U.S. patent application number 10/772168 was filed with the patent office on 2004-07-22 for cellular arrays for the identification of altered gene expression.
Invention is credited to Gonye, Gregory E., Hanafey, Michael K., Larossa, Robert A., Rafalski, J. Antoni, Van Dyk, Tina K..
Application Number | 20040142373 10/772168 |
Document ID | / |
Family ID | 22729032 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040142373 |
Kind Code |
A1 |
Gonye, Gregory E. ; et
al. |
July 22, 2004 |
Cellular arrays for the identification of altered gene
expression
Abstract
The present invention relates to the generation and use of a
cellular array or a cellular array in combination with other
genome-registered arrays (an array of arrays) for the determination
of gene function and/or perturbation mode of action. Each cellular
array consists of a number of microbial strains. Each strain
comprises one reporter gene fusion made up of a gene or gene
fragment operably linked to a reporter gene. Each gene or gene
fragment has been "registered" or mapped to a specific location in
the genome of the organism. The genome-registered collection of the
invention may be used to determine alterations in gene expression
under a variety of conditions. Such collections are amenable to
rapid assay and may be used to confirm, correct or augment data
generated from DNA micro array technology
Inventors: |
Gonye, Gregory E.;
(Wilmington, DE) ; Hanafey, Michael K.;
(Wilmington, DE) ; Larossa, Robert A.; (Chadds
Ford, PA) ; Rafalski, J. Antoni; (Wilmington, DE)
; Van Dyk, Tina K.; (Wilmington, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY
LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
22729032 |
Appl. No.: |
10/772168 |
Filed: |
February 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772168 |
Feb 4, 2004 |
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09832419 |
Apr 11, 2001 |
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6716582 |
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60197348 |
Apr 14, 2000 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 1/6897
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for determining gene function between at least two
genome-registered collections comprising: (a) assembling at least
two genome-wide scale, genome-registered collections; (b)
perturbing each collection from (a) with at least one perturbation;
(c) measuring the response of each collection to each perturbation
of (b); (d) analyzing the results of the at least one perturbation
to identify patterns of similarities and differences between the at
least two genome-registered collections.
2. A method according to claim 1 wherein the perturbation is
selected from the group consisting of radiation, humidity,
alterations in temperature, alterations in carbon source,
alterations in energy source, alterations in nitrogen source,
alterations in phosphorus source, alterations in sulfur source,
alterations in trace element sources, a change in pH, the presence
other organisms, the presence of chemicals, the presence of toxins,
and abnormal levels of normal metabolites.
3. A method for generating a genome-registered collection of
reporter gene fusions comprising the steps of: (a) generating a set
of gene fusions comprising: 1) a reporter gene or reporter gene
complex operably linked to 2) a genomic fragment from an organism
of which at least 15% of the genomic nucleotide sequence is known;
(b) introducing in vitro the reporter gene fusions from step (a)
into a host organism; (c) registering the reporter gene fusions on
the basis of sequence homology to the genomic sequence of the
organism; (d) repeating (a), (b), and/or (c) until reporter gene
fusions have been made to at least 15% of the known genomic
nucleotide sequence of said organism.
4. A method according to claim 3 wherein the gene fusions of step
(a) are generated either in vivo or in vitro.
5. A method for generating a genome-registered collection of
reporter gene fusions comprising: (a) generating random nucleic
acid fragments from the DNA of an organism of which at least 15% of
the nucleotide sequence is known; (b) operably linking the random
nucleic acid fragments generated in (a) to a vector containing a
promoterless reporter gene or reporter gene complex; (c)
introducing the vector (b) containing the gene fusions into a host
organism; (d) determining the nucleic acid sequence of the distal
and the proximal ends of the random nucleic fragments relative to
the reporter gene or reporter gene complex; (e) registering the
sequenced fusions of step (d) on the basis of sequence homology to
the genomic sequence of the host organism; (d) repeating (a), (b),
and/or (c) until reporter gene fusions have been made to at least
15% of the known genomic nucleotide sequence of said organism
6. A method according to claim 5 wherein the random nucleic acid
fragments of step (a) are generated by method selected from the
group consisting of restriction enzyme digestion, physical shearing
of the genome and polymerase chain reaction.
7. A method for generating a genome-registered collection of
reporter gene fusions comprising: (a) providing a genome from an
organism wherein at least 15% of the nucleotide sequence is known;
(b) providing a series of amplification primers having homology to
specific known regions of the genome of (a); (c) amplifying
portions of the genome of (a) with the primers of (b) to create a
collection of nucleic acid amplification products; (d) operably
linking the amplification products of (c) to a vector containing a
promoterless reporter gene or reporter gene complex; (e)
introducing the reporter gene fusions into a said organism; (f)
repeating (a)-(e) until, until reporter gene fusions have been made
to at least 15% of the known genomic nucleotide sequence of said
organism.
8. A method for generating a genome-registered collection of
reporter gene fusions comprising steps of: (a) introducing one or
more transposons into the genome of an organism of which at least
15% of the nucleotide sequence is known, each transposon containing
a promoterless reporter gene or reporter gene complex; (b)
determining the nucleic acid sequence of the junction between the
proximal end of the genomic DNA and the transposon containing the
reporter gene or reporter gene complex and registering the reporter
gene fusions relative to the genomic sequence of the organism, (c)
repeating (a) and (b) until reporter gene fusions have been made to
at least 15% of the known genomic nucleotide sequence of said
organism
9. A method according to any one of claims 1, 3, 5, 7 or 8 wherein
organism is selected from the group consisting of prokaryotes and
fungi.
10. A method according to claim 9 wherein the prokaryote is an
enteric bacterium.
11. A method according to claim 10 wherein the enteric bacterium is
selected from the group consisting of Escherichia and
Salmonella.
12. A method according to one of claims 1, 3, 5, 7 or 8 wherein the
reporter gene or reporter gene complex is selected from the group
consisting of luxCDABE, lacZ, gfp, cat, galK, inaZ, luc, luxAB,
bgaB, nptII, phoA, uidA and xylE.
13. A method according to one of claims 1, 3, 5, 7 or 8 wherein at
least 50% of the genomic nucleotides sequence is known.
14. A method for identifying a profile of inducing conditions for a
reporter gene fusion comprising: (a) obtaining a gene expression
profile of an organism under induced and non-induced conditions
wherein induced genes are identified; (b) providing a
genome-registered collection of reporter gene fusions, said fusions
registered to the genome of the organism of (a); (c) selecting the
reporter gene fusions of (b) that correspond to the induced genes
of (a) to create a subset of the genome-register collection; (d)
contacting the subset of the genome-register collection of (c) with
the inducing conditions of (a) to identify at least one
representative reporter gene fusion whose expression was altered in
a similar manner as in (a); (e) contacting the at least one
representative reporter gene fusion of (d) in a high throughput
manner with a multiplicity of different inducing conditions to
identify a profile of inducing conditions for that reporter gene
fusion.
15. A method according to claim 14 wherein at least 15% of the
genomic nucleotide sequence of said organism is known.
16. A method for identifying a profile of inducing conditions for a
reporter gene fusion comprising: (a) obtaining a gene expression
profile for each of mutant strain and a parental strain organism
under induced and non-induced conditions wherein induced genes are
identified; (b) providing a genome-registered collection of
reporter gene fusions, said fusions registered to the genome of the
organism of (a); (c) selecting the reporter gene fusions of (b)
that correspond to the induced genes of (a) to create a subset of
the genome-register collection; (d) contacting the subset of the
genome-register collection of (c) with the inducing conditions of
(a) to identify at least one representative reporter gene fusion
whose expression was altered in a similar manner as in (a); (e)
contacting the at least one representative reporter gene fusion of
(d) in a high throughput manner with a multiplicity of different
inducing conditions to identify a profile of inducing conditions
for that reporter gene fusion.
17. A method to validate results from comprehensive genome analysis
comprising the steps of: (a) analyzing a genome-wide, gene
expression assay of an organism treated with a condition or
chemical of interest to identify genes with altered expression; (b)
selecting from a genome-registered collection of reporter gene
fusions those reporter gene fusions containing promoter regions
operably linked to genes corresponding to the altered genes from
(a) or genes co-regulated with genes corresponding to the altered
genes from (a); (c) testing expression of the reporter gene fusions
selected from (b) with the conditions or chemicals of interest used
in (a); and (d) comparing the gene expression results from (c) to
the gene expression result of (a).
18. A method to determine operon structure comprising steps of: (a)
selecting a subset of reporter gene fusions from a
genome-registered collection of reporter gene fusions that map to
the region of a possible operon; (b) assaying the subset for the
reporter gene function; and (c) determining a putative operon
structure based on the quantities of reporter gene function.
19. A method for constructing a cellular array containing reporter
gene fusions comprising: (a) generating a set of gene fusions
comprising: 1) a reporter gene or reporter gene complex operably
linked to 2) a genomic fragment from an organism of which at least
15% of the genomic nucleotide sequence is known; (b) selecting a
non-redundant subset of reporter gene fusions from the set of (a)
representative of at least 15% of known or suspected promoter
regions from a genome-registered collection of reporter gene
fusions, each containing a known or suspected promoter region
operably linked to a reporter gene or reporter gene complex; and
(c) fixing the non-redundant subset of reporter gene fusions of (b)
in an array format.
20. A method for measuring gene expression responses to
perturbation comprising: (a) constructing at least 2 identical
cellular arrays, each cellular array comprising a reporter gene
fusion comprising: 1) a reporter gene or reporter gene complex
operably linked to 2) a genomic fragment from an organism of which
at least 15% of the genomic nucleotide sequence is known; wherein
at least one cellular array is a control array and at least one
cellular array is an experimental array; (b) contacting the
experimental array of (a) with a perturbing condition; (c)
comparing the differences between the gene expression activity of
the control and the experimental array wherein gene expression
response to a perturbing condition is determined.
21. The method of claim 20 wherein the cellular array is fixed in a
manner selected from the group consisting of, fixed on a solid
medium, and arrayed in liquid medium.
22. The method of claim 20 wherein the perturbing condition is
selected from the group consisting of radiation, humidity,
alterations in temperature, alterations in carbon source,
alterations in energy source, alterations in nitrogen source,
alterations in phosphorus source, alterations in sulfur source,
alterations in trace element sources, a change in pH, the presence
other organisms, the presence of chemicals, the presence of toxins,
and abnormal levels of normal metabolites.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/197,348 filed Apr. 14, 2000.
FIELD OF THE INVENTION
[0002] This invention is in the field of bacterial gene expression.
More specifically, this invention describes a method to monitor
transcriptional changes on a genome-wide scale using a
genome-registered gene fusion collection.
BACKGROUND OF THE INVENTION
[0003] DNA array analysis is a powerful method for comprehensive
genome analysis of gene expression. Currently, this approach is the
only available method for massively parallel analyses that allow
the expression of each gene of a bacterial genome to be
characterized simultaneously (Richmond et al., (1999) Nucleic Acids
Res. 27:3821-3835., 17, 25; Tao et al., (1999), J. Bacteriol.
181:6425-6440; Wilson et al., (1999) Proc. Natl. Acad. Sci. U.S.A.
96:12833-12838).
[0004] Richmond et al. ((1999) Nucleic Acids Research,
27:3821-3835) has recently reported genome-wide expression
profiling of E. coli at the single ORF level of resolution. Changes
in RNA levels after exposure to heat shock or IPTG were analyzed
using comprehensive low density blots of individual ORFs on a nylon
matrix and comprehensive high density arrays of individual ORFs
spotted on glass slides. The results of the two methods were
compared. Richmond et al. states that radioactive probe/spot blots
are inferior to fluorescent probe/micro-arrays. Moreover, the
comparison of heat shock treatment between the two methods is
fundamentally flawed since the RNA analyzed with spot blots were
derived from broth grown cultures while those analyzed with
micro-arrays were derived from cells grown in defined media.
Despite the power of this new methodology, there are several
problems that limit the reliability of results. For example,
artifacts may arise during the isolation of microbial RNA (Tao et
al., (1999) J. Bacteriol. 181:6425-6440) or from cross
hybridization to paralogous genes (Richmond et al., (1999) Nucleic
Acids Res. 27:3821-3835, 17, 25).
[0005] Another limitation of DNA array methodology is that RNA must
be isolated, converted into DNA by reverse transcriptase with
concomitant incorporation of fluorescent labels. These steps make
it unlikely that facile high throughput screens could be developed
based on DNA array technology. Thus, there exists a need for a
method that adapts results from DNA array technology into high
throughput screens. For the reasons mentioned above and others,
alternative genome-wide expression profiling method as well as
rapid methods to independently verify results from DNA array
experiments are needed.
[0006] Gene fusion technology is an established method for gene
expression monitoring. For example, the initial discovery of the
SOS (DNA damage responsive) regulon of E. coli was done by Kenyon
and Walker ((1980) Proc. Natl. Acad. Sci. U.S.A. 77:2819-2823) by
comparing the transcriptional responses of Escherichia coli to
mitomycin C (MMC), a DNA damaging agent that intercalates into and
forms a covalent attachment with double-stranded DNA. While these
early experiments attempted to scan the bulk of the E. coli genome
by using a transposon that put the lacZYA operon under the control
of many promoter regions, it was not known if the entire genome had
been surveyed because of the random nature of transposition and
unknown location of the majority of transposition events.
Accordingly, additional SOS regulon genes have been identified
since these early experiments (Lomba et al., (1997) Microbiol Lett
156:119-122; Walker, (1996) In Escherichia coli and Salmonella:
Cellular and Molecular Biology. ASM Press, pp 1400 1416).
