above that
contain one or more modified bases. Thus, DNAs or RNAs comprising
unusual bases, such as inosine, or modified bases, such as
tritylated bases, to name just two examples, are polynucleotides as
the term is used herein.
[0043] The term also includes PNAs (peptide nucleic acids),
phosphorothioates, and other variants of the phosphate backbone of
native nucleic acids. Natural nucleic acids have a phosphate
backbone, artificial nucleic acids may contain other types of
backbones, but contain the same bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are "nucleic
acids" or "polynucleotides" as that term is intended herein.
[0044] A "gene" typically refers to a hereditary unit corresponding
to a sequence of DNA that occupies a specific location on a
chromosome and that contains the genetic instruction for a
characteristic(s) or trait(s) in an organism.
[0045] As used herein, the term "transfection" refers to the
introduction of an exogenous and/or recombinant nucleic acid
sequence into the interior of a membrane enclosed space of a living
cell, including introduction of the nucleic acid sequence into the
cytosol of a cell as well as the interior space of a mitochondria,
nucleus, or chloroplast. The nucleic acid may be in the form of
naked DNA or RNA, it may be associated with various proteins or
regulatory elements (e.g., a promoter and/or signal element), or
the nucleic acid may be incorporated into a vector or a chromosome.
A "transformed" cell is thus a cell transfected with a nucleic acid
sequence. The term "transformation" refers to the introduction of a
nucleic acid (e.g., DNA or RNA) into cells in such a way as to
allow expression of the coding portions of the introduced nucleic
acid.
[0046] As used herein, "transformation" or "transformed" refers to
the introduction of a nucleic acid (e.g., DNA or RNA) into cells in
such a way as to allow expression of the coding portions of the
introduced nucleic acid.
[0047] As used herein a "transformed cell" is a cell transfected
with a nucleic acid sequence. As used herein, a "transgene" refers
to an artificial gene or portion thereof that is used to transform
a cell of an organism, such as a bacterium or a plant.
[0048] As used herein, "transgenic" refers to a cell, tissue, or
organism that contains a transgene.
[0049] As used herein, "exogenous nucleic acid sequence" or
"exogenous polynucleotide" refers to a nucleic acid sequence that
was introduced into a cell, organism, or organelle via
transfection. Exogenous nucleic acids originate from an external
source, for instance, the exogenous nucleic acid may be from
another cell or organism and/or it may be synthetic and/or
recombinant. While an exogenous nucleic acid sometimes originates
from a different organism or species, it may also originate from
the same species (e.g., an extra copy or recombinant form of a
nucleic acid that is introduced into a cell or organism in addition
to or as a replacement for the naturally occurring nucleic acid).
Typically, the introduced exogenous sequence is a recombinant
sequence.
[0050] The term "recombinant" generally refers to a non-naturally
occurring nucleic acid, nucleic acid construct, or polypeptide.
Such non-naturally occurring nucleic acids may include natural
nucleic acids that have been modified, for example that have
deletions, substitutions, inversions, insertions, etc., and/or
combinations of nucleic acid sequences of different origin that are
joined using molecular biology technologies (e.g., a nucleic acid
sequences encoding a "fusion protein" (e.g., a protein or
polypeptide formed from the combination of two different proteins
or protein fragments)), the combination of a nucleic acid encoding
a polypeptide to a promoter sequence, where the coding sequence and
promoter sequence are from different sources or otherwise do not
typically occur together naturally). Recombinant also refers to the
polypeptide encoded by the recombinant nucleic acid. Non-naturally
occurring nucleic acids or polypeptides include nucleic acids and
polypeptides modified by man.
[0051] As used herein, "isolated" means removed or separated from
the native environment. Therefore, isolated DNA can contain both
coding (exon) and noncoding regions (introns) of a nucleotide
sequence corresponding to a particular gene. An isolated peptide or
protein indicates the protein is separated from its natural
environment. Isolated nucleotide sequences and/or proteins are not
necessarily purified. For instance, an isolated nucleotide or
peptide may be included in a crude cellular extract or they may be
subjected to additional purification and separation steps.
[0052] With respect to nucleotides, "isolated nucleic acid" refers
to a nucleic acid with a structure (a) not identical to that of any
naturally occurring nucleic acid or (b) not identical to that of
any fragment of a naturally occurring genomic nucleic acid spanning
more than three separate genes, and includes DNA, RNA, or
derivatives or variants thereof. The term covers, for example but
not limited to, (a) a DNA which has the sequence of part of a
naturally occurring genomic molecule but is not flanked by at least
one of the coding sequences that flank that part of the molecule in
the genome of the species in which it naturally occurs; (b) a
nucleic acid incorporated into a vector or into the genomic nucleic
acid of a prokaryote or eukaryote in a manner such that the
resulting molecule is not identical to any vector or naturally
occurring genomic DNA; (c) a separate molecule such as a cDNA, a
genomic fragment, a fragment produced by polymerase chain reaction
(PCR), ligase chain reaction (LCR) or chemical synthesis, or a
restriction fragment; (d) a recombinant nucleotide sequence that is
part of a hybrid gene, e.g., a gene encoding a fusion protein, and
(e) a recombinant nucleotide sequence that is part of a hybrid
sequence that is not naturally occurring. Isolated nucleic acid
molecules of the present disclosure can include, for example,
natural allelic variants as well as nucleic acid molecules modified
by nucleotide deletions, insertions, inversions, or
substitutions.
[0053] It is advantageous for some purposes that a nucleotide
sequence is in purified form. The term "purified" in reference to
nucleic acid represents that the sequence has increased purity
relative to the natural environment.
[0054] The term "polypeptides" and "protein" include proteins and
fragments thereof. Polypeptides are disclosed herein as amino acid
residue sequences. Those sequences are written left to right in the
direction from the amino to the carboxy terminus. In accordance
with standard nomenclature, amino acid residue sequences are
denominated by either a three letter or a single letter code as
indicated as follows: Alanine (Ala, A), Arginine (Arg, R),
Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),
Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G),
Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine
(Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline
(Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W),
Tyrosine (Tyr, Y), and Valine (Val, V).
[0055] "Variant" refers to a polypeptide that differs from a
reference polypeptide, but retains essential properties. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more modifications (e.g., substitutions, additions,
and/or deletions). A substituted or inserted amino acid residue may
or may not be one encoded by the genetic code. A variant of a
polypeptide may be naturally occurring such as an allelic variant,
or it may be a variant that is not known to occur naturally.
[0056] Modifications and changes can be made in the structure of
the polypeptides of in disclosure and still obtain a molecule
having similar characteristics as the polypeptide (e.g., a
conservative amino acid substitution). For example, certain amino
acids can be substituted for other amino acids in a sequence
without appreciable loss of activity. Because it is the interactive
capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
sequence substitutions can be made in a polypeptide sequence and
nevertheless obtain a polypeptide with like properties.
[0057] In making such changes, the hydropathic index of amino acids
can be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a polypeptide
is generally understood in the art. It is known that certain amino
acids can be substituted for other amino acids having a similar
hydropathic index or score and still result in a polypeptide with
similar biological activity. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. Those indices are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0058] It is believed that the relative hydropathic character of
the amino acid determines the secondary structure of the resultant
polypeptide, which in turn defines the interaction of the
polypeptide with other molecules, such as enzymes, substrates,
receptors, antibodies, antigens, and the like. It is known in the
art that an amino acid can be substituted by another amino acid
having a similar hydropathic index and still obtain a functionally
equivalent polypeptide. In such changes, the substitution of amino
acids whose hydropathic indices are within .+-.2 is preferred,
those within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0059] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly, where the biological
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. The following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0060] As outlined above, amino acid substitutions are generally
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions that take
various of the foregoing characteristics into consideration are
well known to those of skill in the art and include (original
residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),
(Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp),
(Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val),
(Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr),
(Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this
disclosure thus contemplate functional or biological equivalents of
a polypeptide as set forth above. In particular, embodiments of the
polypeptides can include variants having about 50%, 60%, 70%, 80%,
90%, and 95% sequence identity to the polypeptide of interest.
[0061] As used herein "functional variant" refers to a variant of a
protein or polypeptide (e.g., a variant of a CCD enzyme) that can
perform the same functions or activities as the original protein or
polypeptide, although not necessarily at the same level (e.g., the
variant may have enhanced, reduced or changed functionality, so
long as it retains the basic function).
[0062] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences, as determined by comparing the
sequences. In the art, "identity" also refers to the degree of
sequence relatedness between polypeptide as determined by the match
between strings of such sequences. "Identity" and "similarity" can
be readily calculated by known methods, including, but not limited
to, those described in (Computational Molecular Biology, Lesk, A.
M., Ed., Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, D. W., Ed., Academic Press,
New York, 1993; Computer Analysis of Sequence Data, Part I,
Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey,
1994; Sequence Analysis in Molecular Biology, von Heinje, G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., Eds., M Stockton Press, New York, 1991; and
Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073
(1988).
[0063] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. The percent identity between two
sequences can be determined by using analysis software (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group,
Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol.
Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The
default parameters are used to determine the identity for the
polypeptides of the present disclosure.
[0064] By way of example, a polypeptide sequence may be identical
to the reference sequence, that is be 100% identical, or it may
include up to a certain integer number of amino acid alterations as
compared to the reference sequence such that the % identity is less
than 100%. Such alterations are selected from: at least one amino
acid deletion, substitution, including conservative and
non-conservative substitution, or insertion, and wherein said
alterations may occur at the amino- or carboxy-terminal positions
of the reference polypeptide sequence or anywhere between those
terminal positions, interspersed either individually among the
amino acids in the reference sequence or in one or more contiguous
groups within the reference sequence. The number of amino acid
alterations for a given % identity is determined by multiplying the
total number of amino acids in the reference polypeptide by the
numerical percent of the respective percent identity (divided by
100) and then subtracting that product from said total number of
amino acids in the reference polypeptide.
[0065] The term "expression" as used herein describes the process
undergone by a structural gene to produce a polypeptide. It can
refer to transcription or the combination of transcription and
translation. Expression generally refers to the transcription of a
gene to produce messenger RNA, as used herein expression may refer
to the entire process of "expression" of a nucleic acid to produce
a polypeptide (e.g., transcription plus translation). If
"expression" is used in reference to a polypeptide, it indicates
that the polypeptide is being produced via expression of the
corresponding nucleic acid.
[0066] As used herein, the term "over-expression" and
"up-regulation" or "increasing" production of a polypeptide refers
to the expression of a nucleic acid encoding a polypeptide (e.g., a
gene) in a modified cell at higher levels (therefore producing an
increased amount of the polypeptide encoded by the gene) as
compared to a "wild type" cell (e.g., a substantially equivalent
cell that is not modified in the manner of the modified cell) under
substantially similar conditions. Thus, to over-express or increase
expression of thaxtomin refers to increasing or inducing the
production of the thaxtomin dipeptide by one or more enzymes
encoded by the thaxtomin biosynthetic genes, which may be done by a
variety of approaches, such as, but not limited to: increasing the
transcription of the genes (such as by placing the genes under the
control of a constitutive promoter) responsible for synthesis of
thaxtomin, or increasing the translation of such genes, inhibiting
or eliminating a repressor of thaxtomin production (e.g., CebR or
.beta.-glucosidase enzyme), or a combination of these and/or other
approaches.
[0067] Conversely, "under-expression" and "down-regulation" refers
to expression of a polynucleotide (e.g., a gene) at lower levels
(producing a decreased amount of the polypeptide encoded by the
polynucleotide) than in a "wild type" cell. As with
over-expression, under-expression can occur at different points in
the expression pathway, such as by decreasing the number of gene
copies encoding for the polypeptide; removing, interrupting, or
inhibiting (e.g., decreasing or preventing) transcription and/or
translation of the gene (e.g., by the use of antisense nucleotides,
suppressors, knockouts, antagonists, etc.), or a combination of
such approaches. "Suppression" refers to the inhibition of
production and/or activity functional gene product. Thus, the
suppression of a gene or protein may indicate that the expression
of the gene and/or activity of the encoded peptide has been
inhibited such as by transcription and/or translation being
inhibited, thus resulting in low to no production of the encoded
protein, or production of a non-functional product, or production
of an interfering nucleic acid that otherwise suppresses activity
of the target protein.
[0068] Similarly, with respect to a gene product, such as a
protein, "reduced activity" indicates that the activity of the
protein is reduced relative to activity in a "wild type cell". Such
reduction in activity can be the result of
inhibition/suppression/down-regulation/under-expression of the gene
encoding the protein, the result of inhibition of translation of
the messenger RNA into a functional gene product, or the result of
production of a non-functional protein with reduced or no activity,
or the direct suppression of the protein activity (e.g., preventing
binding to a target), or the like. "Reduced production" of a gene
product (e.g., a protein), such as by suppression, interruption, or
other inhibition of transcription or translation, may result in
reduced activity, but "reduced activity" of a protein or other gene
product may result from other causes other than "reduced
production", such as set for the above.
[0069] As used herein a "mutation" refers to a heritable change in
genetic material, which may include alteration of single base pairs
of a nucleic acid, or the deletion, insertion, or rearrangement of
larger sections of genes or chromosomes. An "engineered mutation"
refers to a mutation created by human design (e.g., the mutation
did not spontaneously occur by natural causes and/or was the result
of intentional human manipulation). A "genetically modified"
organism is an organism whose genetic material has been altered by
one or more engineered mutations (e.g., human induced
mutations).
[0070] The term "null mutation" refers to a mutation in which the
gene product (e.g., the protein encoded by the gene) is either not
produced (or produced at significantly reduced levels, so as to be
negligible) or is non-functional. Typically, a null mutation will
involve a mutation of the native gene, such that the gene is not
transcribed into RNA, the RNA product cannot be translated, or the
protein produced by gene expression is non-functional.
[0071] The term "plasmid" as used herein refers to a
non-chromosomal double-stranded DNA sequence including an intact
"replicon" such that the plasmid is replicated in a host cell. A
plasmid may include exogenous nucleic acid sequences and/or
recombinant sequences.
[0072] As used herein, the term "vector" or "expression vector" is
used in reference to a vehicle used to introduce an exogenous
nucleic acid sequence into a cell. A vector may include a DNA
molecule, linear or circular, which includes a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription and translation upon introduction
into a host cell or host cell organelles. Such additional segments
may include promoter and terminator sequences, and may also include
one or more origins of replication, one or more selectable markers,
an enhancer, a polyadenylation signal, etc. As such, expression
vectors typically contain recombinant nucleic acid sequences having
different sequences linked together to effect expression of a
target sequence. Expression vectors are generally derived from
yeast DNA, bacterial genomic or plasmid DNA, or viral DNA, or may
contain elements of more than one of these.
[0073] As used herein, the term "expression system" includes a
biologic system (e.g., a cell based system) used to express a
polynucleotide to produce a protein. Such systems generally employ
a plasmid or vector including the polynucleotide of interest (e.g.,
an exogenous nucleic acid sequence, a recombinant sequence, etc.),
where the plasmid or expression vector is constructed with various
elements (e.g., promoters, selectable markers, etc.) to enable
expression of the protein product from the polynucleotide.
Expression systems use the host system/host cell transcription and
translation mechanisms to express the product protein. Common
expression systems include, but are not limited to, bacterial
expression systems (e.g., E. coli), yeast expression systems, viral
expression systems, animal expression systems, and plant expression
systems.
[0074] As used herein, the term "promoter" or "promoter region"
includes all sequences capable of driving transcription of a coding
sequence. In particular, the term "promoter" as used herein refers
to a DNA sequence generally described as the 5' regulator region of
a gene, located proximal to the start codon. The transcription of
an adjacent coding sequence(s) is initiated at the promoter region.
The term "promoter" also includes fragments of a promoter that are
functional in initiating transcription of the gene.
[0075] The term "operably linked" indicates that the regulatory
sequences necessary for expression of the coding sequences of a
nucleic acid are placed in the nucleic acid molecule in the
appropriate positions relative to the coding sequence so as to
effect expression of the coding sequence. This same definition is
sometimes applied to the arrangement of coding sequences and
transcription control elements (e.g. promoters, enhancers, and
termination elements), and/or selectable markers in an expression
vector.
[0076] As used herein, the term "selectable marker" refers to a
gene whose expression allows one to identify cells that have been
transformed or transfected with a vector containing the marker
gene. For instance, a recombinant nucleic acid may include a
selectable marker operably linked to a gene of interest and a
promoter, such that expression of the selectable marker indicates
the successful transformation of the cell with the gene of
interest.
[0077] The terms "native," "wild type", or "unmodified" in
reference to a polypeptide/protein/enzyme, polynucleotide, cell, or
organism, are used herein to provide a reference point for a
variant/mutant of a polypeptide/protein/enzyme, polynucleotide,
cell, or organism prior to its mutation and/or modification
(whether the mutation and/or modification occurred naturally or by
human design).
[0078] As used herein, "thaxtomin" or "thaxtomin compound" refers
to one or more compounds from a family of cyclic dipeptide
phytotoxins, 4-nitroindol-3-yl-containing 2,5-dioxopiperazines,
generated by some species of Streptomyces bacteria (and possibly by
other actinomycetes) and exhibiting toxicity to various plant
species. Thaxtomin compounds of the present disclosure have the
general formula of Formula I below, and variants thereof. At least
5 thaxtomin compounds have been characterized, including thaxtomin
A, A ortho analog, B, C, and D, and up to at least 12 different
variants identified. Thaxtomin A, the most abundant of the
thaxtomins and also believed to be the most physiologically active,
has the chemical formula C.sub.22H.sub.22N.sub.4O.sub.6 (chemical
structure illustrated in FIG. 10). The thaxtomins can cause plant
cell necrosis of various plant species and can induce the formation
of scabs on potato tubers have been isolated from S. scabies. As
used herein "thaxtomin" and "thaxtomin compound" refers generally
to any of the members of this chemical group. Much of the
discussion of thaxtomin in the present disclosure is in reference
to thaxtomin A; however, as thaxtomin A may be a precursor to other
thaxtomin compounds and/or the production of thaxtomin A is
interwoven with production of other thaxtomin compound's, to the
extend the methods and compositions of the present disclosure also
modulate the production of other thaxtomin compounds, this is also
intended to fall within the scope of the present disclosure. The
general structure of a thaxtomin is shown below as Formula I, where
R1 and R3 are independently selected from methyl or H and where R2,
R4, R5, and R6 are each independently selected from hydroxyl or
H.
##STR00001##
[0079] The terms "thaxtomin-inducing conditions" indicates certain
environmental conditions (e.g., natural or cell culture conditions)
known to induce thaxtomin production in wild-type Streptomyces
bacterial species known to be capable of thaxtomin production. For
instance, wild type Streptomyces are induced to produce thaxtomin
in the presence of certain products of cellulose degradation, such
as, but not limited to, cellobiose, as well as xylan-degradation
products (Wach et al. 2007), such as, but not limited to suberin
(Lauzier et al. 2008). In embodiments, "thaxtomin-inducing
conditions" may include specific conditions or cell culture media
(such as but not limited to, Oat Bran Broth (OBB), Oat Bran Agar
(OBA), etc.) known to induce thaxtomin production in cell culture
of wild-type Streptomyces species (such as, but not limited to S.
scabies, S. acidiscabies, and S. turgidiscabies). In embodiments,
"thaxtomin-inducing conditions" may also include a standard cell
culture growth medium supplemented with a known thaxtomin-inducing
compound, such as, but not limited to cellobiose.
[0080] The term "cebR gene" as used in the present disclosure
indicates a nucleic acid sequence encoding a CebR protein in a
Streptomyces or other thaxtomin-producing Actinomycete species that
is a modulator of thaxtomin production. In embodiments, "cebR
genes" include the cebR gene of S. scabies (SEQ ID NO: 1) and
variants and/or homologs (e.g., orthologs and paralogs) thereof
retaining the function of modulation of thaxtomin production. In
embodiments "cebR genes" include nucleic acids having a sequence of
SEQ ID NO: 1 as well as sequences having about 60% or more, about
70% or more, about 80% or more, about 90% or more, about 95% or
more, or about 99% or more (including any intervening ranges)
sequence identity with SEQ ID NO: 1. In embodiments, cebR genes
include nucleic acids having a sequence identity to SEQ ID NO: 1 of
about 60% or more and having a sequence coverage to SEQ ID NO: 1 of
about 70% or more, where sequence coverage indicates the percent of
the total length of nucleic acids that are aligned.
[0081] The term "bglC gene" as used in the present disclosure
indicates a nucleic acid sequence encoding a .beta.-glucosidase
enzyme in a Streptomyces or thaxtomin-producing Actinomycete
species that is a modulator of thaxtomin production. In
embodiments, "bglC genes" include the bglC gene of S. scabies (SEQ
ID NO: 3) and variants and/or homologs (e.g., orthologs and
paralogs) thereof retaining the function of modulating thaxtomin
production. In embodiments "bglC genes" include nucleic acids
having a sequence of SEQ ID NO: 3 as well as sequences having about
60% or more, about 70% or more, about 80% or more, about 90% or
more, about 95% or more, or about 99% or more (including any
intervening ranges) sequence identity with SEQ ID NO: 3. In
embodiments, bglC genes include nucleic acids having a sequence
identity to SEQ ID NO: 3 of about 60% or more and also having a
sequence coverage of about 70% with SEQ ID NO: 3.
Discussion
[0082] In accordance with the purpose(s) of the present disclosure,
as embodied and broadly described herein, embodiments of the
present disclosure, in some aspects, relate to genetically modified
Streptomyces bacteria capable of increased thaxtomin production,
genetically modified Streptomyces bacteria with reduced activity of
a CebR protein encoded by a cebR gene and/or reduced activity of a
.beta.-glucosidase enzyme encoded by the bglC gene, genetically
modified Streptomyces bacteria including a mutation of a native
cebR gene and/or a native bglC gene, methods of increasing
thaxtomin production in Streptomyces bacteria, methods of
suppressing CebR and/or BglC activity, methods of producing
thaxtomin, and thaxtomin produced by the methods of the present
disclosure.
[0083] Streptomyces is a very large genus of Gram-positive, high
G+C content bacteria that are mostly saprophytes and best known for
the production of pharmaceutically- and agriculturally-important
secondary metabolites (Hopwood 2007). Although several hundred
species are known to date, only a handful are phytopathogenic
(Loria et al. 2006). The best studied pathogens are Streptomyces
scabies, S. acidiscabies, S. turgidiscabies and S. ipomoeae, which
cause raised or pitted scab lesions on economically-important root
and tuber crops like potato, radish, beet, peanut, and sweet potato
(FIG. 1A, 1B). The primary virulence determinant of S. scabies, S.
acidiscabies and S. turgidiscabies is the phytotoxin thaxtomin A
(Loria et al. 2008) (FIG. 10). It is member of a family of nitrated
dipeptides formed by non-ribosomal peptide synthases out of the
main components tryptophan, phenylalanine and nitric oxide derived
from arginine (Loria et al. 2008; Barry et al. 2012).
[0084] Thaxtomin A (and other toxic thaxtomin compounds) primarily
targets the cell wall in dividing and expanding plant cells through
an alteration of expression of cell wall biosynthesis-related genes
and depletion of cellulose synthase complexes from the plasma
membrane. This causes extensive cell wall remodeling, characterized
by reduced incorporation of crystalline cellulose into the plant
cell wall, and is compensated by an increased amount of pectins and
hemicelluloses (Scheible et al. 2003; Bischoff et al. 2009). Data
have shown that thaxtomin provokes the same effects on plants,
qualitatively as well as quantitatively, as the synthetic cellulose
biosynthesis inhibitor isoxaben, making thaxtomin an excellent
candidate as a natural herbicide (Heim et al. 1990; Bischoff et al.
2009). In 2001, King and Lawrence reported a study in collaboration
with James A. Gray from Dow Agrosciences, Inc. to evaluate the
potential of thaxtomin for use as a commercial herbicide. The
biological properties of this novel phytotoxin raised an interest
in using thaxtomin as a biological compound to control weeds
(Marrone Bio Innovations 2009, 2010; Novozymes Biologicals 2011,
2012); however, thaxtomin production in wild type Streptomyces
requires specialized cell culture media (such as media supplemented
with cellobiose or other thaxtomin-inducing compounds), which can
be expensive. Thus, the present disclosure provides genetically
modified Streptomyces bacteria with the ability to produce
thaxtomin compounds at an increase over wild type bacteria and in
cell media in which wild type bacteria cannot produce thaxtomin, or
produce it in only trace amounts, as well as methods for producing
thaxtomin using such genetically modified bacteria.
[0085] Acquisition of genes required for virulence is only one step
on the way to pathogenicity. Indeed, more subtle genetic changes
are involved in adapting the expression of newly acquired genes to
the environment and the life cycle of the recipient microorganism.
For instance, a limited number of mutations in intergenic
regulatory regions can transform a harmless strain into a pathogen.
The distribution of cis-acting elements in the gene is an element
involved in development of a strain-specific transcriptional
response. These DNA motifs are targeted by transcription factors,
which themselves are informed of the presence of environmental
signals through direct interaction with membrane sensors or
indirect association with elicitor transporters. The production of
thaxtomin A itself is under transcriptional regulatory control
including at least five global regulators belonging to the bid gene
family involved in secondary metabolism and/or morphological
differentiation of Streptomyces (Bignell et al. 2014) in addition
to the thaxtomin biosynthesis pathway-specific transcriptional
activator, TxtR. The multiplicity of global and specific regulators
associated with thaxtomin production suggests that S. scabies may
respond to multiple triggers that originate from plant material
such as xylan-degradation products (Wach et al. 2007), suberin
(Lauzier et al. 2008), and cellobiose, a product of cellulose
degradation and the best-known elicitor of thaxtomin biosynthesis
(Wach et al. 2007; Johnson et al. 2009) by directly targeting TxtR
(Joshi et al. 2007).