[0007] LaRossa et al. (U.S. Pat. No. 5,683,868) has transformed E.
coli with at construct comprised of luxCDABE operably linked to a
variety of stress promoters. They have used the microorganisms to
detect a variety of environmental insults such as Ethanol,
CdCl.sub.2 and toluene. The presence of sublethal concentration of
insults is indicated by an increase in bioluminescence. However, in
order to generate the transformed host, the stress promoters has to
be identified and characterized. Furthermore, this method is
limited to the stress response only.
[0008] Ashby and Rine (U.S. Pat. No. 5,569,588) reported a method
to measure the transcriptional responsiveness of an organism to a
candidate drug by detecting reporter gene product signals from
separately isolated cells of a target organism on genome-wide
bases. Each cell contains a recombinant construct with the reporter
gene operatively linked to a different endogenous transcriptional
regulatory element of the target organism When cells were treated
with a candidate drug, the transcriptional responsiveness of the
organism to the candidate drug was measured by the detecting the
reporter signal from each cells. However, this method is useful
with the organism only after the majority of transcriptional
regulatory elements of the target organism are known and mapped.
Furthermore, the reporter signals are measured only after cells
reached homeostasis in the presence of drug. The initial
transcriptional responses to chemicals are not considered.
[0009] The Lux-A Collection of random E. coli genomic DNA fused to
the luxCDABE had been used to screen for those gene fusions for
which expression was induced by treatment with the herbicide
sulfometuron methyl. The DNA sequence of 19 of these sulfometuron
methyl inducible gene fusions (smi-lux) was determined and used to
identify the promoter controlling expression of the luxCDABE
reporter (Van Dyk et al., (1998) J. Bacteriol. 180:785-792); the
remaining 8047 gene fusions remained unidentified.
[0010] LaRossa and Van Dyk (U.S. Pat. No. 6,025,131) developed a
method for the identification of gene regulatory regions,
responsive to a particular cellular stress, such as that produced
by herbicides or crop protection chemicals by randomly fusing
regulatory regions to a bacterial luminescent gene complex where
contacting the fusion in a suitable host with a cellular insult
producing a cellular stress results in detection of that cellular
stress by an increase in cellular luminescence. However, this
method was limited to the perturbations in liquid media;
luminescent responses were not detected on solid medium following
overnight growth in the presence of a chemical stress. Furthermore,
it did not allow regulatory region activity analysis in genome-wide
scale.
[0011] The problem to be solved therefore is to provide a way to
measure and follow the changes in gene expression using a
genome-registered collection of reporter gene fusions in a manner
that allows detection of initial transcriptional responses, and
provide a way to cross-validate the results from other method
(i.e., microarray) as well as to determine promoter and operon
structure of genes, and further provide a way to test cellular
responses to various environmental and genetic changes in high
throughput manner.
SUMMARY OF THE INVENTION
[0012] A new method for the use of genome registered collection of
reporter gene fusion is disclosed. Fragments of genomic DNA of host
organism were fused to promoterless reporter gene. The reporter
gene fusions were generated using restriction enzyme digestion,
physical shearing of the genomic DNA, PCR, and transposition
techniques. The reporter gene complexes were genome registered
against the host genome on the basis of homology. Gene expression
of each reporter gene complex is measure as reporter gene activity.
The present invention provides a means to measure the changes in
gene expression profiles in genome wide scale under various
conditions in high throughput manner. In addition to being a
stand-alone high throughput method, the present invention also
provides a way to validate other genome-wide assays such as DNA
microarray. The present invention also provides a method to confirm
the response of several promoters to a particular insult (a
condition or chemical of interest) as well as to identify a number
of previously unknown operons responsive to that insult. Comparison
of the gene expression patterns of two samples differing in one
variable is also possible using this method. The present invention
also provides the method to use an array of arrays by generating
gene expression profiles that yield information relevant to
understanding gene function and modes of chemical action. Such
information can be gained by analysis of genetic alterations
resulting in loss of function, reduced levels of gene products, or
over-expression of gene products. Thus, an array of arrays can be
used to enhance both mode of action studies and functional
genomics.
[0013] In this invention, the sequencing and genome-registering of
the majority of the Lux-A Collection members were completed. Lux
fusions in the Lux-A Collection were fragments of E. coli genomic
DNA fused to promoterless luxCDABE gene. The genome-registered
collection of lux fusions were examined for the biological
responses measured by changes in bioluminescence. The present
invention provides a means to measure the changes in gene
expression profiles under various conditions.
[0014] In addition to being a stand-alone high throughput method,
the present invention also provides the way to validate or detect
false-positive result from other genome wide assays such as
microarray.
[0015] The present invention provides a method to confirm the
response of several promoters to a particular insult as well as to
identify a number of previously unknown operons responsive to that
insult.
[0016] The present invention provides a method for comparing the
gene expression patterns of two samples differing in one variable.
The variables may include but not limited to genotype, media,
temperature, depletion or addition of nutrient, addition of an
inhibitor, physical assault, biological assaults, irradiation,
heat, cold, elevated or lowered pressure, desiccation, low or high
ionic strength, and growth phases.
[0017] The present invention also provides the method to use an
"array of arrays" by generating gene expression profiles that yield
information relevant to understanding gene function and modes of
chemical action. Such information can be gained by analysis of
genetic alterations resulting in loss of function, reduced levels
of gene products, or over-expression of gene products. Thus, an
array of arrays can be used to enhance both modes of action studies
and functional genomics.
[0018] Thus the invention provides a method for identifying altered
gene expression between at least two genome-registered collections
comprising:
[0019] (a) assembling at least two genome-wide scale,
genome-registered collections;
[0020] (b) perturbing each collection from (a) with at least one
perturbation;
[0021] (c) measuring the response of each collection to each
perturbation of (b);
[0022] (d) analyzing the results of the at least one perturbation
to identify genetic differences between the at least two
genome-registered collections.
[0023] Additionally the invention provides a method for generating
a genome-registered collection of reporter gene fusions comprising
the steps of:
[0024] (a) generating a set of gene fusions comprising:
[0025] 1) a reporter gene or reporter gene complex operably linked
to
[0026] 2) a genomic fragment from an organism of which at least 15%
of the genomic nucleotide sequence is known;
[0027] (b) introducing in vitro the reporter gene fusions from step
(a) into a host organism;
[0028] (c) registering the reporter gene fusions on the basis of
sequence homology to the genomic sequence of the organism;
[0029] (d) repeating (a), (b), and/or (c) until reporter gene
fusions have been made to at least 15% of the known genomic
nucleotide sequence of said organism.
[0030] Similarly the invention provides a method for generating a
genome-registered collection of reporter gene fusions
comprising:
[0031] (a) generating random nucleic acid fragments from the DNA of
an organism of which at least 15% of the nucleotide sequence is
known;
[0032] (b) operably linking the random nucleic acid fragments
generated in (a) to a vector containing a promoterless reporter
gene or reporter gene complex;
[0033] (c) introducing the vector (b) containing the gene fusions
into a host organism;
[0034] (d) determining the nucleic acid sequence of the distal and
the proximal ends of the random nucleic fragments relative to the
reporter gene or reporter gene complex;
[0035] (e) registering the sequenced fusions of step (d) on the
basis of sequence homology to the genomic sequence of the host
organism;
[0036] (d) repeating (a), (b), and/or (c) until reporter gene
fusions have been made to at least 15% of the known genomic
nucleotide sequence of said organism. Generation of the random
nucleic acid fragments of step may incorporate restriction enzyme
digestion, physical shearing of the genome and polymerase chain
reaction.
[0037] In another embodiment the invention provides a method for
generating a genome-registered collection of reporter gene fusions
comprising steps of:
[0038] (a) introducing one or more transposons into the genome of
an organism of which at least 15% of the nucleotide sequence is
known, each transposon containing a promoterless reporter gene or
reporter gene complex;
[0039] (b) determining the nucleic acid sequence of the junction
between the proximal end of the genomic DNA and the transposon
containing the reporter gene or reporter gene complex and
registering the reporter gene fusions relative to the genomic
sequence of the organism,
[0040] (c) repeating (a) and (b) until reporter gene fusions have
been made to at least 15% of the known genomic nucleotide sequence
of said organism.
[0041] Alternatively the invention provides a method for
identifying a profile of inducing conditions for a reporter gene
fusion comprising:
[0042] (a) obtaining a gene expression profile of an organism under
induced and non-induced conditions wherein induced genes are
identified;
[0043] (b) providing a genome-registered collection of reporter
gene fusions, said fusions registered to the genome of the organism
of (a);
[0044] (c) selecting the reporter gene fusions of (b) that
correspond to the induced genes of (a) to create a subset of the
genome-register collection;
[0045] (d) contacting the subset of the genome-register collection
of (c) with the inducing conditions of (a) to identify at least one
representative reporter gene fusion whose expression was altered in
a similar manner as in (a);
[0046] (e) contacting the at least one representative reporter gene
fusion of (d) in a high throughput manner with a multiplicity of
different inducing conditions to identify a profile of inducing
conditions for that reporter gene fusion.
[0047] In another embodiment the invention provides a method for
generating a genome-registered collection of reporter gene fusions
comprising:
[0048] (a) providing a genome from an organism wherein at least 15%
of the nucleotide sequence is known;
[0049] (b) providing a series of amplification primers having
homology to specific known regions of the genome of (a);
[0050] (c) amplifying portions of the genome of (a) with the
primers of (b) to create a collection of nucleic acid amplification
products;
[0051] (d) operably linking the amplification products of (c) to a
vector containing a promoterless reporter gene or reporter gene
complex;
[0052] (e) introducing the reporter gene fusions into a said
organism;
[0053] (f) repeating (a)-(e) until, until reporter gene fusions
have been made to at least 15% of the known genomic nucleotide
sequence of said organism.
[0054] In another embodiment the invention provides a method for
identifying a profile of inducing conditions for a reporter gene
fusion comprising:
[0055] (a) obtaining a gene expression profile for each of mutant
strain and a parental strain organism under induced and non-induced
conditions wherein induced genes are identified;
[0056] (b) providing a genome-registered collection of reporter
gene fusions, said fusions registered to the genome of the organism
of (a);
[0057] (c) selecting the reporter gene fusions of (b) that
correspond to the induced genes of (a) to create a subset of the
genome-register collection;
[0058] (d) contacting the subset of the genome-register collection
of (c) with the inducing conditions of (a) to identify at least one
representative reporter gene fusion whose expression was altered in
a similar manner as in (a);
[0059] (e) contacting the at least one representative reporter gene
fusion of (d) in a high throughput manner with a multiplicity of
different inducing conditions to identify a profile of inducing
conditions for that reporter gene fusion.
[0060] Similarly it is an object of the invention to provide a
method to validate results from comprehensive genome analysis
comprising the steps of:
[0061] (a) analyzing a genome-wide, gene expression assay of an
organism treated with a condition or chemical of interest to
identify genes with altered expression;
[0062] (b) selecting from a genome-registered collection of
reporter gene fusions those reporter gene fusions containing
promoter regions operably linked to genes corresponding to the
altered genes from (a) or genes co-regulated with genes
corresponding to the altered genes from (a);
[0063] (c) testing expression of the reporter gene fusions selected
from (b) with the conditions or chemicals of interest used in (a);
and
[0064] (d) comparing the gene expression results from (c) to the
gene expression result of (a).
[0065] The invention additionally provides a method to determine
operon structure comprising steps of:
[0066] (a) selecting a subset of reporter gene fusions from a
genome-registered collection of reporter gene fusions that map to
the region of a possible operon;
[0067] (b) assaying the subset for the reporter gene function;
and
[0068] (c) determining a putative operon structure based on the
quantities of reporter gene function.
[0069] Alternatively the invention provides a method for
constructing a cellular array containing reporter gene fusions
comprising:
[0070] (a) generating a set of gene fusions comprising:
[0071] 1) a reporter gene or reporter gene complex operably linked
to
[0072] 2) a genomic fragment from an organism of which at least 15%
of the genomic nucleotide sequence is known;
[0073] (b) selecting a non-redundant subset of reporter gene
fusions from the set of (a) representative of at least 15% of known
or suspected promoter regions from a genome-registered collection
of reporter gene fusions, each containing a known or suspected
promoter region operably linked to a reporter gene or reporter gene
complex; and
[0074] (c) fixing the non-redundant subset of reporter gene fusions
of (b) in an array format.
[0075] In a preferred embodiment the invention provides a method
for measuring gene expression responses to perturbation
comprising:
[0076] (a) constructing at least 2 identical cellular arrays, each
cellular array comprising a reporter gene fusion comprising:
[0077] 1) a reporter gene or reporter gene complex operably linked
to
[0078] 2) a genomic fragment from an organism of which at least 15%
of the genomic nucleotide sequence is known;
[0079] wherein at least one cellular array is a control array and
at least one cellular array is an experimental array;
[0080] (b) contacting the experimental array of (a) with a
perturbing condition;
[0081] (c) comparing the differences between the gene expression
activity of the control and the experimental array wherein gene
expression response to a perturbing condition is determined.
[0082] Organisms amenable to the present method include prokaryotes
and fungi and particularly enteric bacterium.
[0083] Reporters useful in the present method include luxCDABE,
lacZ, gfp, cat, galK, inaZ, luc, luxAB, bgaB, nptII, phoA, uidA and
xylE.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 is a diagram of fusions to the lac operon from the
Lux-A Collection. The direction of the arrowhead indicates if the
DNA of the cloned chromosomal segment is oriented such that a
promoter, if present, would be driving expression of genes on the
direct (arrowhead to the right) or complementary (arrowhead to the
left ) strand.
[0085] FIG. 2 describes kinetics of induction of increased
bioluminescence from E. coli strain DPD3232 containing a
yebF-luxCDABE fusion treated with various concentrations of
MMC.
[0086] FIGS. 3(A-C) describes generally the Luxarray 0.5
luminescence during solid phase growth. FIG. 3A describes the cell
placement to replicate 96 well spacing to fit a microplate
luminometer. In the white light on the left, each replicate of the
twelve spots is outlined. The bioluminescent image of the same
plate is shown on the right. FIG. 3B describes the signal collected
for each clone as a function of time. Each cycle was about 20 min.