[0086] Thus, as described in the examples below, proteins involved
in regulation of thaxtomin regulation were identified, and
genetically modified Streptomyces bacteria were produced with
mutations affecting the activity of these proteins (e.g., by
mutation in the genes encoding the proteins and/or by inhibition of
the protein itself), resulting in modification of thaxtomin
production. In exemplary embodiments, genetically modified
Streptomyces bacteria have increased production of thaxtomin
compounds, such as, but not limited to, thaxtomin A, and/or are
capable of thaxtomin production in non-inducing conditions and on
non-inducing media (e.g., conditions in which wild type
Streptomyces do not produce thaxtomin or produce only trace amounts
of thaxtomin).
[0087] As described in greater detail below, two genes were
identified in Streptomyces species that produce proteins involved
in the thaxtomin production pathway and whose disruption causes an
increase in thaxtomin production in the modified bacterium and/or
the ability to produce thaxtomin in non-inducing conditions. The
genes include a cebR gene (e.g., SEQ ID NO: 1) and a bglC gene
(e.g., SEQ ID NO: 3), which encode a CebR protein (e.g., SEQ ID NO:
2) and a .beta.-glucosidase enzyme (e.g., SEQ ID NO: 4),
respectively.
[0088] CebR is a repressor of thaxtomin biosynthesis in
Streptomyces bacteria and responds directly to the presence of
cellobiose (and possibly other inducing compounds), which binds and
represses CebR thereby inducing thaxtomin production by the
bacterium. CebR and variants and homologs of cebR are found in
thaxtomin-producing Streptomyces bacterium. The sequence of cebR
from S. scabies is illustrated in SEQ ID NO: 1. The present
disclosure includes cebR sequences of SEQ ID NO: 1 as well as
variants and homologs thereof defined above that encode a CebR
protein that represses thaxtomin production, such as, but not
limited to, cebR genes, variants, or homologs having about 60%
sequence identity or more (with a sequence coverage of about 70% or
more), with SEQ ID NO: 1.
[0089] The mechanism by which the bglC encoded enzyme acts to
induce thaxtomin production is less clear, but like CebR,
inactivation or reduced activity of the bglC .beta.-glucosidase
enzyme induces thaxtomin production. In embodiments, reduced
activity of CebR and/or the bglC .beta.-glucosidase enzyme
increases thaxtomin production so that the affected bacterium
constitutively produces thaxtomin, even in the absence of
thaxtomin-inducing conditions. The sequence of an exemplary bglC
gene (bglC from S. scabies) is illustrated in SEQ ID NO: 3.
However, the scope of the present disclosure includes bglC genes
having SEQ ID NO: 3 as well as variants and homologs thereof, as
defined above, that encode a .beta.-glucosidase enzyme, where
disruption of the enzyme induces thaxtomin production, such as, but
not limited to, bglC genes, variants, or homologs having about 60%
or more sequence identity (with a sequence coverage about 70% or
more) with SEQ ID NO: 3.
[0090] Although the methods of the present disclosure are generally
described with respect to Streptomyces bacterium, since, to date,
Streptomyces is the only species known to produce toxic thaxtomin
compounds, to the extent that other species of Actinomycetes
acquired thaxtomin biosynthetic genes via horizontal transfer or
otherwise, the genetically modified bacteria and methods of the
present disclosure are also applicable to such Actinomycete species
with the acquired thaxtomin production capabilities and are
included within Streptomyces in the scope of the present
application. Thus for any of the genetically modified bacteria
described herein and any methods of thaxtomin production described
herein, it is understood that the scope of the present application
also includes genetically modified Actinomycetes other than
Streptomyces that also have or have acquired thaxtomin production
capabilities.
[0091] The genetically modified bacteria of the present disclosure,
methods of increasing thaxtomin compounds in Streptomyces bacteria,
methods of producing thaxtomin, and thaxtomin produced by methods
of the present disclosure are described in greater detail in the
discussion below and following examples.
[0092] Genetically Modified Bacteria
[0093] Embodiments of the present disclosure include genetically
modified Streptomyces bacteria including a mutation that reduces
activity of a CebR protein encoded by a cebR gene, such that the
genetically modified Streptomyces has increased production of a
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacterium. Embodiments of the present disclosure also
include genetically modified Streptomyces bacteria including a
mutation that reduces activity of a .beta.-glucosidase enzyme
encoded by a bglC gene, such that the genetically modified
Streptomyces has increased production of a thaxtomin compound as
compared to a corresponding wild type Streptomyces bacterium.
Embodiments also include genetically modified Streptomyces bacteria
including a mutation that reduces activity of a CebR protein
encoded by a cebR gene, a mutation that reduces activity of a
.beta.-glucosidase enzyme encoded by a bglC gene, or both
mutations, such that the genetically modified Streptomyces has
increased production of a thaxtomin compound as compared to a
corresponding wild type Streptomyces bacterium.
[0094] In embodiments, the Streptomyces bacterium that is
genetically modified is a Streptomyces species in which the
corresponding wild type Streptomyces bacterium is capable of
producing one or more thaxtomin compounds under standard
thaxtomin-inducing conditions. In other words, the wild type
Streptomyces bacterium can produce thaxtomin compounds in
conditions as described above where Streptomyces species, such as
S. scabies has been demonstrated to produce thaxtomin (e.g., Oat
Bran Broth (OBB), Oat Bran Agar (OBA), and other cell culture
mediums containing cellobiose or other thaxtomin inducer). The
present disclosure also includes genetically modified bacteria from
other actinomycetes that have acquired the ability to produce
thaxtomin. In embodiments, the bacterium is selected from the group
of Streptomyces species including, but not limited to, Streptomyces
scabies, S. acidiscabies, and S. turgidiscabies. In specific
embodiments the Streptomyces bacterium is a genetically modified
Streptomyces scabies bacterium.
[0095] As discussed above, some wild type Streptomyces species
(e.g., Streptomyces scabies, S. acidiscabies, and S.
turgidiscabies, etc.) produce thaxtomin compounds under certain
conditions, defined herein as thaxtomin-inducing conditions, but
cannot produce thaxtomin constitutively or in all conditions, or in
the absence of certain triggers/inducers (for example, but not
limited to, cellobiose). In embodiments, the genetically modified
Streptomyces bacteria of the present disclosure produce one or more
thaxtomin compounds in the absence of at least one carbohydrate
that reduces CebR DNA-binding activity (such as, but not limited
to, cellobiose, cellotriose, and cellohexaose). In embodiments, the
carbohydrate is selected from cellobiose, cellotriose, and
cellohexaose. In an embodiment, the carbohydrate is cellobiose. The
genetically modified Streptomyces bacteria of the present
disclosure may produce one or more thaxtomin compounds, such as,
but not limited to: thaxtomin A, thaxtomin, B, thaxtomin C,
thaxtomin D, and the like. In embodiments, the genetically modified
Streptomyces bacteria of the present disclosure may produce at
least thaxtomin A, which is believed to be the most physiologically
active and may be a precursor to other thaxtomin compounds.
[0096] The genetically modified Streptomyces bacteria of the
present disclosure can be genetically modified in various ways in
order to reduce the activity of a CebR protein or
.beta.-glucosidase enzyme encoded by a cebR gene or a bglC gene,
respectively, such that the genetically modified Streptomyces has
increased production of a thaxtomin compound as compared to a
corresponding wild type Streptomyces bacterium. In general, any
genetic modification of the bacterium that results in reduced
activity of the target proteins (CebR and .beta.-glucosidase) are
intended to be included in the scope of the present disclosure. In
embodiments, the genetic modification can include a mutation of the
native genetic material of the bacterium (e.g., a mutation such as,
but not limited to, an insertion, deletion, or rearrangement of the
native (e.g., wild type) genetic material of the bacterium). In
other embodiments, the genetic modification can include a mutation
resulting from introduction of exogenous genetic material (e.g.,
nucleic acid sequence) into the bacterium (e.g., via
transfection).
[0097] Thus, in embodiments, the genetically modified bacterium
includes a mutation of a native cebR and/or bglC gene, where the
mutation reduces production or functionality of a protein encoded
by the gene. In embodiments, the mutation of the native cebR and/or
bglC gene may inhibit expression of the gene or the functionality
of a resulting gene product. For example, the mutation may inhibit
transcription of the cebR and/or bglC gene into mRNA, may inhibit
translation of the mRNA into the CebR protein and/or
.beta.-glucosidase enzyme, may completely remove the cebR and/or
bglC gene (which also inhibits transcription), may include a
mutation of the cebR and/or bglC gene that still allows
transcription but results in a non-functional CebR protein, and the
like.
[0098] In embodiments, the mutation of cebR and/or bglC (e.g., the
"target" gene) is a null mutation (e.g., the target gene is removed
from the genome or transcription of the gene is otherwise virtually
completely suppressed). In embodiments, the null mutation is
obtained by replacing the target gene with a deletion cassette. In
embodiments, the replacement gene in the deletion cassette can be
any gene other than the gene being replaced. In embodiments, the
replacement gene provides a detectable signal (such as, but not
limited to, antibiotic resistance, fluorescence, color change,
etc.) to allow for detection of mutant cells (e.g., cells
containing the replacement gene in place of the target gene). In
embodiments, the deletion cassette is included in a vector,
plasmid, or other system useful for effecting transformation and
genetic recombination. In embodiments, primers are used that have
sequences including regions homologous to flanking regions of the
target gene as well as of the replacement gene in the deletion
cassette in order to facilitate the replacement of the target gene
with the replacement gene. In embodiments of the present
disclosure, the deletion cassette includes an apramycin resistance
gene having a nucleotide sequence of SEQ ID NO: 42. In some
embodiments where the target gene is a cebR gene having a
nucleotide sequence of SEQ ID NO: 1 a deletion cassette including
SEQ ID NO: 42 and forward and reverse primers having sequence ID
NOS: 8 and 9 can be used to genetically modify Streptomyces
bacteria of the present disclosure to produce cebR null mutants. In
some embodiments where the target gene is a bglC gene having a
nucleotide sequence of SEQ ID NO: 3, a deletion cassette including
SEQ ID NO: 42 and forward and reverse primers having sequence ID
NOS: 28 and 29 can be used to genetically modify Streptomyces
bacteria of the present disclosure to produce cebR null
mutants.
[0099] In embodiments of the present disclosure, the genetically
modified bacterium has reduced activity of CebR and/or
.beta.-glucosidase because it does not produce functional CebR
and/or .beta.-glucosidase, encoded by cebR or bglC, respectively,
either due to lack of production (e.g., lack of transcription
and/or translation) or due to production of a non-functional
protein.
[0100] While some genetic mutations, discussed above, reduce
activity of CebR and/or .beta.-glucosidase and thereby increase
thaxtomin production by including a mutation of the encoding gene
itself (e.g., cebR and/or bglC), other mutations of the genetically
modified bacteria of the present disclosure may include an
exogenous nucleic acid sequence that reduces the activity of the
target proteins, such as by suppressing expression and/or by
interacting with the protein itself to inhibit activity. In
embodiments, the mutation of the genetically modified Streptomyces
bacterium of the present disclosure includes an exogenous nucleic
acid sequence introduced into the bacterium, wherein the exogenous
nucleic acid sequence reduces activity of a CebR protein encoded by
the cebR gene. In embodiments, the exogenous nucleic acid sequence
may include one or more RNAi sequences (e.g., an miRNA sequence
and/or siRNA sequence) that inhibit expression of cebR and/or bglC,
thereby resulting in increased thaxtomin production by the
genetically modified bacterium.
[0101] Other methods known to those of skill in the art for
reducing the activity of a target protein can be used within the
scope of the present disclosure to provide genetically modified
Streptomyces bacteria having a mutation that reduces the activity
of a CebR protein encoded by a cebR gene and or a or
.beta.-glucosidase encoded by a bglC gene, such that the
genetically modified Streptomyces has increased production of a
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacterium.
[0102] Methods of Increasing Production of a Thaxtomin Compound in
Streptomyces
[0103] The present disclosure also provides methods of increasing
production of thaxtomin compound in a Streptomyces bacterium (or
other Actinomycete that has acquired the ability to product
thaxtomin). In general, methods of the present disclosure for
increasing thaxtomin production in a Streptomyces bacterium include
suppressing the activity of a CebR protein encoded by a cebR gene,
suppressing the activity of a .beta.-glucosidase enzyme encoded by
a bglC gene, or both. In embodiments, the method includes providing
a Streptomyces bacterium from a species capable of producing one or
more thaxtomin compounds under standard thaxtomin-inducing
conditions and genetically modifying the Streptomyces bacterium by
creating a mutation in the bacterium that results in reduced
activity of a CebR protein encoded by a cebR gene and/or of a
.beta.-glucosidase enzyme encoded by a bglC gene, such that the
genetically modified Streptomyces has increased production of a
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacterium. As discussed above, genetically modifying
the bacterium can be achieved by any of the approaches discussed
above. For example, the method may include genetically modifying
the bacterium by creating a mutation in the genome (e.g., native
genetic material) of the bacterium and/or creating a mutation by
introducing an exogenous nucleic acid sequence into the bacterium
(e.g., via an expression vector or other expression system).
[0104] As described above, in embodiments the mutation may be a
mutation of a native cebR and/or bglC gene that reduces the
production or functionality of a CebR protein or .beta.-glucosidase
enzyme encoded by the respective mutated gene. Suppressing the
activity of the CebR protein and/or the .beta.-glucosidase enzyme,
in embodiments, can include genetically modifying the Streptomyces
bacterium to inhibit expression of the cebR gene and/or bglC gene
encoding the CebR protein and/or .beta.-glucosidase enzyme,
respectively. In embodiments, the mutation can be a null mutation
of the cebR and/or bglC gene, such as by inserting a deletion
cassette into the genome to remove/replace the cebR and/or bglC
gene, thereby inhibiting production of CebR protein or
.beta.-glucosidase enzyme encoded by the removed/replaced gene. In
embodiments, the deletion cassette can be as described above.
[0105] In embodiments of methods of increasing thaxtomin production
in Streptomyces bacteria, the bacteria are modified by creating a
mutation in the bacterium by introducing an exogenous nucleic acid
sequence into the bacterium, where the exogenous nucleic acid
sequence, or its expression product, reduces activity of the target
protein (e.g., cebR encoded by cebR and/or .beta.-glucosidase
enzyme encoded by bglC). In embodiments, as discussed above, the
exogenous nucleic acid sequence includes an RNAi sequence that
suppresses production (e.g., by suppressing translation of mRNA
sequences encoding the target protein) or function/activity of the
target CebR or .beta.-glucosidase enzyme.
[0106] Other methods known to those of skill in the art can be used
to introduce a mutation into the Streptomyces bacteria that results
in decreased activity of cebR and/or .beta.-glucosidase and
consequently increased thaxtomin production. Such methods are
intended to be included in the scope of the present
application.
[0107] Methods of Producing Thaxtomin
[0108] The present disclosure also includes methods of producing
thaxtomin. Embodiments of such methods include culturing
genetically modified Streptomyces bacteria, where the genetically
modified Streptomyces bacteria comprise a mutation of a native cebR
gene, a mutation of a native bglC gene, or both (such as described
above), where the mutation reduces production or functionality of a
CebR repressor encoded by the cebR gene, a .beta.-glucosidase
enzyme encoded by the bglC gene, or both, so that the modified
Streptomyces bacteria produce thaxtomin. In the methods of
producing thaxtomin of the present disclosure, the genetically
modified Streptomyces bacteria exhibit increased production of
thaxtomin compound as compared to a corresponding wild type
Streptomyces bacteria. As described above, embodiments of the
genetically modified Streptomyces bacteria of the present invention
constitutively produce thaxtomin in environmental conditions (e.g.,
standard growth medium) where wild type Streptomyces bacteria would
not be able to produce thaxtomin or may only produce trace amounts.
In embodiments of the methods of the present disclosure for
producing thaxtomin, the thaxtomin produced by the genetically
modified Streptomyces bacteria is collected and/or extracted from
the cell culture. After collection/extraction of the thaxtomin from
the cell culture, the thaxtomin may be further extracted/separated
from the culture media, and/or the extracted thaxtomin may then be
subject to further isolation and/or purification steps as needed or
desired.
[0109] The isolated and/or purified thaxtomin compound isolated
from the genetically modified Streptomyces bacteria of the present
disclosure can then be used for various purposes, such as in the
production of certain herbicides. Thus, the methods of the present
disclosure also include methods of making herbicides including
thaxtomin by producing thaxtomin according to the methods of the
present disclosure and using the thaxtomin to produce the
herbicide. The present disclosure also includes thaxtomin compounds
produced by the methods of making thaxtomin of the present
disclosure described above.
[0110] Additional details regarding the methods, compositions, and
organisms of the present disclosure are provided in the Examples
below. The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present disclosure to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
[0111] It should be emphasized that the embodiments of the present
disclosure, particularly, any "preferred" embodiments, are merely
possible examples of the implementations, merely set forth for a
clear understanding of the principles of the disclosure. Many
variations and modifications may be made to the above-described
embodiment(s) of the disclosure without departing substantially
from the spirit and principles of the disclosure. All such
modifications and variations are intended to be included herein
within the scope of this disclosure, and protected by the following
claims.
[0112] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed herein. Efforts have been made to ensure
accuracy with respect to numbers (e.g., amounts, temperature,
etc.), but some errors and deviations should be accounted for.
Unless indicated otherwise, parts are parts by weight, temperature
is in .degree. C., and pressure is at or near atmospheric. Standard
temperature and pressure are defined as 20.degree. C. and 1
atmosphere.
[0113] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. In an embodiment, the term "about" can include
traditional rounding according to significant figures of the
numerical value. In addition, the phrase "about `x` to `y`"
includes "about `x` to about `y`".
EXAMPLES
[0114] Now having described the embodiments of the disclosure, in
general, the examples describe some additional embodiments. While
embodiments of the present disclosure are described in connection
with the example and the corresponding text and figures, there is
no intent to limit embodiments of the disclosure to these
descriptions. On the contrary, the intent is to cover all
alternatives, modifications, and equivalents included within the
spirit and scope of embodiments of the present disclosure.
Example 1
[0115] The present example describes the identification of CebR,
the cellulose/cellooligossaccharide and cellobiose utilization
regulator, as master repressor of thaxtomin biosynthesis in
thaxtomin-producing Streptomyces species. The example describes a
modified Streptomyces strain capable of constitutive production of
thaxtomin compounds regardless of the presence of cellobiose or
other cellulose degradation products capable of inducing thaxtomin
production. The results demonstrate how and why (the molecular
mechanism) the inactivation of cebR results in the constitutive
production of thaxtomin independently of cellobiose supply in the
culture medium. The presence of CebR-binding sites associated with
thaxtomin biosynthetic genes in other thaxtomin-producing
streptomycetes (except Streptomyces ipomoeae), or other
thaxtomin-producing actinomycetes, allow application of these
methods beyond the species S. scabies 87-22.
Results & Discussion
[0116] Identification of CebR DNA-Binding Sites Associated with the
Thaxtomin Biosynthetic Genes
[0117] The identification of transcription factors involved in the
control of thaxtomin was performed by scrutinizing the genome of
Streptomyces scabies for the occurrence of putative cis-acting
elements of well-characterized DNA-binding proteins. Position
weight matrices were generated from the compilation of DNA motifs
bound by a series of regulators that were selected due to their
notorious direct or indirect implication in secondary metabolite
biosynthesis and/or morphological differentiation in
streptomycetes. The PREDetector software (Hiard et al. 2007)
identified two 14-bp palindromic sequences similar to CebR boxes
(henceforth cbs for CebR-binding sites) and physically associated
with the thaxtomin biosynthetic genes, txtA and txtB, and the
pathway-specific regulatory gene, txtR (FIG. 2). The bioinformatic
method used to identify DNA motifs in the chromosome of S. scabies
is described in the material and methods section.
[0118] The cGGGAGCGCTCCCA sequence (cbs.sup.txtR-A) (SEQ ID NO: 5)
lies in the intergenic region between txtR and txtA at positions
-786 nt and -900 nt upstream of txtR and txtA, respectively,
whereas the gGGGAGCGCTCCCA sequence (cbs.sup.txtB) (SEQ ID NO: 6)
lies at position +1507 nt within the coding sequence of txtB (FIG.
2). Both sequences display only one single mismatch with the
reported 14-bp cbs palindromic consensus sequence TGGGAGCGCTCCCA
(cbs.sup.cebR-E) (SEQ ID NO: 7) (Marushima et al. 2009). FIG. 2
also illustrates that a 14-bp sequence, TGGAAGCGCTCCCA
(cbs.sup.bglC) (SEQ ID NO: 43), lies -14 nt upstream of bglC with
only a single bp mismatch with cbs.sup.cebR-E (SEQ ID NO: 7).
[0119] CebR has been identified as the repressor of
cellulose/cellooligosaccharides/cellobiose utilization in
streptomycetes (Schlosser et al. 2000; Marushima et al. 2009) and,
in addition, it has also been shown to trigger morphogenesis in
Streptomyces griseus (Marushima et al. 2009). In S. griseus,
binding of cellobiose to CebR relieves its grip on the specific cbs
and allows for transcription of the downstream genes.
Interestingly, cellobiose is also known as the best elicitor of
thaxtomin production in S. scabies, S. acidiscabies and S.
turgidiscabies (Joshi et al. 2007; Wach et al. 2007).
[0120] Inspection of the full length chromosome of S. scabies
further revealed the presence of palindromic cbs within the
intergenic region between cebR (scab57761) itself and cebE
(scab57751, cbs.sup.cebE-R) encoding the orthologue of the
streptomycete cellobiose-binding component of an ABC-type
transporter (FIG. 2), as well as upstream of bglC (scab57721,
cbs.sup.bglC) encoding the orthologue of the .beta.-glucosidase
associated with the cellobiose/cellotriose-specific ABC transporter
in streptomycetes and which cleaves the .beta.31.fwdarw.4
glycosidic bond linking the two glucose molecules.
[0121] Electromobility gel shift assays (EMSAs) were performed in
order to assess binding of the S. scabies CebR regulator
(CebR.sup.sca) to the in silico predicted cbs. The scab57761 gene
was cloned into pET-22b and overexpressed in Escherichia coli
Rosetta (DE3) cells. The resulting recombinant His-tagged CebR
(CebR-His6) was purified from the IPTG-induced E. coli cytoplasmic
extracts by Nickel-NTA affinity column. EMSAs performed with 34-bp
double stranded, Cy5-labelled probes (SEQ ID NOs: 18 & 19
(cbs.sup.txtR-A), SEQ ID NOs: 20 & 21 (cbs.sup.cebE-R) SEQ ID
NOs: 22 & 23 (cbs.sup.txtB), and SEQ ID NOs: 24 & 25
(cbs.sup.bglC)) centered on the putative cbs showed that
cbs.sup.txtR-A and cbs.sup.txtB were strongly bound by
CebR.sup.sca, even with a 20-fold excess of non-specific DNA (FIG.
3). Under the same conditions, strong CebR-binding to
cbs.sup.cebE-R and cbs.sup.bglC, which were used as positive
controls of genes known to be directly controlled by CebR
(Schlosser 2000; Marushima 2009), was observed (FIG. 3). EMSAs
performed with CebR pre-incubated with cellobiose and
cellooligosaccharides revealed that cellobiose was the best
allosteric effector, inhibiting CebR from binding to its DNA
targets (FIG. 4).
[0122] Quantification of the shifted bands presented in FIG. 4
revealed that cellobiose (lane 3), cellotriose (lane 4), and
cellohexaose (lane 7) were able to impair the CebR DNA-binding
ability by 56%, 20%, and 12%, respectively. Experimental in vitro
validations of predicted cis-acting elements allowed proposing a
signaling pathway from cellobiose sensing and transport to
thaxtomin biosynthesis (FIG. 5). The putative working model
suggests that cellobiose is transported via the ABC-type
transporter CebEFG and that the energy for this active transport is
most-likely provided by ATP hydrolysis through the multiple sugar
importer MsiK (Schlosser et al. 1997). Once inside the cell,
cellobiose (and to a lesser extent cellotriose) prevents
CebR-binding to cbs.sup.txtR-A and cbs.sup.txtB allowing expression
of txtA, txtB and txtR. Consequently, TxtR complexed to cellobiose
would be able to increase the expression levels of txtA and txtB,
the thaxtomin biosynthetic genes.
[0123] Deletion of cebR Results in Constitutive Production of
Thaxtomin
[0124] The signaling cascade presented in FIG. 5 suggests a role
for CebR in the production of thaxtomin in plant pathogenic
streptomycetes. In order to evaluate the role of this regulator in
controlling the expression levels of the thaxtomin biosynthetic
genes txtA and txtB, cebR null mutants were generated in S. scabies
(.DELTA.cebR), and its thaxtomin production was assessed on various
media. Quantitative real-time PCR (qRT-PCR) on RNA extracted from
S. scabies wild-type (strain 87-22) and its cebR null mutant showed
that the inactivation of cebR resulted in overexpression of txtA,
txtB and txtR, respectively (FIG. 6). This result confirms that,
despite the unusual position of cbs.sup.txtR-A and cbs.sup.txtB,
further upstream of the target txtA and txtR genes and within the
coding region of txtB (FIG. 2), respectively, the identified DNA
motives are truly functional cis-acting elements.