FIG. 3C describes signals collected from 8 replicates of same
clone.
[0087] FIGS. 4(A-D) describes generally the Luxarray 0.5
perturbation with nalidixic acid (NA). FIG. 4A represents results
from strains containing the parental plasmid and two non-responding
reporters, osmY, and lacZYA and FIG. 4B represents the results from
three DNA damage responsive reporters, uvrA, recA, dinG.
[0088] FIG. 5 describes High density Luxarray 0.5 perturbation with
nalidixic acid. The squares represent reporter gene response from
nalidixic acid treated cultures and the circles represent reporter
gene activity from untreated cultures.
[0089] FIG. 6 describes graphical representation of selection
criteria implemented to select Luxarray 1.0 Clones.
[0090] FIG. 7 describes bioluminescence captured from Luxarray 1.0
reporters after 14 hr growth on LB media by cooled CCD array camera
(FluorChem 8000, AlphaInnotech).
[0091] FIG. 8 describes the predicted promoters in a gene cluster
of 20 genes for typeI extracellular polysaccharide in E. coli and
the promoter activity of luxCDABE gene fusions from the region
encoding production of type I extracellular polysaccharide. The E.
coli genes in this region are shown with the top set of arrows.
Above this line are the predicted promoters regions from two
sources (P, Blattner et al., (1997) Science 277:1453-1462); P,
Thieffry et al. (1998) Bioinformatics 14:391-400). The two
promoters supported by DNA array and gene fusion data presented
here are shown in bold type. The b designation and common name for
each gene is shown. The ratios of the deduced mRNA level in the
rpoC mutant strain to the deduced mRNA level in the rpoC.sup.+
strain determined by the micro array method are shown on the next
line. The mapped location of the chromosomal inserts of selected
luxCDABE gene fusions in this region are shown with the second set
of thicker arrows. Below these are the lux clone identification
number and bioluminescence data (RLU/10.sup.9 cfu) for each plasmid
in the rpoC.sup.+ E. coli host strain and the host strain carrying
an rpoC mutation. In the case of overlapping gene fusions, the
shorter one is listed first.
[0092] FIGS. 9(A-D) represents generally the induction of the
promoter b1728 by nalidixic acid (NA) in lexA+ host (FIG. 9A) in
comparison to the promoter in lexind host (FIG. 9C), and induction
of the promoter b1728 by mitomicyn C (MC) in lexA+ host (FIG. 9B)
in comparison to the promoter in lexind host (FIG. 9D).
[0093] FIG. 10 represents uhpT-lux upregulation by limonene.
[0094] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions which form a part of this application.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The present invention relates to the generation and use of a
cellular array or a cellular array in combination with other
genome-registered arrays (an array of arrays) for the determination
of gene function and/or perturbation mode of action. Each cellular
array consists of a number of microbial strains. Each strain
comprises one reporter gene fusion made up of a gene or gene
fragment operably linked to a reporter gene. Each gene or gene
fragment has been "registered" or mapped to a specific location in
the genome of the organism. Cellular arrays of the present
invention are those that contain reporter gene fusions to at least
15% of the genome of the organism being analyzed. Cellular arrays
containing these reporter gene fusions are referred to herein as
"registered collections"
[0096] The genome-registered collection of the invention may be
used to determine alterations in gene expression under a variety of
conditions. Such collections are amenable to rapid assay and may be
used to confirm, correct or augment data generated from DNA micro
array technology.
[0097] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0098] "Open reading frame" is abbreviated ORF. The term "ORF" is
refers to a gene that specifies a protein.
[0099] "Polymerase chain reaction" is abbreviated PCR.
[0100] As used herein, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0101] The term "primer" refers to an oligonucleotide (synthetic or
occurring naturally), which is capable of acting as a point of
initiation of nucleic acid synthesis or replication along a
complementary strand when placed under conditions in which
synthesis of a complementary stand is catalyzed by a polymerase.
Wherein the primer contains a sequence complementary to a region in
one strand of a target nucleic acid sequence and primes the
synthesis of a complementary strand, and a second primer contains a
sequence complementary to a region in a second strand of the target
nucleic acid and primes the synthesis of complementary strand;
wherein each primer is selected to hybridize to its complementary
sequence, 5' to any detection probe that will anneal to the same
strand.
[0102] A "cellular array" means a set of strains differing from one
another only in the chromosomal fragment fused to a reporter gene
or reporter gene complex.
[0103] The term "array of arrays" refers to a collection of
cellular arrays wherein each individual cellular array contains
reporter gene fusions responsive to a specific inducing condition
or set of conditions.
[0104] The terms "global scale" or "genome-wide scale" when applied
to reporter gene fusions refer to a minimum 15% representation of
the transcription units of an organism. For example, there is
predicted to be 2328 transcription units (t.u.s) in E. Coli
(RegulonDB Database v.3.0,
http://www.cifn.unam.mx/Computational_Biology/E.coli-predictions/).
Thus a set of unique fusions representing 329 t.u.s (15% of 2328)
is at "global scale". The term "global scale" or "genome-wide
scale" when applied to mutations or overexpression refers to a
minimum 15% representation of the open reading frames of an
organism.
[0105] The term "genome register" refers to a procedure of
precisely locating or mapping a defined nucleic acid sequence
within a genome.
[0106] The term "genome-registered collection" refers to a set of
strains containing reporter gene fusions, loss of gene function
mutations, altered gene function mutations, or overexpressed genes
that have been "registered" or mapped by homology to the nucleic
acid sequence of the genome of the organism. These
genome-registered collections include reporter gene fusions to at
least 15%, more preferably at least 20%, and most preferably at
least 50% of all known or predicted promoter regions, loss of gene
function mutations in at least 15%, more preferably at least 20%,
and most preferably at least 50%, of all known or predicted ORFs,
altered gene function mutations in at least 15%, more preferably at
least 20%, and most preferably at least 50%, of all known or
predicted ORFs, or overexpression of in at least 15%, more
preferably at least 20%, and most preferably at least 50%, of all
known or predicted ORFs.
[0107] As used herein the term "known" as applied to a gene or ORF
within the context of a genome means that the sequence of the gene
or ORF is known and should not be limited to knowledge of the
function of that gene or ORF.
[0108] The term "reporter gene fusion" refers to a chimeric gene
consisting, in one part, of a gene or genes that are useful to
detect transcription and/or translation initiated in the other
part.
[0109] The term "reporter gene complex" refers to any set of two or
more genes that together are useful to generate a measurable
signal.
[0110] The term "reporter gene activity" or "reporter construct
activity" refers to the accumulation of the limiting component(s)
of the reporter system in use which results in the measurable
signal associated with that reporter system. The measured activity
is the sum result of the de novo production, ongoing degradation or
deactivation, and/or activation of the limiting component(s).
[0111] The term "Lux-A Collection" refers to a specific set of E.
coli strains containing plasmid-borne luxCDABE fusions.
[0112] The term "lux fusion" or "luxCDABE fusion" refers to a
chimeric gene consisting, of a genomic sequence joined to luxCDABE
genes.
[0113] The term "congenic strains" refer to two or more strains
that differ from one another by a single mutation or a strain
differing in only one gene to denote genes whose expression differ
as a function of the allele.
[0114] The term "DNA microarray" or "DNA chip" means assembling PCR
products of a group of genes or all genes within a genome on a
solid surface in a high density format or array. General methods
for array construction and use are available (see Schena M, Shalon
D, Davis R W, Brown P O., Quantitative monitoring of gene
expression patterns with a complementary DNA microarray. Science.
Oct. 20, 1995; 270(5235): 467-70. and
http://cmgm.stanford.edu/pbrown/mguide/index.html). A DNA
microarray allows the analysis of gene expression patterns or
profile of many genes to be performed simultaneously by hybridizing
the DNA microarray comprising these genes or PCR products of these
genes with cDNA probes prepared from the sample to be analyzed. DNA
microarray or "chip" technology permits examination of gene
expression on a genomic scale, allowing transcription levels of
many genes to be measured simultaneously. Briefly, DNA microarray
or chip technology comprises arraying microscopic amounts of DNA
complementary to genes of interest or open reading frames on a
solid surface at defined positions. This solid surface is generally
a glass slide, or a membrane (such as nylon membrane). The DNA
sequences may be arrayed by spotting or by photolithography (see
http://www.affymetrix.com/). Two separate fluorescently-labeled
probe mixes prepared from the two sample(s) to be compared are
hybridized to the microarray and the presence and amount of the
bound probes are detected by fluorescence following laser
excitation using a scanning confocal microscope and quantitated
using a laser scanner and appropriate array analysis software
packages. Cy3 (green) and Cy5 (red) fluorescent labels are
routinely used in the art, however, other similar fluorescent
labels may also be employed. To obtain and quantitate a gene
expression profile or pattern between the two compared samples, the
ratio between the signals in the two channels (red:green) is
calculated with the relative intensity of Cy5/Cy3 probes taken as a
reliable measure of the relative abundance of specific mRNAs in
each sample. Materials for the construction of DNA microarrays are
commercially available (Affymetrix (Santa Clara Calif.) Sigma
Chemical Company (St. Louis, Mo.) Genosys (The Woodlands, Tex.)
Clontech (Palo Alto Calif.) and Corning (Corning N.Y.). In
addition, custom DNA microarrays can be prepared by commercial
vendors such as Affymetrix, Clontech, and Corning.
[0115] The basis of gene expression profiling via micro-array
technology relies on comparing an organism under a variety of
conditions that result in alteration of the genes expressed. A
single population of cells may be exposed to a variety of stresses
that will result in the alteration of gene expression.
Alternatively, the cellular environment may be kept constant and
the genotype may be altered. Typical stresses that result in an
alteration in gene expression profile will include, but is not
limited to conditions altering the growth of a cell or strain,
exposure to mutagens , antibiotics, UV light, gamma-rays, x-rays,
phage, macrophages, organic chemicals, inorganic chemicals,
environmental pollutants, heavy metals, changes in temperature,
changes in pH, conditions producing oxidative damage, DNA damage,
anaerobiosis, depletion or addition of nutrients, addition of a
growth inhibitor, and desiccation. Non-stressed cells are used for
generation of "control" arrays and stressed cells are used to
generate an "experimental", "stressed" or "induced" arrays. Induced
arrays are those that demonstrate "altered gene expression".
[0116] "Altered gene expression" refers to the change in the level
of a transcription or translation products. If the gene is
"up-regulated", the level of transcription or translation products
is elevated. If the gene is "down-regulated" the level of
transcription or translation products is decreased. Conditions
under which gene expression is altered is also known as an
"inducing condition". In some instance a number of different
conditions may result in the same transcriptional effect on a
single gene. Thus a number of inducing conditions will either
up-regulate or down regulate the same gene. This collection of like
inducing conditions is known as a "profile of inducing conditions".
Similarly the term "perturbation" as used herein in reference to a
cellular array or a DNA micro array is any alteration of
environment or genotype the results in altered gene expression.
[0117] The terms "high density" or "comprehensive" micro array
refers to a high-density DNA micro-array containing at least 75% of
the open reading frames of the organism.
[0118] The term "expression profile" refers to the expression of
groups of genes.
[0119] The term "gene expression profile" refers to the expression
of individual gene and suite of individual genes.
[0120] The "comprehensive expression profile" refers to the gene
expression profile of more than 75% of genes in the genome. In
"comprehensive gene expression analysis" at least >75% of gene
expression of the organism is analyzed.
[0121] The term "corresponding to" is used herein to refer to
similar or homologous sequences, whether the exact position is
identical or different from the molecule to which the similarity or
homology is measured. A nucleic acid or amino acid sequence
alignment may include spaces. Thus, the term "corresponding to"
refers to the sequence similarity, and not the numbering of the
amino acid residues or nucleotide bases.
[0122] The terms "print" or "printing" refer to transferring one or
more cultures from one locale, most often a multiwell culture
plate, to a substrate or solid surface by physical contact transfer
or any of a plurality of technologies.
[0123] The term "regulon" refers to groups of operons sharing
similar regulation.
[0124] The term "operon" refers to a unit of bacterial gene
expression and regulation, including structural genes and control
elements in DNA recognized by regulator gene product(s) that in
combination support the production of an mRNA.
[0125] The term "probe" refers to a single-stranded nucleic acid
molecule that can base pair with a complementary single stranded
target nucleic acid to form a double-stranded molecule.
[0126] The term "genotype" refers to the genetic constitution of an
organism as distinguished from its physical appearance.
[0127] The term "genomic DNA" refers to a single complete set of
genetic information carried in the chromosomes of an organism.
[0128] The term "total RNA" refers to non-fractionated RNA from an
organism.
[0129] The terms "protein specifying RNA" or "protein specifying
transcript" refer to RNA derived from an ORF.
[0130] The terms "transposon" and "transposable element" are used
interchangeably and mean a region of nucleic acid that is capable
of moving from one position to another where this movement is
catalyzed by the element itself.
[0131] The term "transposition" means a biochemical reaction that
catalyzes the movement of a transposable element from one site into
different site within a DNA molecule. Transposition can be carried
out in vivo or in vitro.
[0132] The term "in vitro transposition" means a biochemical
reaction initiated outside the cell that catalyzes the movement of
a transposable element from one site into different site within a
DNA molecule.
[0133] The term "in vivo transposition" means a biochemical
reaction that takes place within the cell that catalyzes the
mobilization of a transposon from of site to another within the
genome of the host.
[0134] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor (1989), particularly Chapter 11 and Table 12.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. Hybridization requires that the two nucleic acids
contain complementary sequences, although depending on the
stringency of the hybridization, mismatches between bases are
possible. The appropriate stringency for hybridizing nucleic acids
depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of Tm for hybrids of nucleic acids having
those sequences. The relative stability (corresponding to higher
Tm) of nucleic acid hybridizations decreases in the following
order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). Furthermore, the skilled artisan will recognize
that the temperature and wash solution salt concentration may be
adjusted as necessary according to factors such as length of the
probe.