[0125] Moreover, the search for similar sequences in other
thaxtomin-producing Streptomyces pathogens confirmed the occurrence
of cbs in their thaxtomin biosynthetic regions. In S. acidiscabies,
the oldest pathogenic species that displays a similar response to
cellobiose, three cbs signatures were identified within the
thaxtomin biosynthetic cluster i.e. one upstream of txtA (-787 nt),
one within txtA (+32 nt) and one within txtB (+1559 nt). In S.
turgidiscabies one cbs was found within txtA (+32 nt) and one
within txtB (+1508 nt). A third cbs was found at position -1444 nt
upstream of txtR. No cbs associated with thaxtomin production could
be identified in S. ipomoeae which, in contrast to the previously
mentioned pathogenic species, appears to produce only thaxtomin C
and is not responsive to cellobiose or other oligosaccharides in
terms of toxin production (Guan et al. 2012). Therefore, the
occurrence of cbs upstream of or within the txt genes correlates
with the observed cellobiose-dependent production of thaxtomin A in
pathogenic Streptomyces species.
[0126] Thaxtomin production levels of both the S. scabies and the
cebR null mutant were also assessed on Oat Bran Agar (OBA), an
undefined complex medium known to induce thaxtomin production
(Johnson et al. 2007, incorporated herein by reference), and ISP-4.
On OBA the cebR mutant overproduced thaxtomin, which is bright
yellow in color, compared to the wild-type (FIG. 7A). When streaked
out on plates containing the International Streptomyces Project
(ISP) mediumISP-4, the S. scabies strain where the gene coding for
the CebR repressor had been deleted (.DELTA.cebR) produced
thaxtomin without the addition of the cellobiose inducer (FIG. 7A).
The same results were observed with liquid cultures (Oat Bran
Broth, OBB).
[0127] Since ISP-4 medium (containing soluble starch as carbon
source) is not known to induce toxin production (other than
occasional trace amounts that could result from contamination) in
S. scabies, this result demonstrates that the mutant of S. scabies
was able to produce thaxtomin A independent of the presence the
elicitor cellobiose. Extraction of thaxtomin from the plates and
analysis by HPLC confirmed the overproduction of the toxin by the
cebR mutant on ISP-4 (FIG. 7B). .DELTA.cebR complemented with the
S. scabies cebR gene and its upstream region restores the
cellobiose-dependent induction of thaxtomin, demonstrating that the
phenotype of the mutant is caused by the chromosomal deletion of
orf scab57761 (FIGS. 8A-B).
[0128] The production of thaxtomin by both strains grown on ISP-4
with and without cellobiose supply was compared. As shown in FIGS.
8A and 8B, addition of cellobiose triggers a significant increase
of thaxtomin production in a wild-type or .DELTA.cebR background,
respectively. The further increase in toxin production by the
mutant upon addition of cellobiose is most likely due to the
function of cellobiose as ligand of the transcriptional activator
TxtR, which drives the expression of the thaxtomin biosynthetic
genes txtA and txtB (Joshi et al. 2007) (FIG. 5).
Conclusion
[0129] The present data suggest that the methods of the present
disclosure for increasing thaxtomin production through the deletion
of cebR is, in addition to S. scabies, is applicable to S.
turgidiscabies and S. acidiscabies, as well as in any other
streptomycete or actinomycete that would possess a cbs associated
with thaxtomin biosynthetic or regulatory genes.
Material and Methods
[0130] Bacterial Strains, Media, Chemicals, and Culture
Conditions
[0131] All strains and plasmids used in this study are described in
Table 1. Escherichia coli strains were cultured in Luria-Bertani
(LB) medium at 37.degree. C. Streptomyces strains were routinely
grown on International Streptomyces Project medium 4 (ISP-4; BD
Biosciences), Soy Flour Mannitol agar (SFM; Kieser et al. 2000), or
in Tryptic Soy Broth (TSB; BD Biosciences) at 28.degree. C. Where
required, the medium was supplemented with antibiotics at the
following concentrations: apramycin (Apr, 100 .mu.g/ml), kanamycin
(Kan, 50 .mu.g/ml), chloramphenicol (Cml, 25 .mu.g/ml),
thiostrepton (Thio, 25 .mu.g/ml), and/or nalidixic acid (NA, 50
.mu.g/ml). Spore suspensions were prepared from 7- to 10-day-old
ISP-4 plates and maintained as 20% glycerol stocks at -80.degree.
C. Cellobiose, cellotriose, cellotetraose, cellopentose, and
cellohexaose were purchased at Megazyme (Ireland).
[0132] CebR Regulon Prediction in Streptomycetes
[0133] Creation of the position weight matrix (PWM) and the
computational prediction of the CebR regulon in S. scabies were
performed with the PREDetector software (Hiard et al. 2007) as
described previously (Craig et al. 2012, incorporated herein by
reference for PWM and computational prediction). DNA motifs known
to be bound by CebR in S. griseus (Marushima et al. 2009) were used
as a training set to generate the PWM (PWM-CebR.sup.gri). This
PWM-CebR.sup.gri was used to scan the genome of Streptomyces
species for similar DNA motifs using a cut-off score of 8 (with a
reliability threshold of 10).
[0134] Generation of the cebR and bglC Gene Deletions in S. scabies
87-22
[0135] Deletion mutants in S. scabies 87-22 were created using the
REDIRECT PCR targeting methodology (Gust et al. 2003, incorporated
herein by reference) replacing the selected gene by an antibiotic
deletion cassette. These cassettes including an oriT and an
antibiotic resistance gene (aac(3)/V for apramycin resistance,
Table 1), and flanked by FRT sites (FLP-recombinase recognition
targets) were generated by PCR using primers with gene-specific
homology extensions (Table 2), and pIJ773 (Table 1) as template.
The gel-purified deletion cassettes were electroporated into the E.
coli BW25113 strain harboring the arabinose-inducible X RED
expression plasmid, pIJ790, and cosmid 833 containing the gene of
interest. Transformants were recovered on apramycin selective
medium, and correct gene replacement in the cosmid was confirmed by
PCR and sequencing. The resulting mutated cosmid was then
transferred into S. scabies 87-22 via intergeneric conjugation
after passage through the E. coli ET12567 strain harboring pUZ8002
(Table 1). Exconjugants were selected for resistance to apramycin,
and sensitivity to kanamycin. Genomic DNA was extracted from
Streptomyces cultures grown in TSB medium using the MasterPure.TM.
Gram Positive DNA Purification Kit (Epicentre Biotechnologies)
according to the manufacturer's instructions and verification of
the mutant isolates was performed by PCR.
[0136] Analysis of Thaxtomin A Production
[0137] Mycelial suspensions of S. scabies strains were prepared
from 48-72 h-old TSB-grown cultures by pelleting the mycelia,
washing twice with sterile water, and resuspending in 1 ml sterile
water to obtain an optical density at 600 nm (OD.sub.600 mn) of
1.0. For analysis of thaxtomin production, OBB medium (Johnson et
al. 2007, incorporated herein by reference) as well as TDM medium
with cellobiose or glucose as the only carbon source (modified from
Johnson et al. 2007) was used. Three times 50 ml medium in 250 ml
flasks were inoculated with 200 .mu.l of mycelial suspension of
OD.sub.600 nm 1.0. After incubation for 7 days at 28.+-.2.degree.
C. with shaking at .about.250 rpm, a 10 ml culture sample was taken
from each culture. Thaxtomin A was purified from the supernatant
and analyzed by HPLC as described previously (Johnson et al. 2007).
The pellets of the culture samples were dried and weighed as a
measure for bacterial growth.
[0138] Samples of 50 .mu.l from a mycelial suspension of OD.sub.600
nm 1.0 were plated out on small petri dishes (5 cm diameter)
containing 12.5 ml solid medium. After incubation for 7 days at
28.degree. C., plates were visually inspected for thaxtomin
production based on the typical yellow pigmentation due to secreted
thaxtomin. Experiments were repeated using different biological
replicates of the Streptomyces strains with three technical
replicates per strain.
[0139] His-Tagged CebR Production in E. coli and Protein
Purification
[0140] The orf encoding SCAB57761 was amplified by PCR using the
primers SCAB_57761+3 NdeI and SCAB_57761+1056_EcoRI (Table 2). The
corresponding PCR product was subsequently cloned into the
pJET1.2/blunt Cloning Vector, yielding pSAJ001. After DNA
sequencing, a NdeI-EcoRI DNA fragment was excised from pSAJ001 and
cloned into pET-22b digested with the same restriction enzymes. The
resulting construct, pSAJ002, was transformed to E. coli BL21(DE3)
competent cells. E. coli cells carrying pSAJ002 were grown at
37.degree. C. in 250 ml LB medium containing 100 .mu.g/ml of
ampicillin until the culture reached an optical density at 600 nm
(OD.sub.600) of 0.6. Production of Hiss-tagged CebR was induced
overnight (.about.20 h) at 16.degree. C. by addition of 1 mM
isopropyl-.beta.-d-thiogalactopyranoside (IPTG). Cells were
collected by centrifugation and ruptured by sonication in lysis
buffer (50 mM Tris-HCl buffer; pH 7.5; supplemented with the
Complete Protease Inhibitor Cocktail, EDTA-free, Roche). Soluble
proteins were first loaded onto a pre-equilibrated Ni.sup.2+
-nitrilotriacetic acid (NTA)-agarose column (5 ml bed volume) to
remove most of the E. coli proteins. The flow-through potentially
containing proteins not bound during this first purification was
reloaded onto a second Ni-NTA-agarose column. Both columns were
washed with 25 ml of washing buffer (Tris-HCl 50 mM, NaCl 200 mM).
Hiss-tagged CebR was eluted at around 150 mM imidazole. Fractions
containing the pure protein were pooled and desalted using a HiTrap
Desalting column (GE Healthcare) with 20 mM Tris-HCl buffer.
[0141] Protein Identification by LC-ESI-MS/MS
[0142] The band containing the Hiss-tagged CebR protein was excised
from the SDS-PAGE gel stained with Coomassie Blue, reduced,
alkylated and digested within the gel slice using trypsin. The
protein digest was independently analyzed on a Liquid Chromatograph
(nano Ultimate 3000-Dionex)-ESI-ion trap (AmaZon Speed EDT-Bruker
Daltonics), in positive ion mode. Spectra were interpreted using
Data analysis vs 4.0 (Bruker). Database searches were performed
using the Mascot server vs. 2.2.04 and Protein Scape vs. 3.0
(Bruker) on NCBI, restricted to bacterial taxonomies.
[0143] Electromobility Gel Shift Assays (EMSAs)
[0144] EMSAs with 34-bp double-stranded probes (generated by PCR,
Table 2) were performed using Cy5-labeled cbs probes (6 nM and 30
nM final concentration) and Hiss-tagged CebR at a final
concentration between 0.08 and 3.2 .mu.M in a total reaction volume
of 50 .mu.l. All reactions were carried out in EMSA buffer (10 mM
Tris-HCl, pH 7.5, 1 mM dithiothreitol [DTT], 0.25 mM CaCl.sub.2,
0.5 mM MgCl.sub.2, 50 mM KCl, and 2% glycerol) containing excess of
nonspecific DNA (salmon sperm DNA). After 15 min of incubation at
room temperature, reaction mixtures were loaded onto a 1% (wt/vol)
agarose gel. Bound and unbound probes were separated by gel
electrophoresis for 30 min at 100 V at room temperature, and
fluorescent DNA was visualized using a Typhoon Trio+ variable-mode
imager (FRFC 2.4506.08).
[0145] Real-Time Quantitative RT-PCR
[0146] RNA was prepared from 72-h old mycelia grown on ISP-4 plates
at 28.degree. C. using the RNeasy Mini Kit (Qiagen) according to
the manufacturer's instructions. PCR reactions on the purified RNA
were performed to verify the absence of genomic DNA. cDNA synthesis
was performed on 1 .mu.g of DNAse-treated (Turbo DNA-free Kit,
Ambion) RNA using the iScript.TM. cDNA Synthesis Kit (BioRad).
Quantitative Real-Time PCR (qPCR) was carried out in 10 .mu.l
containing 4 .mu.l of SsoAdvanced.TM. Sybr.RTM. Green Supermix
(BioRad), 4 .mu.l of 1/10 diluted cDNA and 0.5 pmol of each
gene-specific primer (Table 2), and subjected to the following PCR
protocol: 3 min at 95.degree. C., 40 cycles of 30 s at 95.degree.
C. followed by 45 s at 60.degree. C. A melting curve analysis
(samples were heated from 60.degree. C. to 95.degree. C.) was
performed after each qPCR run to verify specific amplification. The
murX, hrdB and gyrA genes (Joshi et al., 2007) were used to
normalize the amount of RNA in the samples. Each measurement was
performed in triplicate with three different cebR mutant
isolates.
Example 2
[0147] The present example demonstrates that inactivation of bglC
encoding the enzyme that hydrolyses cellobiose (the best allosteric
effector of CebR) also results in a mutant strain that
constitutively produces thaxtomin.
[0148] Deletion of the .beta.-Glucosidase Gene Accompanying the
Cellobiose/Cellotriose Transporter
[0149] Upon active transport of cellobiose/cellotriose into the
cell, catabolism of the incoming cellobiose is accomplished by a
.beta.-glucosidase that cleaves the .beta.1.fwdarw.4 glycosidic
bond linking the two glucose molecules. Deletion of the
.beta.-glucosidase gene (scab57721) associated with the
cellobiose/cellotriose-specific ABC transporter in S. scabies
resulted in increased thaxtomin production on most of the media
tested. The mutant overproduced thaxtomin in OBB cultures (FIG. 9A)
as well as on OBA plates (FIG. 9B), while the addition of
cellobiose did not increase the thaxtomin levels of the mutant
compared to the wild type strain (FIG. 9B). Even on Thaxtomin
Defined Medium (TDM, Johnson et al. 2007) with glucose as the only
carbon source, the .beta.-glucosidase deletion mutant produced up
to 50% of wild type levels on TDM with cellobiose (FIG. 10).
Overproduction was also measured on ISP-4. Moreover, addition of
cellobiose to the ISP-4 medium further increased the thaxtomin
production level of the .beta.-glucosidase mutant (FIG. 11).
[0150] Materials and methods are as described above for Example 1.
Generation of the bglC gene deletions in S. scabies 87-22 was
performed as described above for the cebR gene deletions. Briefly,
deletion mutants in S. scabies 87-22 were created using the
REDIRECT PCR targeting methodology (Gust et al. 2003, incorporated
herein by reference) replacing the selected gene by an antibiotic
deletion cassette.
[0151] It should be emphasized that the above-described embodiments
of the present disclosure are merely possible examples of
implementations, and are merely set forth for a clear understanding
of the principles of this disclosure. Many variations and
modifications may be made to the above-described embodiment(s) of
the disclosure without departing substantially from the spirit and
principles of the disclosure. All such modifications and variations
are intended to be included herein within the scope of this
disclosure and protected by the following claims.
Tables
TABLE-US-00001 [0152] TABLE 1 Bacterial strains, plasmids, and
cosmids used in this study Strain or Source or plasmid
Description.sup..dagger. reference Streptomyces strains 87-22 S.
scabies wild type strain (Loria et al. (wild type) 1995)
.DELTA.scab57761 87-22 derivative with a deletion of the This study
(.DELTA.cebR) scab57761 gene (Apr.sup.R) .DELTA.scab57721 87-22
derivative with a deletion of the This study (.DELTA..beta.-gluc)
scab57721 gene (Apr.sup.R) E. coli strains Rosetta (DE3) Host for
heterologous expression of Novagen cebR from the pET-22b vector
DH5.alpha. General cloning host Gibco-BRL BW25113 Host for the
REDIRECT PCR targeting (Gust et al. system 2003) ET12567 dam.sup.-,
dcm.sup.-, hsdS.sup.-; non-methylating (MacNeil et al. host for
transfer of DNA into 2003) Streptomyces spp. (Cml.sup.R, Tet.sup.R)
Plasmids or cosmids pIJ790 .lamda. Red plasmid (t.sup.S, Cml.sup.R)
(Gust et al. 2003) pUZ8002 Supplies transfer functions for (Kieser
et al. mobilization of oriT-containing vectors 2000) from E. coli
to Streptomyces (Kan.sup.R) pIJ773 Template for the REDIRECT PCR
(Gust et al. targeting system, contains the 2003) [aac(3)IV + oriT]
disruption cassette (Amp.sup.R, Apr.sup.R) SuperCos1 Cosmid cloning
vector (Amp.sup.R, Kan.sup.R) Stratagene Cosmid 833 SuperCos1
derivative containing the This study S. scabies 87-22
cellobiose/cellotriose- specific ABC-transporter locus (Kan.sup.R,
Amp.sup.R) pJET1.2/blunt Plasmid used for efficient cloning Thermo
Cloning Vector of PCR products (Amp.sup.R) scientific pSAJ001
pJET1.2 derivative containing the This study S. scabies 87-22 cebR
gene (Amp.sup.R) pET-22b Expression vector used for production
Novagen of His.sub.6-tagged CebR in E. coli BL21 (DE3). pSAJ002
pET-22b derivative containing the This study S. scabies 87-22 cebR
gene inserted into the Ndel-EcoRI sites (Amp.sup.R) pAU3-45 pSET152
(a .PHI.C31-derived integration (Bignell et al. vector) derivative
containing the tsr 2005) gene inserted into the (blunted) Nhel site
(Apr.sup.R, Thio.sup.R) pRLIF8 pAU3-45 derivative containing the
This study scab57761 gene and its promoter inserted into the Xbal
site (Apr.sup.R, Thio.sup.R) .sup..dagger.Apr.sup.R, apramycin
resistance; Cml.sup.R, chloramphenicol resistance; Tet.sup.R,
tetracylcin resistance; t.sup.s, temperature sensitive; Kan.sup.R,
kanamycin resistance; Amp.sup.R, ampicillin resistance; Thio.sup.R,
thiostrepton resistance
TABLE-US-00002 TABLE 2 List of oligonucleotides used in this study
Sequence (5'.fwdarw.3')* Primer (SEQ ID NO) Use imf196
gattccacgccagcgcggtagtgacgggagac scab57761 Redirect
gaccatgattccggggatccgtcgacc deletion cassette (SEQ ID NO: 8) imf197
caagcgcttcgtcatccaggtcgatctgggtcgc scab57761 Redirect
actcatgtaggctggagctgcttc deletion cassette (SEQ ID NO: 9) imf198
ctcccacgagtgatgtgttg PCR verification of (SEQ ID NO: 10)
.DELTA.scab57761 imf199 ccgtgtccttcttcatggtg PCR verification of
(SEQ ID NO: 11) .DELTA.scab57761 DRB21 gtctggcagttccaggagtc murX
gene expression (SEQ ID NO: 12) analysis (qPCR) DRB22
aggtgttccaccacaggaag murX gene expression (SEQ ID NO: 13) analysis
(qPCR) DRB23 ggacatccagacgcagtaca gyrA gene expression (SEQ ID NO:
14) analysis (qPCR) DRB24 Ctcggtgttgagcttctcct gyrA gene expression
(SEQ ID NO: 15) analysis (qPCR) DRB9 tggtcgaggtcatcaacaag hrdB gene
expression (SEQ ID NO: 16) analysis (qPCR) DRB10
tggacctcgatgaccttctc hrdB gene expression (SEQ ID NO: 17) analysis
(qPCR) DRB13 gagcgactgtccttcatgg txtA gene expression (SEQ ID NO:
18) analysis (qPCR) DRB14 cgtcgtccagtaccacgag txtA gene expression
(SEQ ID NO: 19) analysis (qPCR) DRB48 cggctacttcccgatggat txtB gene
expression (SEQ ID NO: 20) analysis (qPCR) DRB49
ctcgatgtcactcctggtca txtB gene expression (SEQ ID NO: 21) analysis
(qPCR) DRB60 ggatgcgatccacttctgat txtR gene expression (SEQ ID NO:
22) analysis (qPCR) DRB61 cgcaccgatatgttgtgttc txtR gene expression
(SEQ ID NO: 23) analysis (qPCR) imf200 ctgggttacgtcccgaacac
scab57761 gene (SEQ ID NO: 24) expression analysis (qPCR) imf201
ccttgaggatgtcggagaag scab57761 gene (SEQ ID NO: 25) expression
analysis (qPCR) imf274 aaatctagaccagcgtgatcttggtcttg PCR
complementation (SEQ ID NO: 26) of .DELTA.scab57761 imf275
aaatctagaccgtgtccttcttcatggtg PCR complementation (SEQ ID NO: 27)
of .DELTA.scab57761 imf304 cgcgccccgtaacccgtcgcgcctatcgtgcgc
scab57721 Redirect cgggtgattccggggatccgtcgacc deletion cassette
(SEQ ID NO: 28) imf305 ccacgcggggatgttgtgtgtgccgcaccggcc scab57721
Redirect cggtcatgtaggctggagctgcttc deletion cassette (SEQ ID NO:
29) imf306 acttcttctggcccttcgtg PCR verification of (SEQ ID NO: 30)
.DELTA.scab57721 imf307 gcgggctcctacgactactg PCR verification of
(SEQ ID NO: 31) .DELTA.scab57721 SCAB_57761 + 3_NdeI
catatggtgacaggccacggggc Cloning of scab57761 (SEQ ID NO: 32) in
pET-22b SCAB_57761 + 1056_EcoRI gaattcggaagaatcccgccccacc Cloning
of scab57761 (SEQ ID NO: 33) in pET-22b SCAB_31791-909 Cy5
tgtcaataagcgggagcgctcccacagcgctct EMSA probe cbs.sup.txtR-A c (SEQ
ID NO: 34) SCAB_31801-796 gagagcgctgtgggagcgctcccgcttattgac EMSA
probe cbs.sup.txtR-A a (SEQ ID NO: 35) SCAB_57751c-140 Cy5
ccaggtactgtgggagcgctcccacgagtgatg EMSA probe cbs.sup.cebR-5 t (SEQ
ID NO: 36) SCAB_57761-506 acatcactcgtgggagcgctcccacagtacctg EMSA
probe cbs.sup.cebR-5 g (SEQ ID NO: 37) SCAB_31781c + 1530 Cy5
ctcccccagggggagcgctcccactgcgctgta EMSA probe cbs.sup.txtB (SEQ ID
NO: 38) SCAB_31781c + 1497 tacagcgcagtgggagcgctccccctggggga EMSA
probe cbs.sup.txtB g (SEQ ID NO: 39) SCAB_57721 + 10
ggttcaggcatggaagcgctcccattggtggtc EMSA probe cbs.sup.bglC g (SEQ ID
NO: 40) SCAB_57721-24_Cy5 cgaccaccaatgggagcgcttccatgcctgaa EMSA
probe cbs.sup.bglC cc (SEQ ID NO: 41) *Non-homologous extensions
are underlined, while engineered restriction sites are indicated in
bold.