[0135] The term "complementary" is used to describe the
relationship between nucleotide bases that are capable to
hybridizing to one another. For example, with respect to DNA,
adenosine is complementary to thymine and cytosine is complementary
to guanine. Accordingly, the instant invention also includes
isolated nucleic acid fragments that are complementary to the
complete sequences as reported in the accompanying Sequence Listing
as well as those substantially similar nucleic acid sequences.
[0136] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence, unless mentioned otherwise. For example, "reporter
genes" used in gene fusion does not include regulatory (promoter)
sequences unless specified otherwise. "Native gene" refers to a
gene as found in nature with its own regulatory sequences.
[0137] The terms "chimeric gene", "gene fusion", or "fusion" refer
to any non-native gene or genes comprising two or more genomic or
artificial DNA fragments that are not found in nature. Accordingly,
a chimeric gene, gene fusion or fusion may comprise regulatory
sequences and coding sequences the are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source but arranged in a manner that different than that
is found in nature. "Endogenous gene" refers to a native gene in
its natural location in the genome of an organism. A "foreign" gene
refers to a gene not normally found in the host organism, but that
is introduced into the host organism by gene transfer. Foreign
genes can comprise native genes inserted into a non-native
organism, or chimeric genes. A "transgene" is a gene that has been
introduced into the genome by a transformation procedure.
[0138] The term "coding sequence" refers to a DNA sequence that
codes for a specific amino acid sequence. "Suitable regulatory
sequences" refer to nucleotide sequences located upstream (5'
non-coding sequences), within, or downstream (3' non-coding
sequences) of a coding sequence, and which influence the
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences may include
promoters, translation leader sequences, introns, and
polyadenylation recognition sequences.
[0139] The term "promoter" refers to a DNA sequence to which RNA
polymerase can bind to initiate the transcription. In general, a
coding sequence is located 3' to a promoter sequence. Promoters may
be derived in their entirety from a native gene, or be composed of
different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental conditions. Promoters which cause a gene to be
expressed in most cell types at most times are commonly referred to
as "constitutive promoters". It is further recognized that since in
most cases the exact boundaries of regulatory sequences have not
been completely defined, DNA fragments of different lengths may
have identical promoter activity. "Promoter region" is promoter and
adjacent areas whose function may be modulate promoter
activity.
[0140] The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor.
[0141] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA (mRNA)" refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene (U.S. Pat. No. 5,107,065). The complementarity of an
antisense RNA may be with any part of the specific gene transcript,
i.e., at the 5' non-coding sequence, 3' non-coding sequence,
introns, or the coding sequence. "Functional RNA" refers to
antisense RNA, ribozyme RNA, or other RNA that is not translated
yet has an effect on cellular processes.
[0142] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0143] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0144] The term "transformation" refers to the acquisition of new
genes in a cell after the incorporation of nucleic acid.
[0145] The terms "plasmid", "vector", and "cassette" refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA molecules. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitate
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0146] The term "restriction endonuclease" or "restriction enzyme"
refers to an enzyme which binds and cuts within a specific
nucleotide sequence within double stranded DNA.
[0147] The term "bioluminescence" refers to the phenomenon of light
emission from any living organism.
[0148] The term "relative light unit" is abbreviated "RLU" and
refers to a measure of light emission as measured by a luminometer,
calibrated against an internal standard unique to the luminometer
being used.
[0149] The term "stress" or "environmental stress" refers to the
condition produced in a cell as the result of exposure to an
environmental insult.
[0150] The terms "insult" or "environmental insult" refers to any
substance or environmental change that results in an alteration of
normal cellular metabolism in a bacterial cell or population of
cells. Environmental insults may include, but are not limited to,
chemicals, environmental pollutants, heavy metals, changes in
temperature, changes in pH as well as agents producing oxidative
damage, DNA damage, anaerobiosis, changes in nitrate availability
or pathogenesis.
[0151] The term "stress response" refers to the cellular response
resulting in the induction of either detectable levels of stress
proteins or in a state more tolerant to exposure to another insult
or an increased dose of the environmental insult.
[0152] The term "stress gene" refers to any gene whose
transcription is induced as a result of environmental stress or by
the presence of an environmental insults.
[0153] The terms "log phase", "log phase growth", "exponential
phase", or "exponential phase growth" refer to cell cultures of
organisms growing under conditions permitting the exponential
multiplication of the cell number.
[0154] Standard recombinant DNA and molecular cloning techniques
used here are well known in the art and are described by Sambrook,
J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989); and by Silhavy, T. J.,
Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions,
Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.
(1984); and by Ausubel, F. M. et al., Current Protocols in
Molecular Biology, published by Greene Publishing Assoc. and
Wiley-Interscience (1987).
[0155] In this invention, a random set of fragments of E. coli
genome was generated by partially digesting E. coli genome with the
restriction enzyme Sau3A1, and separating the fragments by size
fractionation. The fraction with an average size of 1.8 KB was
isolated and ligated to the plasmid pDEW201 which contains the
origin of replication and bla from pBR322, four transcription
terminators upstream of the promoterless luxCDABE gene. The
ligation products were transformed into E. coli XL2Blue cells.
Approximately 24000 resulting transformants were pooled and used AS
the source of heterologous plasmid DNA. This plasmid DNA pool was
used to transform E. coli DPD1675 cells. A total of 8066 individual
transformants were isolated and labeled as Lux-A Collection. A
homology search for the sequence from beginning and end of clones
from Lux-A Collection was performed against complete E. coli
sequence. The location and orientation of each chimeric gene fusion
with respect to the E. coli genome was determined based on the
computed homology. Additional sequences for the collection can be
added by more random fusions or inverting the orientation of DNA in
the fusion to put the promoter regions in the right orientation for
the inserted DNA by using the method that is routine in the
art.
[0156] Preferred organisms for use in the present invention are
those whose genomes are being sequenced or that have completely
sequenced genomes and are amenable to introduction of gene fusions
by transduction, transformation, or conjugation. They include but
are not limited to the 113 organisms listed on the WEB page,
http://www.ncbi.nlm.nih.gov/PMGifs/- Genomes/bact.html as of Feb.
15, 2000. In addition to the organisms mentioned above, any
microbial organism with at least 15%, preferably 20%, and most
preferably 50% of its genome sequence known that is amenable to
introduction of gene fusions by transduction, transformation, or
conjugation can be used in this invention to generate reporter gene
fusions.
[0157] Within the context of the present invention prokaryotes and
fungi having at least 15% of their genome sequence are particularly
suitable. Of the prokaryotes, enteric bacteria such as Escherichia
and Salmonella are preferred and E. coli is most preferred as it is
well characterized and its genome has been sequenced. it is most
preferred prokaryotic organism for this invention.
[0158] It will be appreciated that for the purposes of the present
invention that the teachings with respect to E. coli are
particularly adaptable to any of the enteric bacteria. Enteric
bacteria are members of the family Enterobacteriaceae, and include
such members as Escherichia, Salmonella, and Shigella. They are
gram-negative straight rods, 0.3-1.0.times.1.0-6.0 .mu.m, motile by
peritrichous flagella, except for Tatumella, or nonmotile. They
grow in the presence and absence of oxygen and grow well on
peptone, meat extract, and (usually) MacConkey's media. Some grow
on D-glucose as the sole source of carbon, whereas others require
vitamins and/or mineral(s). They are chemoorganotrophic with
respiratory and fermentative metabolism but are not halophilic.
Acid and often visible gas is produced during fermentation of
D-glucose, other carbohydrates, and polyhydroxyl alcohols. They are
oxidase negative and, with the exception of Shigella dysenteriae 0
group 1 and Xenorhabdus nematophilus, catalase positive. Nitrate is
reduced to nitrite except by some strains of Erwinia and Yersina.
The G+C content of DNA is 38-60 mol % (T.sub.m, Bd). DNAs from
species from species within most genera are at least 20% related to
one another and to Escherichia coli, the type species of the
family. Notable exceptions are species of Yersina, Proteus,
Providenica, Hafnia and Edwardsiella, whose DNAs are 10-20% related
to those of species from other genera. Except for Erwinia
chrysanthemi all species tested contain the enterobacterial common
antigen (Bergy's Manual of Systematic Bacteriology, D. H. Bergy, et
al., Baltimore: Williams and Wilkins, 1984).
[0159] As for the reporter gene or gene complex, luxCDABE is most
preferred for its sensitivity and simplicity of assay conditions.
Other reporter genes well known in the art also can be used in this
invention. The preferred reporter genes include but are not limited
to lacZ, gfp, cat, galK, inaZ, luc, luxAB, bgaB, nptII, phoA, uidA,
and xylE.
[0160] Methods to introduce fusions into such strains are well
known in the art. Fusions can be introduced by transduction,
transformation or conjugation as appropriate for each specific
organism. They can be constructed in vitro by techniques including
but not limited to transposon (e.g., Tn7) mediated transposition,
ligating of random fragments to a reporter gene construct
containing a vector, or ligating of PCR products to the same
vector. They can be constructed by in vivo transposition where the
transposon is introduced into the cell by transduction,
transformation, or conjugation. Kits for in vitro transposition are
commercially available (see for example The Primer Island
Transposition Kit, available from Perkin Elmer Applied Biosystems,
Branchburg, N.J., based upon the yeast Ty1 element (including the
AT2 transposon); The Genome Priming System, available from New
England Biolabs, Beverly, Mass.; based upon the bacterial
transposon Tn7; and the EZ::TN Transposon Insertion Systems,
available from Epicentre Technologies, Madison, Wis., based upon
the Tn5 bacterial transposable element.
[0161] The preferred methods to generate a set of fragments of the
genome include but are not limited to, the partial digestion with a
restriction enzyme, physical shearing, polymerase chain reaction
(PCR) amplification, the combination of restriction enzyme
digestion and PCR, the combination of restriction enzyme digestion
and physical shearing, the combination of physical shearing and
PCR, and the combination of all three. Above methods for generating
fragments are well known in the art. For example PCR is well
described in (U.S. Pat. No. 4,683,202 (1987, Mullis, et al.) and
U.S. Pat. No. 4,683,195 (1986, Mullis, et al.), and restriction
enzyme digestion and physical shearing is well described in
Sambrook et al., supra.
[0162] When using PCR to generate fragments several approaches may
be taken. In one instance random primers may be used to amplify
portions of the genome. In this situation the fragments,
(amplification products) generated will be random in nature. In the
alternative, where portions of the sequence of the genome are known
primers may be designed to specific loci within the genome. This is
a directed fragment generation approach.
[0163] Furthermore, chimeric reporter gene fusions can be also made
by using transposition at the genome level. The above-described
methods for generating transpositions are well known in the
art.
[0164] After the sequencing and genome-registering the majority of
the Lux-A Collection members were completed the biological
responses of the strain collection containing luxCDABE fusions to
members of well-characterized regulatory circuits were
examined.
[0165] Thus, gene fusions to the lac operon, and members of the
heat shock, SOS, SoxRS, and OxyR regulons were selected from the
Lux-A Collection and the responses to known inducers of each of
these global regulatory circuits were determined.
[0166] Appropriate biological responses were demonstrated for each
of regulons mentioned above. The Lux-A Collection contained 3
members that are fusions of luxCDABE to the lac operon (FIG. 1).
These cultures were grown in the presence of glucose and
bioluminescence was measured and compared to the cultures grown in
the absence of glucose. Cultures grown in the absence of glucose
were much more highly bioluminescent than the ones in the presence
of glucose.
[0167] Four out of twelve heat shock promoters were found as lux
fusions. A total of 6 fusion containing strains were isolated and
tested for the stress response in the presence of various
concentration of ethanol. The heat shock regulon gene fusions from
the Lux-A Collection confirmed induction of heat shock response by
ethanol.
[0168] For SOS regulon, 7 lux fusion were found in the Lux-A
Collection. When these stains were tested with DNA damaging
chemical nalidixic acid, increased level of bioluminescence was
observed. However, treatment with ethanol did not induce
bioluminescence in the same culture.
[0169] In a similar fashion, although SoxR/S and OxyR regulons were
not fully represented in the Lux-A Collection, the biologically
appropriate response was observed.
[0170] Thus, while the Lux-A Collection is not comprehensive in
containing a fusion controlled by each promoter in E. coli, it
nonetheless provides a genomic-wide overview of transcriptional
responses to imposed stresses and can be adapted to optimize
responses.
[0171] The effect of mitomycin C (MMC), a known DNA damaging agent,
was tested with DNA microarray and the resulting gene expression
pattern was compared with the gene expression data from the treated
cells of the Lux-A Collection. As expected, the expression of the
known SOS genes was elevated in micro array data. However the
expression of several SOS regulon genes was elevated less than
2-fold (Table 12) and as such were within a large group of 792
genes the expression of which was elevated by 20% or more. Because
most of these are likely due to artifacts in the array data rather
than to actual biologically relevant responses, the group of genes
with less than two fold increase in expression were considered MMC
non-inducible genes. Had these experiments been conducted using a
compound with an otherwise unknown mode of action, some
biologically relevant gene expression events would have been missed
with the DNA micro array approach. To test if strains carrying
luxCDABE gene fusions would yield the expected positive result, the
three gene fusions that were available in the Lux-A Collection of
reporter gene fusions were tested for mitomycin C responses. In all
three cases, mitomycin C induced increased bioluminescence. Thus,
this demonstrates that negative results from DNA microarrays can be
questioned by contradictory positive results with corresponding
gene fusions.