TABLE-US-00003 Additional nucleic acid and protein sequences cebR
(scab57761) (SEQ ID NO: 1)
ATGGTGACAGGCCACGGGGCACGGGGCCGGAGCGGTGGGCGGCCGACGTT
GGAGGAGGTCGCCGCACGGGCCGGAGTGGGCCGGGGGACGGTGTCCCGGG
TGATCAACGGCTCGCCCCGGGTGAGCGACGCGACCCGCGCGGCGGTCGAG
GCGGCCGTCGCGGAGCTGGGTTACGTCCCGAACACGGCGGCCCGCGCGCT
CGCGGCGAACCGTACCGACGCGATCGCGATGGTCGTGCCCGAACCGGAGA
CCCGCTTCTTCTCGGAGCCGTACTTCTCCGACATCCTCAAGGGTGTCGGA
GCGCAACTGTCCGACACCGAGATGCAGCTCCTGCTGATCTTCGCGGGCAA
CGACCGGGAGCGCCGGCGCCTCGCCCAGTACCTGGCCGCGCACCGCGTCG
ACGGTGTCCTCCTGGTCTCCGTCCACGCGGACGACCCGCTCCCCGATCTG
CTGTCGCAACTGGAAATCCCGGCCGTCATCAGCGGCCCCCGCTCCGAGCA
CGAGACGCTCCCCTCGGTCGACTCCGACAACTACGGCGGCGGCCGCTCGG
CGGTCGAGCACCTCATCGCACGGGGGCGCGCCCGGATCGCCACGATCACC
GGCCGGCTGGACGTCTACGGCGCCCAGCGGCGCATCGAGGGCTACCGCGA
CGCCCTGGAGGACGCGGGCCGCGAGGTGGACGAGCGCCTGATCGCCCCCG
GTGACTTCACGGAGGAGGGCGGCCGCCGAGCGATGCGCGAACTCCTGGCC
CGCTGCCCCGACCTCGACGCGGTCTTCGCCGAGTCGGACGTCATGGCCGC
CGGCGCCCGCCAGGTGCTCCGCGAGGAGGGCCGCCGCATACCCGACGACG
TGGCGCTGGTCGGCTACGACGACTCGGCGATCGCCCGCCACATGGACCCG
CCGCTCACCAGCGTCCGCCAGCCGATAGAGGAGATGGGCCGCGCGATGAT
CGACCTCCTCCTGGACGAGATCGCGGACCGCCGCCCGGCGGTGTCGAGGG
GCTTGGAACGACGCCAGGTGGTGCTGCCGACGGAGCTGGTGGGGCGGGAT TCTTCCTGA CebR
(SCAB57761) (SEQ ID NO: 2)
MVTGHGARGRSGGRPTLEEVAARAGVGRGTVSRVINGSPRVSDATRAAVE
AAVAELGYVPNTAARALAANRTDAIAMVVPEPETRFFSEPYFSDILKGVG
AQLSDTEMQLLLIFAGNDRERRRLAQYLAAHRVDGVLLVSVHADDPLPDL
LSQLEIPAVISGPRSEHETLPSVDSDNYGGGRSAVEHLIARGRARIATIT
GRLDVYGAQRRIEGYRDALEDAGREVDERLIAPGDFTEEGGRRAMRELLA
RCPDLDAVFAESDVMAAGARQVLREEGRRIPDDVALVGYDDSAIARHMDP
PLTSVRQPIEEMGRAMIDLLLDEIADRRPAVSRGLERRQVVLPTELVGRD SS bglC
(scab57721) (SEQ ID NO: 3)
ATGCCTGAACCCGTGAATCCGGCCACCCCGGTGACCTTTCCTCCCGCCTT
CCTCTGGGGCGCGGCCACCTCCGCGTACCAGATCGAGGGGGCGGTGCGGG
AGGACGGCCGTACGCCCTCCATCTGGGACACCTTCAGTCACACGCCGGGC
AAGACCGCCGGCGGCGAGAACGGTGACATCGCTGTCGACCACTACCACCG
CTACCGCGACGACGTGGCGATGATGGCGGACCTGGGCCTCAACGCGTACC
GCTTCTCCGTCTCCTGGTCGCGGGTGCAGCCGACGGGGCGGGGCCCGGCC
GTCCAGAAGGGGCTCGACTTCTACCGACGGCTGGTCGACGAGCTGCTGGC
CAAGGGCATCAAGCCCGCCGTCACCCTCTACCACTGGGACCTCCCGCAGG
AGCTGGAGGACGCCGGCGGCTGGCCCGAGCGGGACATCGTGCACCGGTTC
GCCGAGTACGCGCGGATCATGGGCGAGGCGCTCGGCGACCGCGTCGAGCA
GTGGATCACCCTCAACGAGCCGTGGTGCACCGCGTTCCTGGGCTACGGCT
CCGGGGTGCACGCGCCGGGCCGTACGGACCCGGTGGCGTCCCTGCGCGCG
GCCCACCATCTGAACGTGGCGCACGGCCTCGGCGTCTCGGCGCTGCGGTC
GGCGATGCCCGCCCGCAACTCGATCGCGGTGAGCCTCAACTCCTCGGTGG
TGCGGCCGATCACCAGCTCCCCGGAGGACCGGGCCGCGGCCCGGAAGATC
GACGACCTCGCGAACGGCGTCTTCCACGGACCGATGCTGCACGGGGCCTA
CCCGGAGACCCTGTTCGCCGCGACCTCGTCGCTGACGGACTGGTCGTTCG
TGCGGGACGGTGACGTGGCGACGGCCCATCAGCCGCTGGACGCTCTGGGG
CTGAACTACTACACGCCGGCGCTGGTCGGCGCGGCGGACGCCGGCCTGGA
GGGCCCCCGCGCGGACGGCCACGGGGCGAGCGAGCACTCGCCGTGGCCGG
CCGCGGACGACGTCCTGTTCCACCAGACCCCGGGCGAGCGTACGGAGATG
GGCTGGACCATCGACCCGACGGGCCTGCACGAGCTGATCATGCGGTACGC
GCGGGAGGCTCCGGGCCTGCCGATGTACGTGACGGAGAACGGCGCCGCGT
ACGACGACAAGATGGACGCGGACGGCCGTGTCCACGACCCCGAGCGCATC
GCCTACCTGCACGGCCACCTGCGGGCGGTCCGGCGCGCGATCGCCGAGGG
GGCGGACGTGCGCGGGTACTACCTGTGGTCCCTGATGGACAACTTCGAGT
GGGCGTACGGCTACGGCAAGCGCTTCGGCGCGGTGTACGTCGACTACGCG
ACCCTGACCCGCACACCGAAGTCGAGCGCGCACTGGTACGGGCAGGCGGC
GAAGACGGGCGCCCTCCCGCCGCTGGCGCCGGCGCCGGCGTAG BglC (SCAB57721) (SEQ
ID NO: 4) MPEPVNPATPVTFPPAFLWGAATSAYQIEGAVREDGRTPSIWDTFSHTPG
KTAGGENGDIAVDHYHRYRDDVAMMADLGLNAYRFSVSWSRVQPTGRGPA
VQKGLDFYRRLVDELLAKGIKPAVTLYHWDLPQELEDAGGWPERDIVHRF
AEYARIMGEALGDRVEQWITLNEPWCTAFLGYGSGVHAPGRTDPVASLRA
AHHLNVAHGLGVSALRSAMPARNSIAVSLNSSVVRPITSSPEDRAAARKI
DDLANGVFHGPMLHGAYPETLFAATSSLTDWSFVRDGDVATAHQPLDALG
LNYYTPALVGAADAGLEGPRADGHGASEHSPWPAADDVLFHQTPGERTEM
GWTIDPTGLHELIMRYAREAPGLPMYVTENGAAYDDKMDADGRVHDPERI
AYLHGHLRAVRRAIAEGADVRGYYLWSLMDNFEWAYGYGKRFGAVYVDYA
TLTRTPKSSAHVVYGQAAKTGALPPLAPAPA Apramycin resistance gene deletion
cassette (SEQ ID NO: 42)
ATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATTCTCTAGAAAG
TATAGGAACTTCGAAGTTCCCGCCAGCCTCGCAGAGCAGGATTCCCGTTG
AGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGC
GGGTGGGCCTACTTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACC
AAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAA
AAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTA
TGCAGCGGAAAATGCAGCTCACGGTAACTGATGCCGTATTTGCAGTACCA
GCGTACGGCCCACAGAATGATGTCACGCTGAAAATGCCGGCCTTTGAATG
GGTTCATGTGCAGCTCCATCAGCAAAAGGGGATGATAAGTTTATCACCAC
CGACTATTTGCAACAGTGCCGTTGATCGTGCTATGATCGACTGATGTCAT
CAGCGGTGGAGTGCAATGTCGTGCAATACGAATGGCGAAAAGCCGAGCTC
ATCGGTCAGCTTCTCAACCTTGGGGTTACCCCCGGCGGTGTGCTGCTGGT
CCACAGCTCCTTCCGTAGCGTCCGGCCCCTCGAAGATGGGCCACTTGGAC
TGATCGAGGCCCTGCGTGCTGCGCTGGGTCCGGGAGGGACGCTCGTCATG
CCCTCGTGGTCAGGTCTGGACGACGAGCCGTTCGATCCTGCCACGTCGCC
CGTTACACCGGACCTTGGAGTTGTCTCTGACACATTCTGGCGCCTGCCAA
ATGTAAAGCGCAGCGCCCATCCATTTGCCTTTGCGGCAGCGGGGCCACAG
GCAGAGCAGATCATCTCTGATCCATTGCCCCTGCCACCTCACTCGCCTGC
AAGCCCGGTCGCCCGTGTCCATGAACTCGATGGGCAGGTACTTCTCCTCG
GCGTGGGACACGATGCCAACACGACGCTGCATCTTGCCGAGTTGATGGCA
AAGGTTCCCTATGGGGTGCCGAGACACTGCACCATTCTTCAGGATGGCAA
GTTGGTACGCGTCGATTATCTCGAGAATGACCACTGCTGTGAGCGCTTTG
CCTTGGCGGACAGGTGGCTCAAGGAGAAGAGCCTTCAGAAGGAAGGTCCA
GTCGGTCATGCCTTTGCTCGGTTGATCCGCTCCCGCGACATTGTGGCGAC
AGCCCTGGGTCAACTGGGCCGAGATCCGTTGATCTTCCTGCATCCGCCAG
AGGCGGGATGCGAAGAATGCGATGCCGCTCGCCAGTCGATTGGCTGAGCT
CATAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC
GAAGCAGCTCCAGCCTACA
REFERENCES
[0153] Barry, S. M., Kers, J. A., Johnson, E. G., Song, L., Aston,
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81-88.
Sequence CWU 1
1
4311059DNAStreptomyces scabies 1atggtgacag gccacggggc acggggccgg
agcggtgggc ggccgacgtt ggaggaggtc 60gccgcacggg ccggagtggg ccgggggacg
gtgtcccggg tgatcaacgg ctcgccccgg 120gtgagcgacg cgacccgcgc
ggcggtcgag gcggccgtcg cggagctggg ttacgtcccg 180aacacggcgg
cccgcgcgct cgcggcgaac cgtaccgacg cgatcgcgat ggtcgtgccc
240gaaccggaga cccgcttctt ctcggagccg tacttctccg acatcctcaa
gggtgtcgga 300gcgcaactgt ccgacaccga gatgcagctc ctgctgatct
tcgcgggcaa cgaccgggag 360cgccggcgcc tcgcccagta cctggccgcg
caccgcgtcg acggtgtcct cctggtctcc 420gtccacgcgg acgacccgct
ccccgatctg ctgtcgcaac tggaaatccc ggccgtcatc 480agcggccccc
gctccgagca cgagacgctc ccctcggtcg actccgacaa ctacggcggc
540ggccgctcgg cggtcgagca cctcatcgca cgggggcgcg cccggatcgc
cacgatcacc 600ggccggctgg acgtctacgg cgcccagcgg cgcatcgagg
gctaccgcga cgccctggag 660gacgcgggcc gcgaggtgga cgagcgcctg
atcgcccccg gtgacttcac ggaggagggc 720ggccgccgag cgatgcgcga
actcctggcc cgctgccccg acctcgacgc ggtcttcgcc 780gagtcggacg
tcatggccgc cggcgcccgc caggtgctcc gcgaggaggg ccgccgcata
840cccgacgacg tggcgctggt cggctacgac gactcggcga tcgcccgcca
catggacccg 900ccgctcacca gcgtccgcca gccgatagag gagatgggcc
gcgcgatgat cgacctcctc 960ctggacgaga tcgcggaccg ccgcccggcg
gtgtcgaggg gcttggaacg acgccaggtg 1020gtgctgccga cggagctggt
ggggcgggat tcttcctga 10592352PRTStreptomyces scabies 2Met Val Thr
Gly His Gly Ala Arg Gly Arg Ser Gly Gly Arg Pro Thr1 5 10 15Leu Glu
Glu Val Ala Ala Arg Ala Gly Val Gly Arg Gly Thr Val Ser 20 25 30Arg
Val Ile Asn Gly Ser Pro Arg Val Ser Asp Ala Thr Arg Ala Ala 35 40
45Val Glu Ala Ala Val Ala Glu Leu Gly Tyr Val Pro Asn Thr Ala Ala
50 55 60Arg Ala Leu Ala Ala Asn Arg Thr Asp Ala Ile Ala Met Val Val
Pro65 70 75 80Glu Pro Glu Thr Arg Phe Phe Ser Glu Pro Tyr Phe Ser
Asp Ile Leu 85 90 95Lys Gly Val Gly Ala Gln Leu Ser Asp Thr Glu Met
Gln Leu Leu Leu 100 105 110Ile Phe Ala Gly Asn Asp Arg Glu Arg Arg
Arg Leu Ala Gln Tyr Leu 115 120 125Ala Ala His Arg Val Asp Gly Val
Leu Leu Val Ser Val His Ala Asp 130 135 140Asp Pro Leu Pro Asp Leu
Leu Ser Gln Leu Glu Ile Pro Ala Val Ile145 150 155 160Ser Gly Pro
Arg Ser Glu His Glu Thr Leu Pro Ser Val Asp Ser Asp 165 170 175Asn
Tyr Gly Gly Gly Arg Ser Ala Val Glu His Leu Ile Ala Arg Gly 180 185
190Arg Ala Arg Ile Ala Thr Ile Thr Gly Arg Leu Asp Val Tyr Gly Ala
195 200 205Gln Arg Arg Ile Glu Gly Tyr Arg Asp Ala Leu Glu Asp Ala
Gly Arg 210 215 220Glu Val Asp Glu Arg Leu Ile Ala Pro Gly Asp Phe
Thr Glu Glu Gly225 230 235 240Gly Arg Arg Ala Met Arg Glu Leu Leu
Ala Arg Cys Pro Asp Leu Asp 245 250 255Ala Val Phe Ala Glu Ser Asp
Val Met Ala Ala Gly Ala Arg Gln Val 260 265 270Leu Arg Glu Glu Gly
Arg Arg Ile Pro Asp Asp Val Ala Leu Val Gly 275 280 285Tyr Asp Asp
Ser Ala Ile Ala Arg His Met Asp Pro Pro Leu Thr Ser 290 295 300Val
Arg Gln Pro Ile Glu Glu Met Gly Arg Ala Met Ile Asp Leu Leu305 310
315 320Leu Asp Glu Ile Ala Asp Arg Arg Pro Ala Val Ser Arg Gly Leu
Glu 325 330 335Arg Arg Gln Val Val Leu Pro Thr Glu Leu Val Gly Arg
Asp Ser Ser 340 345 35031443DNAStreptomyces scabies 3atgcctgaac