[0172] The genes not previously known to be upregulated with
mitomycin C, provide an opportunity to further examine the
correlation of DNA array and gene fusion experimental data. For
this class of genes, four fusions were available in the Lux-A
Collection of reporter gene fusions. The corresponding luxCDABE
fusions to these four genes provided no evidence of increased gene
expression induced by MMC. Thus, the positive results from the
array were classified as false-positive.
[0173] One gene fusion in the Lux-A Collection of gene fusions was
found to have a genomic fragment that when inverted would result in
a fusion to yebF, a gene observed to be upregulated by mitomycin C
in DNA micro array experiments. When the genomic DNA fragment was
released and inserted back into the vector, bioluminescence from
some of the transformed colonies were found to be highly inducible
by another DNA damaging agent, nalidixic acid, strongly suggesting
that the inversion of DNA might have occurred. Furthermore,
induction of bioluminescence by MMC was demonstrated as shown in
FIG. 2.
[0174] The induction of increased gene expression from the yebF-lux
fusion not only validates the data from the DNA array study, but
furthermore, allows facile dose response and kinetic analyses and
provides a biosensor strain for a high throughput screen. It is
possible that the DNA damage response reported by yebF-lux fusion
might be mediated by the well-characterized SOS response because
there is a LexA box upstream of yebG (Lomba et al., (1997) FEMS
Microbiol Lett 156:119-122), which is just upstream of yebF.
However, there is also suggested to be a promoter that drives
transcription of the yebF gene (Blattner et al., (1997) Science
277:1453-1462), therefore the induction of yebF expression by MMC
was unexpected. Thus, these results demonstrate the concept that a
previously unknown gene expression event can be discovered with a
DNA microarray, then a corresponding lux gene fusion can be used to
validate and extend the results. Furthermore, such a lux fusion
strain can form the basis of a high throughput screen based on gene
expression changes.
[0175] The yebF-luxCDABE gene fusion mentioned in the Example 3 can
be used as a high throughput screen for compounds other than MMC
and nalidixic acid that result in DNA damage. The use of the
luxCDABE bioluminescent reporter fusion allows facile detection of
reporter gene activity in a manner that does not require cell lysis
or addition of enzymatic substrates. Thus development of a high
throughput screen only requires an instrument to quantitate light
production (of which there are many available commercially) and a
source of bacterial cell cultures containing the gene fusion of
choice. Such bacterial cell cultures can be supplied by one of
several methods. A basic way to use bacterial strains containing
gene fusions is simply with freshly grown bacterial cell cultures.
An alternative to daily cultivation is the use of frozen culture
aliquots, such as has been successfully demonstrated with an E.
coli bioluminescent sensor that is competent for gene expression
assays immediately after thawing, having been previously stored at
-80.degree. C. using a cryoprotectant such as glycerol (Sticher et
al., (1997) Appl. Environ. Microbiol. 63:4053-4060). Two other
common methods of handling bacteria, lyophilization and continuous
culture, have also proven to be useful sources of lux fusion
strains for testing purposes. Lyophilization has been successfully
applied to metal responsive and heat shock responsive cellular
biosensor strains (Corbisier et al., (1996) Environ. Toxicol. Water
Qual. 11:171-177; Tauriainen et al., (1998) Biosensors &
Bioelectronics 13:931-938; Tauriainen et al., (1997) Appl. Environ.
Microbiol. 63:4456-4461; Van Dyk and Wagner (1998) U.S. Pat. No.
5,731,163; Wagner and Van Dyk (1998), Methods in Molecular Biology:
Bioluminescence Methods and Protocols, vol. 102. Humana Press Inc
p.: 123-127). Continuous cultivation of E. coli bioluminescent
biosensors in mini bioreactors has been shown to yield reproducible
detection of stress responses (Gu et al., (1996), Biotechnol. Prog.
12:393-397; Gu et al., (1999) Biosens. Bioelectron.
14:355-361).
[0176] Other possible methods to supply cellular biosensors for
high throughput screens are immobilization by entrapment in a
carrier material or use of a bioluminescent bioreporter integrated
circuit (BBIC). Strontium alginate immobilization of a lux fusion
containing bacterial strain has been demonstrated for use as a
probe for waste streams (Heitzer et al., (1994) Appl. Environ.
Microbiol. 60:1487-1494; Matrubutham et al., (1997) Appl.
Microbiol. Biotechnol. 47:604-609; Webb et al, (1997) Biotechnol.
Bioeng. 54:491-502). In another example of immobilization, calcium
alginate beads, harboring an SOS responsive lux fusion strain,
stored in a CaCl.sub.2 solution at 4.degree. C. were found to give
useful DNA damage induction responses for up to one month after
formation (Davidov et al., in press). Likewise, calcium alginate
immobilization of a copper-responsive biosensor results in superior
stability of the biosensor relative to the immobilization of the
same biosensor in agarose (de Lorenzo et al., (1999) Anal. Chim.
Acta 387:235-244). Furthermore, combining of immobilized cells and
light detection equipment is possible for sensors that produce
visible light as a signal. In one such case, an E. coli luc fusion
strain is immobilized on the end of fiber optic monitoring device
(Ikariyama et al., (1997) Anal. Chem. 69:2600-2605). Finally, in
the BBIC approach, which takes advantage of the cellular signal
generated by the five gene luxCDABE reporter, a bioluminescent
biosensor strain is deposited onto a micro-luminometer fabricated
within an integrated circuit; the light produced by the biosensor
is detected by the integrated circuit, which then processes and
communicates the results (Simpson et al., (1998) Soc. Opt. Eng.
3328 (Smart Electronics and MEMS):202-212; Simpson et al., (1998)
TIBTECH 16:332-338).
[0177] Therefore, development of a DNA damage responsive high
throughput screen based on the newly discovered yebF-luxCDABE
fusion is readily accomplished by choosing one of the above known
methods to provide an active cellular biosensor and combining it,
if necessary, with an instrument that measures visible light
production. Other promoters from a stress response gene may be used
to generate high throughput screens using a lux fusion. Stress
response gene promoters from both prokaryotic and eukaryotic cells
may be used, however promoters from bacteria are preferred and
promoters from E. coli are most preferred. Suitable stress response
gene promoters may be selected from but are not limited to the list
of genes under the heading "responding genes" given in Table 1
below, and other newly discovered regulatory circuits.
1TABLE 1 REGULATORY REGULATORY RESPONDING STIMULUS GENE(S) CIRCUIT
GENES* Protein rpoH Heat Shock grpE, dnaK, Damage.sup.a lon, rpoD,
groESL, lysU, htpE, htpG, htpI, htpK, clpP, clpB, htpN, htpO, htpX,
etc. DNA Damage.sup.b lexA, recA SOS recA, uvrA, lexA, umuDC, uvrA,
uvrB, uvrC, sulA, recN, uvrD, ruv, dinA, dinB, dinD, dinF etc.
Oxidative oxyR Hydrogen katG, ahp, Damage.sup.c Peroxide etc.
Oxidative soxRS Superoxide micF, sodA, Damage.sup.d nfo, zwf, soi,
etc. Membrane fadR Fatty Acid fabA Damage.sup.e Starvation
Any.sup.f ? Universal uspA Stress Stationary rpoS Resting State
xthA, katE, Phase.sup.g appA, mcc, bolA, osmB, treA, otsAB, cyxAB,
glgS, dps, csg, etc. Amino Acid relA, spoT Stringent his, ilvBN,
Starvation.sup.h ilvGMEDA, thrABC, etc. Carbon cya, crp Catabolite
lac, mal, gal, Starvation.sup.i Activation ara, tna, dsd, hut, etc.
Phosphate phoB, phoM, P Utilization phoA, phoBR, Starvation.sup.j
phoR, phoU phoE, phoS, aphA, himA, pepN, ugpAB, psiD, psiE, psiF,
psiK, psiG, psiI, pstJ, psiN, psiR, psiH, phiL, phiO, etc. Nitrogen
glnB, glnD, N Utilization glnA, hut, Starvation.sup.k glnG, glnL
etc. *Genes whose expression is increased by the corresponding
stimulus and whose expression is controlled by the corresponding
regulatory gene(s). .sup.aNeidhardt and van Bogelen in E. coli and
Salmonella typhimurium; Cellular and Molecular Biology (Neidhardt,
F. C., et al. Eds., pp. 1334-1345, American Society of
Microbiology, Washington, DC (1987)) .sup.bWalker in E. coli and
Salmonella typhimurium; Cellular and Molecular Biology (Neidhardt,
F. C., et al. Eds., pp. 1346-1357, American Society of
Microbiology, Washington, DC (1987)) .sup.cChristman et al. Cell
41: 753-762 (1985); Storz et al. Science 248: 189-194 (1990);
Demple, Ann. Rev. Genet. 25: 315-337 (1991) .sup.dDemple, Ann. Rev.
Genet. 25: 31 337 (1991) .sup.eMagnuson et al. Microbiol. Rev 57:
522-542 (1993) .sup.fNystrom and Neidhardt, J. Bacteriol, 175:
2949-2956 (1993); Nystrom and Neidhardt (Mol. Microbiol. 6:
3187-3198 (1992) .sup.gKolter et al. Ann. Rev. Microbiol. 47:
855-874 (1993) .sup.hCashel and Rudd in E. coli and Salmonella
typhimurium; Cellular and Molecular Biology (Neidhardt, F. C., et
al. Eds., pp. 1410-1438, American Society of Microbiology,
Washington, DC (1987)); Winkler in E. coli and Salmonella
typhimurium; Cellular and Molecular Biology (Neidhardt, F. C., et
al. Eds., pp. 395-411, American Society of Microbiology,
Washington, DC (1987)) .sup.iNeidhardt, Ingraham and Schaecter.
Physiology of the Bacterial Cell: A Molecular Approach, Sinauer
Associates, Sunderland, MA (1990), pp 351-388; Magasanik and
Neidhardt in E. coli and Salmonella typhimurium; Cellular and
Molecular Biology (Neidhardt, F. C., et al. Eds., pp. 1318-1325,
American Society of Microbiology, Washington, DC (1987))
.sup.jWanner in E. coli and Salmonella typhimurium; Cellular and
Molecular Biology (Neidhardt, F. C., et al. Eds., E. coli and
Salmonella typhimurium; Cellular and Molecular Biology (Neidhardt,
F. C., et al. Eds., pp. 1326-1333, American Society of
Microbiology, Washington, DC (1987)) .sup.kRietzer and Magasanik in
E. coli and Salmonella typhimurium; Cellular and Molecular Biology
(Neidhardt, F. C., et al. Eds., pp. 1302-1320, American Society of
Microbiology, Washington, DC (1987)); Neidhardt, Ingraham and
Schaecter. Physiology of the Bacterial Cell: A Molecular Approach,
Sinauer Associates, Sunderland, MA (1990), pp 351-388
[0178] Fusions to lux reporter genes can also be used to support
identification of the operonic structures, newly discovered
regulatory circuits, and promoter sites. Since the random library
of E. coli genomic DNA was fused to the promoterless lux gene, the
strength of bioluminescence in the resulting fusions should depend
on the promoter from the E. coli. For example, if the fusion
contains the complete promoter, lux gene expression would be
stronger than the fusion that contains a partial region of same
promoter. If the promoter is truncated to the point that it is
non-functional, the lux gene expression would be absent. Such
result can support the postulated operonic structures and
dependence of promoter function in host (Example 4).
[0179] The construction and sequencing of a random library of E.
coli genomic fragments in plasmid pDEW201 has generated a
genome-registered set of promoter activity reporter constructs. A
highly parallel, solid phase cellular assay has been developed
utilizing a subset of these reporters by combining bioinformatic
analysis and robotic manufacturing of arrays and high throughput
culture production. This assay has the capacity, utilizing standard
robotic tools, to monitor promoter activity of entire microbial
genomes in duplicate, or alternatively entire genomes of different
microbes on the same array. The output of the assay is a grayscale
image and is compatible with commercial products for image analysis
and data analysis developed specifically of DNA microarray
technologies.
[0180] Briefly, the assay involves creating a nonredundant
collection of clones containing reporter constructs. These cells
are grown to stationary phase and robotically printed at high
density onto a porous membrane (e.g., Biodyne B Nunc). Any contact
or non contact printing robot (e.g., Biomek 2000, Beckman) may be
used for printing. The membrane is in close contact with solid
media. A key aspect of this invention is the ability to move the
membrane from one surface to another surface containing different
media. The ability to measure the luminescence as a function of
regulatory region activity from the cells grown in the solid
surface was surprising and unexpected. Previously, LaRossa and Van
Dyk (U.S. Pat. No. 6,025,131) reported that the method to detect
the activity of regulatory regions as reporter gene activity in
suitable hosts was restricted to the cells grown in the liquid
media. Growth in solid media allows one to grow the cells to a
desired density prior to perturbation then follow the kinetics of
the response. Experimental protocols often involve perturbations
that prohibit long term exposure due to cell death or other
irreversible effects. The ability to move the entire array to new
growth conditions allows one a great variety of experimental
schemes including, but not limited, to pulsed or pulse/chase
exposures, reversibility, and short term kinetic studies. Effects
of perturbants can be determined by comparison of luminescence
generated by treated and control cultures.
[0181] Several important characteristics of the assay system needed
to be evaluated. In particular, growth density and conditions,
sensitivity, reproducibility, and the ability to perturb the
reporters and detect changes were major focus points.
[0182] Using DNA damage in E. Coli as a model system, the assay was
first developed with a small set of well characterized clones to
evaluate the robustness of the approach. The set of 10 clones were
tested for the response to DNA damaging agent, nalidixic acid. The
expected nalidixic induced upregulation of genes in the SOS regulon
was detected as increased bioluminescence (FIG. 4B). The level of
bioluminescence from the strains containing fusions to genes that
are not responsive to DNA damage were not affected by the nalidixic
acid treatment (FIG. 4A).
[0183] The clone set of Luxarray 0.5 was used to develop a highly
parallel solid phase assay by printing the clones at high density.
This invention allows collection of an image of the signal
generated from reporter constructs such that the signal intensity
can be subsequently quantified. This requires not only that the
collection parameters (focal plane, magnification, integration
time, and algorithm) are constant but also that the downstream
image analysis software has the ability to process the images
generated. Chemical perturbation, one of the utility of this
invention, requires physically relocating a membrane from one
culture plate to another. This results in images with minimal X-Y
positional registration. Several commercially available products
can efficiently process these images. ArrayVision.TM. (Imaging
Research, Toronto, Canada) and ImageQuant (Molecular Dynamics,
Sunnyvale, Calif.) are two examples of appropriate software
packages. It is preferred that a cooled CCD camera is used to
capture the data.
[0184] Computational filters were applied to the genome-registered
clone collection to generate a nonredundant set of reporter
constructs representing approximately 28% of the promoters in the
genome. Even at this level of coverage, it is likely that at least
one representative promoter of all the regulons in E. coli is
present in the collection. This large set was used in DNA damage
perturbation experiments to confirm the response of several
promoters. As found with the low density experiments quantified
with the luminometer, the expected responses for each clone were
well demonstrated. The five documented DNA damage-responsive
reporter constructs clearly show an upregulation of expression. In
contrast, light production from the strain carrying the lac
promoter fusion as well as several strains carrying other promoter
fusions was decreased.
[0185] A high density cellular array (Lux array 1.0) in this
invention was also used to identify a number of putative previously
unknown DNA damage-responsive operons. When treated with the DNA
damaging chemical, nalidixic acid, in addition to the expected
promoter activity (i.e., SOS regulon), several previously
undescribed promoters demonstrated a similar behavior. This
invention can be used to monitor transcriptional changes in high
throughput manner.
[0186] This invention also provides a method to identify the
fusions that would be useful except that the orientation of the
chromosomal DNA is inverted relative to the reporter genes, such
that the promoter regions of interest are not operably linked to
the reporter genes. Selecting such fusions and inverting the
orientation of the insert DNA can significantly enhance the utility
of a sequenced collection by adding many more operable linked
fusion to the collections. A simple way to do this is to digest the
plasmid DNA with a single restriction enzyme that cuts just outside
the cloned region and religate the pieces. Although a mixture of
plasmids results from this procedure, in many cases the correctly
oriented plasmid can be found because cells containing it, but not
other possible products, will produce light. This inverting method
was also used to add gene fusions to the Lux-A Collection. Other
methods besides light production can be used (notably PCR) to
identify the orientation or size of the insert.
[0187] Additionally, specific PCR primers can be designed for any
region of interest on the chromosome for which sequence data is
available. For E. coli the entire genome is available. Therefore by
utilizing positional and directional coordinates for known and
predicted promoters, specific primers were designed for the purpose
of generating a PCR product containing each promoter. The actual
promoter was assumed to lie in a 400 basepair genomic fragment
ending at the translational start codon position of the first open
reading frame in the operon. Primer pairs were selected from this
region for each operon such that the "right" primer was forced to
include the start codon of the first open reading frame of the
operon (Primer3, Rozen and, Skaletsky (1996,1997,1998). Code
available at
http://www-genome.wi.mit.edu/genome_software/other/primer3.h- tml).
Acceptable PCR primer pairs were identified for 80% of the operons
using the default Primer3 parameters with an optimal melting
temperature, Tm, of 68.degree. C. Primer pairs for the remaining
operons can be identified by either relaxing one or more
parameters, expanding the search area or by using a different
algorithm. The final version of the primers also includes addition
of appropriate EcoRI and SacI endonuclease cleavage sites (left and
right primers respectively) such that the PCR products can be
directionally cloned into pDEW201.
[0188] Still other methods to generate gene fusions for a
genome-registered collection result in chromosomal rather than
plasmid-borne gene fusions. Transposable genetic elements carrying
reporter genes can be used to generate gene fusions. If this is
done by random transposition into the chromosome of the host
organism, the subsequent gene fusions can be genome-registered with
respect to the chromosomal DNA sequence by determining the sequence
of the junction between the transposable element and the hromosome
(Nichols et al., (1998) J. Bacteriol. 180:6408-6411).
Alternatively, transpositions done in vitro using a defined (and
thus genome-registered) segment of chromosomal DNA can be
recombined by homologous recombination into the host chromosome.
Furthermore, gene fusions formed in vitro by other methods, such as
clones in plasmid DNA, can be recombined into the chromosome of the
host by homologous recombination (Balbas et al., (1996) Gene
172:65-69; Lloyd and Low (1996) In Escherichia coli and Salmonella:
Cellular and Molecular Biology. ASM Press, pp 2236-2255), site
specific recombination (Nash H. (1996) In Escherichia coli and
Salmonella: Cellular and Molecular Biology. ASM Press, pp
2363-2376) or both (Boyd et al., (2000) J. Bacteriol.
182:842-847).
[0189] The preferred methods to generate gene fusions include are
but not limited to the use of plasmid DNA from clones requiring
reorientation as template for the PCR to generate inversion, use of
specific PCR primers when sequence data is available, or use of
transposition to generate gene fusions in chromosome (Kenyon and
Walker (1980) Proc. Natl. Acad. Sci. U.S.A. 77:2819-2823). The most
preferred method to generate genomic fragments is using restriction
enzyme or physical shearing. The genomic fragments are then ligated
to promoterless reporter gene to generate gene fusion. The above
methods to generate fusions are well known in the art.
[0190] This invention also provides a method to use the genome
registered collection of gene fusions in a liquid format to
discover gene fusions useful as biosensors. For example, 39 gene
fusions were found to be upregulated when exposed to limonene for
135 minutes (Example 7). By use of these gene fusion in a strain to
be used as the production organism for limonene, the presence of
limonene, or the optimum conditions for the limonene production can
be measured by the upregulation of bioluminescence.
[0191] Gene expression profiles yield information relevant to
understanding gene function and modes of chemical action. Likewise,
such information can be gained by analysis of genetic alterations
resulting in loss of function, reduced levels, or over-expression
of gene products. Thus, an "array of arrays" can be used to enhance
both mode of action studies and functional genomics. Flow diagrams
I and II depict two ways such arrays of arrays can be used.
[0192] Flow diagram I represents the several tests can be performed
on a given perturbation that changes the environment of the cell.
1
[0193] Flow diagram II describes that as data from arrays of arrays
is analyzed, altered responses of interest can be further analyzed
by selected tools in the array of arrays. 2
[0194] These arrays of arrays can be built by generating large
collections of genome-registered mutations in genes of an organism.
Several methods are available including but not limited to
classical radiation and chemical induced mutagenesis as well as
more modem genomic-based techniques including random in vivo
transposition followed by sequencing of junctions to determine the
gene disrupted, targeted in vitro transposition into individual
genes followed by homologous recombination in vivo to generate
disruptants, and primer oligonucleotide generated deletion
insertion alleles generated in vitro by PCR and subsequently
recombined into the genome. Spontaneous mutants can also be
selected by a variety of methods (LaRossa, R. A. (1996) In
Escherichia coli and Salmonella: Cellular and Molecular Biology.
ASM Press, p. 2527-2587.). Likewise, a large collection of
genome-registered genetic alterations that result in
over-expression can be generated. This can be accomplished in
several ways including but not limited to genetic selection
(LaRossa, R. A. (1996) In Escherichia coli and Salmonella: Cellular
and Molecular Biology. ASM Press, p. 2527-2587), cloning on
multicopy plasmids, placement of the gene to be over-expressed
behind a strong promoter, and placement of the gene to be
over-expressed behind a regulated promoter.
[0195] Perturbations are any alteration of environment or genotype.
Perturbations that change the environment include but are not
limited to physical properties, such as radiation fluence,
radiation spectrum, humidity, substratum, or temperature;
nutritional properties, such as carbon source, energy source,
nitrogen source, phosphorus source, sulfur source, or trace element
sources; biological properties, such as presence of competitors,
predators, commensals, pathogens such as phage and other viruses,
the presence of toxins, or bacterocins; and chemical properties,
such as presence of chelators, inhibitors, toxicants or abnormal
levels of normal metabolites.
[0196] Several tests can be performed on a given perturbation that
changes the environment of the cell (Flow Diagram I). Responses
include patterns of gene expression (e.g., reporter gene
expression, presence or absence of specific protein or
intermediates) and phenotypic effects of genetic alterations; these
responses can be analyzed concomitantly. Examples of phenotypes
that may be screened for in the present method include but are not
limited to metabolic capacity (e.g., carbon source requirement,
auxotroph requirement, amino acid requirement, nitrogen source
requirement, and purine requirement); Resistance to inorganic
chemicals (e.g., acid, arsenate, azide, heavy metals, and
peroxide); Resistance to organic and biological chemicals (e.g.,
antibiotics, Acridine, actinomycin, amino purine, amino
phenylalanine, colicin, ethanol, fluoroacetate, mitomycin C, and
nalidixic acid); Resistance to biological agents (e.g., phages);
Resistance to physical extremes (e.g., temperature, pH,
osmotolerance and radiation). The phenotypes amenable to detection
by the present invention are numerous and a full review may be
found in, Robert LaRossa: Escherichia coli and Salmonella: Cellular
and Molecular Biology (1996) ASM press p. 2527-2587). This will
yield an enhanced empirical matching of one perturbation to
another. For instance if one chemical yields a very similar pattern
of effects in all tests to another chemical, then the likelihood of
a similar mode of action of the two chemicals is high. Secondly,
such concomitant analysis of several patterns will enhance
understanding of gene function. For example, if a group of genes is
regulated similarly by environmental perturbation and genetic
perturbation (i.e., mutation) in this group of genes have similar
phenotypic effects, then similar function can be hypothesized.
[0197] Flow Diagram II depicts that as data from arrays of arrays
is analyzed, altered responses of interest can be further analyzed
by selected tools in the array of arrays. For example, one can
evaluate any perturbation by asking which mutants are
hypersensitive or hyper-resistant to the environmental change. Look
at the gene expression profile of wild type and the altered mutants
in response to the environmental change and in its absence. Another
approach to examine genetic changes is to compare the
genome-registered collection of mutants to the wild type by
examining how growth characteristics vary between the mutants and
wild type with changes in a wide range of environmental parameters.
Differences of interest are then followed up with gene expression
profiling.
[0198] As arrays of arrays are utilized, the massive amount of data
on phenotypes, which results from interactions between genotypes
and the environment and found by changing either the genetic
composition or the culture conditions, will allow interpretation of
the interplay between mutants and gene expression profiles.
Analysis of such interactions will be also useful for discovery of
gene function and determining the modes of chemical action.
Furthermore, these analyses may lead to identification of useful
targets for pharmaceuticals, antimicrobials, or agrochemicals,
development of environmental diagnostic tests, or development of
high throughput screen based on modes or sites of chemical
action.
EXAMPLES
[0199] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usage and conditions.
General Methods
[0200] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, pub. by Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0201] The meaning of abbreviations is as follows: "KB" means
kilobase(s) "hr" means hour(s), "min" means minute(s), "sec" means
second(s), "d" means day(s), "ml" means milliliter(s), ".mu.l"
means microliter(s), "nl" means nanoliter(s), ".mu.g" means
microgram(s), "ng" means nanogram(s), "mM" means millimolar,
".mu.M" means micromolar.
[0202] Media and Culture Conditions:
[0203] Materials and methods suitable for the maintenance and
growth of bacterial cultures were found in Experiments in Molecular
Genetics (Jeffrey H. Miller), Cold spring Harbor Laboratory Press
(1972), Manual of Methods for General Bacteriology (Phillip
Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester,
Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), pp.
210-213, American Society for Microbiology, Washington, D.C. or
Thomas D. Brock in Biotechnology: A Textbook of Industrial
Microbiology, Second Edition (1989) Sinauer Associates, Inc.,
Sunderland Mass. All reagents and materials used for the growth and
maintenance of bacterial cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), DIFCO Laboraoties (Detroit, Mich.), Gibco/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.)
unless otherwise specified.
[0204] LB medium contains following per liter of medium:
Bacto-tryptone (10 g), Bacto-yeast extract (5 g), and NaCl (10
g).
[0205] Vogel-Bonner medium contains the following per liter: 0.2 g
MgSO.sub.4.7H.sub.2O, 2 g citric acid.1H.sub.2O, 10 g
K.sub.2HPO.sub.4 and 3.5 g NaHNH.sub.4PO.sub.4.4H.sub.2O.
[0206] Minimal M9 medium contains following per liter of medium:
Na.sub.2HPO.sub.4 (6 g), KH.sub.2PO.sub.4 (3 g), NaCl (0.5 g), and
NH.sub.4Cl (1 g).
[0207] Above media were autoclaved for sterilization then 10 ml of
0.01 M CaCl.sub.2 and 1 ml of 1 M MgSO.sub.4.7H.sub.2O were added
to M9 mediums. Carbon source and other nutrient were added as
mentioned in the examples. All additions were pre-sterilized before
they were added to the media.
[0208] Molecular Biology Techniques:
[0209] Restriction enzyme digestions, ligations, transformations,
and methods for agarose gel electrophoresis were performed as
described in Sambrook, J., et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).
Polymerase Chain Reactions (PCR) techniques were found in White,
B., PCR Protocols: Current Methods and Applications, Volume
15(1993) Humana Press Inc.
Example 1
Construction, Sequencing and Registering a Random Library of E.
coli Genomic Fragments Fused to a luxCDABE Reporter
[0210] The random library of E. coli genomic fragments in plasmid
pDEW201, which contains the origin of replication and bla from
pBR322, four transcription terminators upstream of the promoterless
P. luminescens luxCDABE genes, and a multiple cloning site that
lies between the terminators and luxCDABE were constructed as
previously described (Van Dyk et al., (1998) J. Bacteriol.