ccgtgaatcc ggccaccccg gtgacctttc ctcccgcctt cctctggggc 60gcggccacct
ccgcgtacca gatcgagggg gcggtgcggg aggacggccg tacgccctcc
120atctgggaca ccttcagtca cacgccgggc aagaccgccg gcggcgagaa
cggtgacatc 180gctgtcgacc actaccaccg ctaccgcgac gacgtggcga
tgatggcgga cctgggcctc 240aacgcgtacc gcttctccgt ctcctggtcg
cgggtgcagc cgacggggcg gggcccggcc 300gtccagaagg ggctcgactt
ctaccgacgg ctggtcgacg agctgctggc caagggcatc 360aagcccgccg
tcaccctcta ccactgggac ctcccgcagg agctggagga cgccggcggc
420tggcccgagc gggacatcgt gcaccggttc gccgagtacg cgcggatcat
gggcgaggcg 480ctcggcgacc gcgtcgagca gtggatcacc ctcaacgagc
cgtggtgcac cgcgttcctg 540ggctacggct ccggggtgca cgcgccgggc
cgtacggacc cggtggcgtc cctgcgcgcg 600gcccaccatc tgaacgtggc
gcacggcctc ggcgtctcgg cgctgcggtc ggcgatgccc 660gcccgcaact
cgatcgcggt gagcctcaac tcctcggtgg tgcggccgat caccagctcc
720ccggaggacc gggccgcggc ccggaagatc gacgacctcg cgaacggcgt
cttccacgga 780ccgatgctgc acggggccta cccggagacc ctgttcgccg
cgacctcgtc gctgacggac 840tggtcgttcg tgcgggacgg tgacgtggcg
acggcccatc agccgctgga cgctctgggg 900ctgaactact acacgccggc
gctggtcggc gcggcggacg ccggcctgga gggcccccgc 960gcggacggcc
acggggcgag cgagcactcg ccgtggccgg ccgcggacga cgtcctgttc
1020caccagaccc cgggcgagcg tacggagatg ggctggacca tcgacccgac
gggcctgcac 1080gagctgatca tgcggtacgc gcgggaggct ccgggcctgc
cgatgtacgt gacggagaac 1140ggcgccgcgt acgacgacaa gatggacgcg
gacggccgtg tccacgaccc cgagcgcatc 1200gcctacctgc acggccacct
gcgggcggtc cggcgcgcga tcgccgaggg ggcggacgtg 1260cgcgggtact
acctgtggtc cctgatggac aacttcgagt gggcgtacgg ctacggcaag
1320cgcttcggcg cggtgtacgt cgactacgcg accctgaccc gcacaccgaa
gtcgagcgcg 1380cactggtacg ggcaggcggc gaagacgggc gccctcccgc
cgctggcgcc ggcgccggcg 1440tag 14434480PRTStreptomyces scabies 4Met
Pro Glu Pro Val Asn Pro Ala Thr Pro Val Thr Phe Pro Pro Ala1 5 10
15Phe Leu Trp Gly Ala Ala Thr Ser Ala Tyr Gln Ile Glu Gly Ala Val
20 25 30Arg Glu Asp Gly Arg Thr Pro Ser Ile Trp Asp Thr Phe Ser His
Thr 35 40 45Pro Gly Lys Thr Ala Gly Gly Glu Asn Gly Asp Ile Ala Val
Asp His 50 55 60Tyr His Arg Tyr Arg Asp Asp Val Ala Met Met Ala Asp
Leu Gly Leu65 70 75 80Asn Ala Tyr Arg Phe Ser Val Ser Trp Ser Arg
Val Gln Pro Thr Gly 85 90 95Arg Gly Pro Ala Val Gln Lys Gly Leu Asp
Phe Tyr Arg Arg Leu Val 100 105 110Asp Glu Leu Leu Ala Lys Gly Ile
Lys Pro Ala Val Thr Leu Tyr His 115 120 125Trp Asp Leu Pro Gln Glu
Leu Glu Asp Ala Gly Gly Trp Pro Glu Arg 130 135 140Asp Ile Val His
Arg Phe Ala Glu Tyr Ala Arg Ile Met Gly Glu Ala145 150 155 160Leu
Gly Asp Arg Val Glu Gln Trp Ile Thr Leu Asn Glu Pro Trp Cys 165 170
175Thr Ala Phe Leu Gly Tyr Gly Ser Gly Val His Ala Pro Gly Arg Thr
180 185 190Asp Pro Val Ala Ser Leu Arg Ala Ala His His Leu Asn Val
Ala His 195 200 205Gly Leu Gly Val Ser Ala Leu Arg Ser Ala Met Pro
Ala Arg Asn Ser 210 215 220Ile Ala Val Ser Leu Asn Ser Ser Val Val
Arg Pro Ile Thr Ser Ser225 230 235 240Pro Glu Asp Arg Ala Ala Ala
Arg Lys Ile Asp Asp Leu Ala Asn Gly 245 250 255Val Phe His Gly Pro
Met Leu His Gly Ala Tyr Pro Glu Thr Leu Phe 260 265 270Ala Ala Thr
Ser Ser Leu Thr Asp Trp Ser Phe Val Arg Asp Gly Asp 275 280 285Val
Ala Thr Ala His Gln Pro Leu Asp Ala Leu Gly Leu Asn Tyr Tyr 290 295
300Thr Pro Ala Leu Val Gly Ala Ala Asp Ala Gly Leu Glu Gly Pro
Arg305 310 315 320Ala Asp Gly His Gly Ala Ser Glu His Ser Pro Trp
Pro Ala Ala Asp 325 330 335Asp Val Leu Phe His Gln Thr Pro Gly Glu
Arg Thr Glu Met Gly Trp 340 345 350Thr Ile Asp Pro Thr Gly Leu His
Glu Leu Ile Met Arg Tyr Ala Arg 355 360 365Glu Ala Pro Gly Leu Pro
Met Tyr Val Thr Glu Asn Gly Ala Ala Tyr 370 375 380Asp Asp Lys Met
Asp Ala Asp Gly Arg Val His Asp Pro Glu Arg Ile385 390 395 400Ala
Tyr Leu His Gly His Leu Arg Ala Val Arg Arg Ala Ile Ala Glu 405 410
415Gly Ala Asp Val Arg Gly Tyr Tyr Leu Trp Ser Leu Met Asp Asn Phe
420 425 430Glu Trp Ala Tyr Gly Tyr Gly Lys Arg Phe Gly Ala Val Tyr
Val Asp 435 440 445Tyr Ala Thr Leu Thr Arg Thr Pro Lys Ser Ser Ala
His Trp Tyr Gly 450 455 460Gln Ala Ala Lys Thr Gly Ala Leu Pro Pro
Leu Ala Pro Ala Pro Ala465 470 475 480514DNAArtificial
SequencecbstxtR-A S. Scabies 5cgggagcgct ccca 14614DNAArtificial
SequencecbstxtB S. scabies 6ggggagcgct ccca 14714DNAArtificial
SequencecbscebR-E S. Scabies 7tgggagcgct ccca 14859DNAArtificial
Sequenceprimer 8gattccacgc cagcgcggta gtgacgggag acgaccatga
ttccggggat ccgtcgacc 59958DNAArtificial Sequenceprimer 9caagcgcttc
gtcatccagg tcgatctggg tcgcactcat gtaggctgga gctgcttc
581020DNAArtificial Sequenceprimer 10ctcccacgag tgatgtgttg
201120DNAArtificial Sequenceprimer 11ccgtgtcctt cttcatggtg
201220DNAArtificial Sequenceprimer 12gtctggcagt tccaggagtc
201320DNAArtificial Sequenceprimer 13aggtgttcca ccacaggaag
201420DNAArtificial Sequenceprimer 14ggacatccag acgcagtaca
201520DNAArtificial Sequenceprimer 15ctcggtgttg agcttctcct
201620DNAArtificial Sequenceprimer 16tggtcgaggt catcaacaag
201720DNAArtificial Sequenceprimer 17tggacctcga tgaccttctc
201819DNAArtificial Sequenceprimer 18gagcgactgt ccttcatgg
191919DNAArtificial Sequenceprimer 19cgtcgtccag taccacgag
192019DNAArtificial Sequenceprimer 20cggctacttc ccgatggat
192120DNAArtificial Sequenceprimer 21ctcgatgtca ctcctggtca
202220DNAArtificial Sequenceprimer 22ggatgcgatc cacttctgat
202320DNAArtificial Sequenceprimer 23cgcaccgata tgttgtgttc
202420DNAArtificial Sequenceprimer 24ctgggttacg tcccgaacac
202520DNAArtificial Sequenceprimer 25ccttgaggat gtcggagaag
202629DNAArtificial Sequenceprimer 26aaatctagac cagcgtgatc
ttggtcttg 292729DNAArtificial Sequenceprimer 27aaatctagac
cgtgtccttc ttcatggtg 292859DNAArtificial Sequenceprimer
28cgcgccccgt aacccgtcgc gcctatcgtg cgccgggtga ttccggggat ccgtcgacc
592958DNAArtificial Sequenceprimer 29ccacgcgggg atgttgtgtg
tgccgcaccg gcccggtcat gtaggctgga gctgcttc 583020DNAArtificial
Sequenceprimer 30acttcttctg gcccttcgtg 203120DNAArtificial
Sequenceprimer 31gcgggctcct acgactactg 203223DNAArtificial
Sequenceprimer 32catatggtga caggccacgg ggc 233325DNAArtificial
Sequenceprimer 33gaattcggaa gaatcccgcc ccacc 253434DNAArtificial
Sequenceprimer 34tgtcaataag cgggagcgct cccacagcgc tctc
343534DNAArtificial Sequenceprimer 35gagagcgctg tgggagcgct
cccgcttatt gaca 343634DNAArtificial Sequenceprimer 36ccaggtactg
tgggagcgct cccacgagtg atgt 343734DNAArtificial Sequenceprimer
37acatcactcg tgggagcgct cccacagtac ctgg 343833DNAArtificial
Sequenceprimer 38ctcccccagg gggagcgctc ccactgcgct gta
333933DNAArtificial Sequenceprimer 39tacagcgcag tgggagcgct
ccccctgggg gag 334034DNAArtificial Sequenceprimer 40ggttcaggca
tggaagcgct cccattggtg gtcg 344134DNAArtificial Sequenceprimer
41cgaccaccaa tgggagcgct tccatgcctg aacc 34421369DNAArtificial
SequenceApramycin resistance gene deletion cassette 42attccgggga
tccgtcgacc tgcagttcga agttcctatt ctctagaaag tataggaact 60tcgaagttcc
cgccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
120agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta
tcctgcccgg 180ctgacgccgt tggatacacc aaggaaagtc tacacgaacc
ctttggcaaa atcctgtata 240tcgtgcgaaa aaggatggat ataccgaaaa
aatcgctata atgaccccga agcagggtta 300tgcagcggaa aatgcagctc
acggtaactg atgccgtatt tgcagtacca gcgtacggcc 360cacagaatga
tgtcacgctg aaaatgccgg cctttgaatg ggttcatgtg cagctccatc
420agcaaaaggg gatgataagt ttatcaccac cgactatttg caacagtgcc
gttgatcgtg 480ctatgatcga ctgatgtcat cagcggtgga gtgcaatgtc
gtgcaatacg aatggcgaaa 540agccgagctc atcggtcagc ttctcaacct
tggggttacc cccggcggtg tgctgctggt 600ccacagctcc ttccgtagcg
tccggcccct cgaagatggg ccacttggac tgatcgaggc 660cctgcgtgct
gcgctgggtc cgggagggac gctcgtcatg ccctcgtggt caggtctgga
720cgacgagccg ttcgatcctg ccacgtcgcc cgttacaccg gaccttggag
ttgtctctga 780cacattctgg cgcctgccaa atgtaaagcg cagcgcccat
ccatttgcct ttgcggcagc 840ggggccacag gcagagcaga tcatctctga
tccattgccc ctgccacctc actcgcctgc 900aagcccggtc gcccgtgtcc
atgaactcga tgggcaggta cttctcctcg gcgtgggaca 960cgatgccaac
acgacgctgc atcttgccga gttgatggca aaggttccct atggggtgcc
1020gagacactgc accattcttc aggatggcaa gttggtacgc gtcgattatc
tcgagaatga 1080ccactgctgt gagcgctttg ccttggcgga caggtggctc
aaggagaaga gccttcagaa 1140ggaaggtcca gtcggtcatg cctttgctcg
gttgatccgc tcccgcgaca ttgtggcgac 1200agccctgggt caactgggcc
gagatccgtt gatcttcctg catccgccag aggcgggatg 1260cgaagaatgc
gatgccgctc gccagtcgat tggctgagct cataagttcc tattccgaag
1320ttcctattct ctagaaagta taggaacttc gaagcagctc cagcctaca
13694314DNAArtificial SequenceNucleotide sequence cbsbglC
43tggaagcgct ccca 14
* * * * *
References