180:785-792; Van Dyk and LaRossa (1998) Methods in Molecular
Biology: Bioluminescence, Methods and Protocols, Humana Press Inc.
vol. 102:85-95).
[0211] Briefly, chromosomal DNA isolated from E. coli strain W3110
(Ernsting et al., (1992) J. Bacteriol. 174:1109-1118) was partially
digested with the restriction enzyme Sau3A1, size fractionated by
agarose gel electrophoreses, and a fraction with an average size of
approximately 1.8 KB isolated. This fraction was ligated to pDEW201
that had previously been digested with BamHI and treated with calf
intestinal alkaline phosphatase. The ligation products were used to
transform ultracompetent E. coli XL2Blue cells (Stratagene) to
ampicillin resistance using the protocol provided by Stratagene.
Preliminary characterization of individual XL2Blue transformants
that were picked in random indicated that a large percentage (16 of
16) contained insert DNA with sizes ranging from 0.9 to 3.0 KB.
Approximately 24,000 of these transformants were pooled and used as
a source of heterogeneous plasmid DNA isolated using Qiagen tip20
columns (Qiagen Corp). This plasmid DNA pool was used to transform
E. coli DPD1675 (Nishimura et al. (1990). Nucleic Acids Res.
18:6169; Van Dyk and LaRossa (1998) Methods in Molecular Biology:
Bioluminescence Methods and Protocols, Humana Press inc. vol.
102:85-95) selecting for ampicillin resistance and using a 30
minute phenotypic expression time to minimize the presence of
siblings. A total of 8066 individual transformants were used to
inoculate the 96-wells of sterile Microtest III.TM. Tissue Culture
Plates (Falcon.RTM.) each containing 190 .mu.l of Vogel-Bonner
medium (Davis et al., (1980) Advanced bacterial genetics. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) with glucose as
a carbon source and supplemented with thiamine, uracil, proline,
and 25 .mu.g/ml of ampicillin. These plates were covered and
incubated overnight without shaking at 37.degree. C. The overnight
cultures in 96-well plates were used for permanent cryogenic
storage in duplicate at -80.degree. C. (Menzel, R., (1989) Anal.
Biochem. 181:40-50). These 8066 individual cultures are called the
Lux-A Collection.
[0212] For DNA sequence analysis, one of the duplicate sets of the
Lux-A Collection was thawed and used as an inoculum for cultures
grown to saturation in Terrific Broth (Gibco-BRL, Inc) containing
100 .mu.g/ml ampicillin in 96 well deep-well plates. Plasmid DNA
was extracted from the cultures using the Qiagen R.E.A.L..TM. prep
method with the following modification: after lysis of cells, the
plates were placed in a boiling water bath for 5 minutes and then
rapidly chilled in an ice-water bath before precipitation with
Buffer 3. This modification prevented degradation of the plasmid
DNA by the nucleases present in the non-endA host strain, DPD1675.
DNA sequencing reactions were performed with approximately 1 .mu.g
of plasmid DNA under standard ABI Prism.TM. DyeTerminator Reaction
Ready conditions with the primers pDEW201.forward (SEQ ID NO: 1,
5'-GGATCGGAATTCCCGGGGAT-3') and pDEW201.reverse (SEQ ID NO:2,
5'-CTGGCCGTTAATAATGAATG-3') to obtain sequence information from
each end of the insert. DNA sequences were determined on
ABI377.TM.-XL 96-lane upgraded Sequencers under 4.times. run
conditions on 5% PAG (polyacrylamide gel) LongRanger.TM. (FMC,
Inc.) gels and analyzed with ABI software. DNA sequences were
transferred to a UNIX based utility for further analysis. A
homology search for the sequence from the beginning and end of each
Lux-A clone (in both orientations) was performed against the
complete E. coli sequence (Genbank accession U00096) using
Pearson's FASTA program (fasta3, Version 3.1t13). The essential
Fasta options were -nQH -m 10 -z 0.
[0213] The essential data about each highly significant alignment
(FASTA score >1000, minimum overlap length >200, and minimum
identity >70%) was stored in a relational database (Sybase
System 11, Sybase Inc.).
[0214] The location of Lux-A clone on the E. coli genome was then
based on the above computed homologies for both the beginning and
end of the insert, using the following rules:
[0215] i) Both distal and proximal ends of the insert must have an
unambiguously high sequence homology with E. coli,
[0216] ii) the relative location and orientation of the matches of
the sequence determined from the beginning and end of the clone
implied a reasonable length for the clone, which were known to fall
in a fairly narrow distribution.
[0217] In many cases the above procedure gave a single probable
location, but in others there were multiple possible locations. The
results were stored in the relational database.
[0218] A table of open reading frame annotations for E. coli was
downloaded using NCBI's Entrez facility, and this data was stored
in the relational database.
[0219] A web based, tabular interface to the data was created. This
allows one to see the Lux-A clones in relation to the functional
annotation. A Java based graphical interface was also created to
make positional and directional relationships easy to visualize.
Thus the Lux-A Collection was registered to the E. coli genome.
Example 2
Validation of Lux-A Collection by Verification of Selected Global
Regulatory Responses
[0220] With the sequencing of Lux-A Collections and registering of
the majority of the LuxA members, it was possible to further
examine the biological responses of strains containing luxCDABE
fusions to other members of well-characterized regulatory circuits.
Gene fusions to the lac operon, and members of the heat shock, SOS,
SoxRS, and OxyR regulons were selected from the Lux-A Collection
and the responses to known inducers of each of these global
regulatory circuits were tested.
[0221] Growth Media and Chemicals.
[0222] A rich liquid medium, LB (Miller, J. H., (1972) Experiments
in molecular genetics. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.), was used. When specified, ampicillin (Amp)
was added at 150 .mu.g/ml. Ethanol (200 proof, Quantum Chemicals)
was diluted directly into LB medium. A stock solution of 20 mg/ml
nalidixic acid (Sigma Chemical Co.) in 1 M NaOH was further diluted
into LB medium. Likewise, a stock solution of 100 mg/ml methyl
viologen (Sigma Chemical Co.) in water was further diluted into LB
medium. A 30% solution of hydrogen peroxide (EM Science) was
diluted into LB medium to the desired concentrations.
[0223] Re-Isolation from Microplates, Growth of Cultures, and
Testing Stress Responses.
[0224] Selected strains were reisolated from the Lux-A Collection
microplates stored at -80.degree. C. by streaking for single
colonies on LB-Amp plates. The purity of the cultures in the wells
was tested by inoculating four single colonies into 100 .mu.l of LB
medium in a 96 well luminometer plate (Microlite, Dynex
Technologies). Consistent bioluminescence of each set of four
isolates provided evidence of clonal purity.
[0225] To test the stress responses, one or two single colonies of
each selected strain were used to inoculate 5.0 ml of LB medium
containing 150 .mu.g/ml of ampicillin. These cultures were grown
overnight at 37.degree. C. then diluted by adding 50 .mu.l or 100
.mu.l of the overnight culture into 10.0 ml of fresh LB medium
(without ampicillin) and grown with agitation at 37.degree. C.
until the culture was in exponential phase with readings on a
Klett-Summerson colorimeter containing the red filter of between 10
and 40. These actively growing cultures were immediately used to
initiate a stress response experiment by adding 50 .mu.l of
cultures to 50 .mu.l of LB medium containing various concentrations
of chemical in the wells a 96 well luminometer plate. This division
of the actively growing culture at the time of chemical addition
ensured identical populations were present when the stress was
imposed. Light production was measured in a Dynex ML3000
luminometer at 37.degree. C. The dimensionless units of light
production, relative light units (RLU), are obtained by comparison
with the light reading from an internal light-emitting diode. The
cycle mode of the ML3000 luminometer, similar to previous
descriptions (Van Dyk et al., Appl. Environ. Microbiol.
60:1414-1420) was used.
[0226] Gene Fusions to the lacZYA Operon.
[0227] Transcription of the very well characterized lac operon is
regulated by both specific and global regulatory circuits.
Specific, negative regulation is mediated by the lacI-encoded
repressor (Choy and Adhya, (1996) In Escherichia coli and
Salmonella: Cellular and Molecular Biology. ASM Press pp
1287-1299). Global regulation in response to glucose availability
is mediated by positive transcriptional activation of cAMP-CRP
(Botsford and Harman (1992) Cyclic AMP in prokaryotes. Microbiol.
Rev. 56:100-122). The Lux-A Collection contains three members that
are fusions of luxCDABE to the lac operon as shown in FIG. 1. To
test for the appropriate response of these gene fusions to glucose,
each was reisolated and tested for bioluminescence when grown in
the presence or absence of glucose in LB medium. The results are
shown in Table 2.
2TABLE 2 Fusions to the lac operon in the Lux-A Collection. RLU of
100 .mu.l overnight cultures Lux Clone LBAmp.sup.150 + 0.4% glucose
LBAmp.sup.150 lux-a.pk034.a6 (a) 0.002 23.0 lux-a.pk034.a6 (b)
0.001 18.8 lux-a.pk050.b9 (a) 0.001 26.0 lux-a.pk050.b9 (b) 0.001
0.007 lux-a.pk065.d4 (a) 0.001 19.1 lux-a.pk065.d4 (a) 0.001
19.6
[0228] Two isolated single colonies of each culture were tested by
growing overnight in the specified medium then measuring the
bioluminescence of 100 .mu.l in an ML3000 luminometer. LBAmp medium
is LB medium containing ampicillin at 150 mg/ml concentration.
[0229] With one exception, each of the isolated colonies from the
cultures in the Lux-A Collection was much more highly
bioluminescent in the absence of glucose than in its presence. One
colony from the lux-a.pk050.b9 that was not highly bioluminescent
might be due to contamination of the culture in that well. Other
putative cross contamination events have not been observed. Each of
the lacZ-luxCDABE fusion strains had at least one reisolated colony
that gave the expected response to glucose. Thus, the appropriate
biological response of these Lux-A Collection members was
verified.
[0230] Heat Shock Regulon Gene Fusions.
[0231] The Lux-A Collection was examined to find fusions to genes
in the .sigma..sup.32-controlled heat shock regulon (Gross, C. A.,
(1996) In Escherichia coli and Salmonella: Cellular and Molecular
Biology, Second ed. ASM Press, pp. 1382-1399). Table 3 shows the
genes or operons for which the Lux-A Collection was searched and
the number of gene fusions found for each gene.
3TABLE 3 Lux-A Collection Gene Fusions to Heat Shock Regulon
members Number of Heat shock gene Fusions in the or operon Lux-A
Collection grpE 0 lon 1 clpPX 0 dnaKJ 0 groESL (mopAB) 0 rpoD 3
htpG 0 clpB 1 htpX 0 htgA (htpY) 0 hslT (ibpA) 0 rfaDFCL (htrM)
1
[0232] While there was not complete representation of the each
member of the heat shock regulon, lux fusions to four of twelve
heat shock promoter (33%) were found. Thus, the Lux-A Collection
contains members that should report on activation of this stress
response.
[0233] In addition, strains with the previously constructed
grpE-luxCDABE gene fusion in parental plasmid pDEW201 (Van Dyk et
al., (1998) J. Bacteriol. 180:785-792) had been placed in selected
wells of the Lux-A Collection plates as a control. One of these was
also selected for testing. Table 4 summarizes the information on
the six strains that were reisolated from wells of the frozen Lux-A
Collection.
4TABLE 4 Heat shock regulon gene fusions Genes in 3% Ethanol Strain
cloned Basal Response Lux clone name insert size fusion to: DNA
RLU* ratio# lux-a. pk057.h1 DPD2241 581 grpE yfjB' grpE' 61.1 5.2
lux-a. DPD2242 2154 lon `clpP clpX 6.71 5.6 pk021.g11 lon`.sup.1
lux-a. pk089.e7 DPD2246 1989 rpoD `dnaG 9.49 2.4 rpoD`.sup.2 lux-a.
pk054.c3 DPD2243 2319 clpB `sfhB yfiH 38.5 4.7 clpB`.sup.3 lux-a.
pk040.e4 DPD2244 2650 rfaDFCL yibB' rfaD 33.1 1.4 rfaF' *From zero
time point of control (untreated) actively growing cultures in
Vogel-Bonner minimal medium that had been obtained during the
screen for sulfometuron methyl responses (Van Dyk et al., J.
Bacteriol. 180: 785-792). #The ratio of the bioluminescence from
the culture treated with 3% ethanol at 40 minutes after ethanol
addition to LB divided by the bioluminescence of the untreated
control in LB at the same time point. .sup.1clpP and clpX are
cotranscribed; thus, there is not expected to be a promoter
upstream of clpX. .sup.2This fragment should contain only the rpoD
specific promoter (Tylor et al., (1984) Cell 38: 371-381)
.sup.3There is no intergenic space between sfhB and yfiH, thus
there is unlikely to be a promoter upstream of yfiH. The mark
(`or`) is used to designate that the E. coli gene in the chimeric
reporter gene fusion is truncated. If the ' is at the start of the
gene name (e.g. 'lacZ), it means the 5' end is missing. If the ' is
at the end of the gene (e.g. lacZ') it means the 3' end of the gene
is not present.
[0234] The light production from each of these strains in the
absence of stress is consistent with each of these chromosomal DNA
fragments containing an active promoter because these values are
much greater than that from a culture carrying the parental plasmid
pDEW201, which had 0.002 RLU under the same conditions (Van Dyk et
al., (1998) J. Bacteriol. 180:785-792). These strains were tested
for their ability to report on the heat shock stress response by
using 5, 4, 3, and 2 (v/v) % ethanol, a known, potent chemical
inducer. For each of the six strains, at least two concentrations
of ethanol resulted in increased bioluminescence as compared with
the control, untreated culture. Table 4 contains the response ratio
for each strain to 3% ethanol at 40 minutes of treatment. These
data demonstrate that these heat shock regulon gene fusions from
the Lux-A Collection report the induction of the heat shock
response.
[0235] SOS Regulon Gene Fusions.
[0236] In a like fashion as described above, the Lux-A Collection
was examined to find fusions to genes in the SOS DNA damage
response regulon (Kim et al., (1997) Proc Natl Acad Sci USA
94(25):13792-7; Walker, G. C. (1996) In Escherichia coli and
Salmonella: Cellular and Molecular Biology. ASM Press pp.
1400-1416). Table 5 shows the genes or operons for which the
randomly generated Lux-A Collection was examined and the number of
gene fusions present for each gene.
5TABLE 5 Quantities of Lux-A Collection Gene Fusions to SOS Regulon
members Number of SOS gene Fusions in the or operon Lux-A
Collection umuDC 0 recA 2 uvrA 1 uvrB 0 sulA (sfiA) 0 uvrD 1 recN 0
polB (din A) 0 dinP (dinB) 1 dinD 0 dinF 1 dinG 1 dinI 0 ruvAB
1
[0237] Of the fourteen SOS regulon genes or operons examined, seven
(50%) were found to be represented by useful fusions in the Lux-A
Collection. Thus, this collection contains members that should
report on activation of the SOS stress response. Table 6 summarizes
the information on the eight strains that were reisolated from
wells of the frozen Lux-A Collection.
6TABLE 6 SOS regulon gene fusions Nalidixic acid Strain Genes in
Insert Basal response Lux clone name insert size fusion to: DNA
RLU* ratio# lux-a. DPD2247 2840 dinF plsB(-)' dgkA(+) 0.224 2.2
pk085.a5 lexA(+) dinF(+)' lux-a. DPD2248 2022 dinG `rhlE(+) ybiA(-)
32.8 2.0 pk0024.f5 dinG(+)` lux-a. DPD2249 2125 dinP
fhiA(-)'mbhA(+) 4.41 5.0 pk055.a3 dinP(+)' lux-a. DPD2250 1789 recA
`mtlB(-) ygaD(-) 97.5 1.8 pk022.d4 recA(-)` lux-a. DPD2251 1782
recA `mtlB(-) ygaD(-) 99.5 2.8 pk085.b5 recA(-)` lux-a. DPD2253
2105 uvrA yjcC(+)' yjcB(-) not 1.4 pk01.b6 ssb(+) uvrA(-)' done
lux-a. DPD2254 1632 uvrD `xerC(+) 15.7 1.2 pk052.b6 yigB(+)
uvrD(+)` lux-a. DPD2293 1890 ruvA(-) `yebC(-) ruvC(-) 14.3 4.6
pk058.f2 yebB(+) ruvA(-)` *From zero time point of control
(untreated) actively growing cultures in Vogel-Bonner minimal
medium that had been obtained during the screen for sulfometuron
methyl responses (Van Dyk et al., (1998) J. Bacteriol.
180-785-792). #The ratio of the bioluminescence from the culture
treated with 5 .mu.g/ml of nalidixic acid at 110 minutes after
nalidixic acid addition to LB divided by the bioluminescence of the
untreated control in LB at the same time point. The mark (`or`) is
used to designate that the E. coli gene in the chimeric reporter
gene fusion is truncated. If the ' is at the start of the gene name
(e.g. 'lacZ), it means the 5' end is missing. If the ' is at the
end of the gene (e.g. lacZ') it means the 3' end of the gene is not
present.
[0238] Each of these strains that were tested for basal
bioluminescence in defined medium had a greater level of light
production than did a strain containing the parental plasmid,
indicating the presence of a promoter driving expression of the
luxCDABE reporter. The ability of these promoter-luxCDABE fusions
to report activation of the SOS stress response was tested using
nalidixic acid, a known, potent chemical inducer. The final
concentrations of nalidixic acid were 1, 5, 25, and 125 .mu.g/ml
for all but strain DPD2293, which was tested at 80, 40, 20, 10, 5,
2.5, and 1.25 .mu.g/ml. For each of the eight strains, at least
three concentrations of nalidixic resulted in increased
bioluminescence. Table 6 gives the response ratio to 5 .mu.g/ml of
nalidixic acid at 110 minutes after addition. These data
demonstrate that induction of the SOS response is reported by
increased bioluminescence for these SOS regulon gene fusion in the
Lux-A Collection.
[0239] Specificity of responses was tested by measuring the effect
of ethanol on these SOS regulon gene fusions. In each case there
was little to no increased bioluminescence induced by 5, 4, or 3%
(v/v) ethanol treatment. For example, the treatment with 3% ethanol
at 40 minutes after addition, a condition which resulted in
increased light production from all the heat shock regulon gene
fusions (Table 4), for strain DPD2249 containing a dinP-luxCDABE
gene fusion yielded a response ratio was 0.80. Similarly, when
strain DPD2243 containing a heat shock regulon clpB-luxCDABE fusion
was tested with nalidixic acid, no increase in bioluminescence was
observed; at 110 minutes after addition of 5 .mu.g/ml nalidixic
acid, the response ratio was 0.40. Thus, the specificity of the
heat shock response to ethanol and the SOS response to nalidixic
acid was shown.
[0240] SoxS-Regulated Oxidative Damage Regulon Gene Fusions.
[0241] The Lux-A Collection was also examined for the presence of
fusions to genes in the SoxR and SoxS regulated oxidative stress
response regulon (Koh et al., (1999) Mol. Gen. Genet. 261:374-380;
Rosner and Storz (1997) Curr. Top. Cell. Regul. 35:163-177; Van Dyk
et al., (1998) J. Bacteriol. 180:785-792). Table 7 below shows
results.
7TABLE 7 Quantities of Lux-A Collection Gene Fusions to the SoxR/S
regulon members Number of SoxR/A regulon Fusions in the gene or
operon Lux-A Collection sodA 0 nfo 0 fumC 0 achA 0 fpr 0 zwf 3 micF
0 acrAB 0 inaA 1 pqiAB 0 ribA 1 poxB 1
[0242] In a similar fashion to the heat shock and SOS regulons, the
SoxR/S regulon was not fully represented in the Lux-A Collection.
Nevertheless, the collection contained gene fusions to 33% of the
SoxR/S regulon gene or operons that would be expected to report on
the activation of this stress response. Table 8 summarizes the
information on the six strains that were reisolated from wells of
the frozen Lux-A Collection.
8TABLE 8 SoxR/S regulon gene fusions Methyl viologen Strain Genes
in Insert Basal response Lux clone name insert size fusion to: DNA
RLU* ratio lux-a. DPD2278 1708 zwf pykA(+)` 5.0 Not tested pk053.b1
yebK(+) zwf'(-)' lux-a. DPD2272 1708 zwf pykA(+)` 16.9 3.6 pk082.g7
yebK(+) zwf'(-)' lux-a. DPD2279 1709 zwf pykA(+)` 4.2 Not tested
pk088.e1 yebK(+) zwf'(-) lux-a. DPD2286 2308 ribA yciM(+)' 5.8 4.7
pk078.d2 o102(+) pgpB(+) ribA(-)' lux-a. DPD2087 1583 inaA 'glpQ
yhaH 9.8 9.0 pk014.a9 inaA` lux-a. DPD3509 1131 poxB `b0872 poxB`
1.5 15.0 pk071.a11 *From zero time point of control (untreated)
actively growing cultures in Vogel-Bonner minimal medium that had
been obtained during the screen for sulfometuron methyl responses
(Van Dyk et al., (1998) J. Bacteriol. 180: 785-792). #The ratio of
the bioluminescence from the culture treated with 250 .mu.g/ml of
methyl viologen at 120 minutes after methyl viologen addition to LB
divided by the bioluminescence of the untreated control in LB at
the same time point. The mark (`or`) is used to designate that the
E. coli gene in the chimeric reporter gene fusion is truncated. If
the ' is at the start of the gene name (e.g. 'lacZ), it means the
5' end is missing. If the ' is at the end of the gene (e.g. lacZ')
it means the 3' end of the gene is not present.
[0243] Like the SOS and heat shock regulon fusions, these had basal
light production greater than the promoterless parental plasmid
indicating the presence of a promoter. Responsiveness of four of
these strains to a known inducer of the SoxR/S regulon, methyl
viologen, was tested at 1000, 500, 250, 125, 62, 31 and 16
.mu.g/ml. Each of these seven methyl viologen concentrations
induced increased bioluminescence from each of the four strains.
Table 8 gives the response ratio for 250 .mu.g/ml at 120 minutes of
treatment for the four tested strains. Here again, the biologically
appropriate response was observed.
[0244] OxyR-Regulated Oxidative Damage Regulon Gene Fusions.
[0245] Fusions to OxyR regulated genes (Rosner and Storz. (1997)
Curr. Top. Cell. Regul. 35:163-177) were found in the Lux-A
Collection as summarized in Table 9.
9TABLE 9 Quantities of Lux-A Collection Gene Fusions to the OxyR
regulon members Number of OxyR regulon Fusions in the gene or
operon Lux-A Collection katG 0 gorA 0 dps 0 ahpCF 3
[0246] Of these four genes or operons controlled by OxyR, fusions
to one (25%) was available in the Lux-A Collection. Table 10
summarizes the information on the three strains, each containing a
fusion of the ahpCF operon regulatory region to the luxCDABE
reporter, that were reisolated from wells of the frozen Lux-A
Collection.
10TABLE 10 OxyR regulon gene fusions H.sub.2O.sub.2 response Genes
in RLU H.sub.2O.sub.2 ratio in insert fusion Insert initial
response pcnB.sup.- Lux clone Strain name size to: DNA screen*
ratio# host.dagger-dbl. lux-a. DPD2283 1184 ahpC dsbG' 92.2 1.3
16.5 pk051.d5 ahpC(+)' lux-a. DPD2284 1184 ahpC dsbG' 68.3 1.6 15.3
pk051.e3 ahpC(+)' lux-a. DPD2285 1182 ahpC dsbG' 47.2 1.2 not done
pk03.d6 ahpC(+)' *From zero time point of control (untreated)
actively growing cultures in Vogel-Bonner minimal medium that had
been obtained during the screen for sulfometuron methyl responses
(Van Dyk et al. (1998) J. Bacteriol. 180: 785-792). #The ratio of
the bioluminescence from the culture treated with 0.002% hydrogen
peroxide at 30 minutes after hydrogen peroxide addition to LB
divided by the bioluminescence of the untreated control in LB at
the same time point. .dagger-dbl.The ratio of the bioluminescence
from the culture of E. coli host strain DPD2228 [F- .DELTA.lac4169
rpsL pcnB80 zad-2084::Tn10] containing the plasmid DNA from the
original Lux-A Collection strain listed treated with 0.002%
hydrogen peroxide at 30 minutes in LB after hydrogen peroxide
addition divided by the bioluminescence of the untreated control in
LB at the same time point. The mark (`or`) is used to designate
that the E. coli gene in the chimeric reporter gene fusion is
truncated. If the ' is at the start of the gene name (e.g. 'lacZ),
it means the 5' end is missing. If the ' is at the end of the gene
(e.g. lacZ') it means the 3' end of the gene is not present.
[0247] The high level of light production from these three strains
containing ahpC-luxCDABE gene fusions indicates that they each
contained a very active promoter. To test if these strains would
report on activation of the OxyR-controlled oxidative stress
response, each was treated with hydrogen peroxide at 0.016, 0.008,
0.004, 0.002, 0.001, 0.0005, 0.00025%. At best, the bioluminescence
was minimally induced. Table 10 shows the response ratio to
treatment with 0.002% hydrogen peroxide at 30 minutes after
treatment.
[0248] Previously, another luxCDABE fusion in plasmid pDEW201 to a
gene in the OxyR regulon, katG had been shown to yield larger
response ratios to hydrogen peroxide when the plasmid was moved to
an E. coli host strain containing a pcnB mutation (Van Dyk et al.,
(2000) in press. In A. Mulchandani and O. A. Sadik (ed.), Recent
Advances in Environmental Chemical Sensors and Biosensors. ACS
Symposium Series). Mutations in this gene result in reduced plasmid
copy number for plasmids with origins of replication like that in
pBR322 (Lopilato et al., (1986) Mol. Gen. Genet. 205:285-290), thus
resulting in reduced basal expression of gene fusions carried on
such plasmids. Accordingly, to test if reducing the copy number of
the ahpC-luxCDABE fusions would also yield more reliable detection
of the OxyR-mediated stress response, plasmid DNA taken from two of
these Lux-A Collection strains was moved by transformation into a
pcnB.sup.31 mutant host strain. The two resulting strains were
tested for responses to hydrogen peroxide at 0.004, 0.002, 0.001,
0.0005, 0.00025, 0.00012, 0.00006%. Each of these seven
concentrations dramatically induced increased bioluminescence from
both ahpC-luxCDABE fusion strains in the pcnB.sup.31 host. The last
column of Table 10 gives the response ratio to treatment with
0.002% hydrogen peroxide at 30 minutes. Thus, the appropriate
biological response from these fusions to a highly expressed gene
was obtained when the copy number of the gene fusion was
reduced.
[0249] In one instance where the response from the plasmid-borne
gene fusion to a highly expressed gene was weak, it was
demonstrated that reduction of plasmid copy number with a pcnB
mutation resulted in more potent induction. Other fusions to highly
expressed genes can also be moved to a host strain with reduced
copy number, such a pcnB mutant, or integrated into the chromosome
at a gene dosage of one (Elsemore, D. A. (1998) Methods in
Molecular Biology: Bioluminescencent Protocols., vol. 102,
p:97-104, Humana Press, Inc).
Example 3
Use of a Genome-Registered Collection of Reporter Gene Fusions to
Confirm or Question Results from DNA Array Analysis and to Develop
High throughput Screens Based on Gene Expression
[0250] DNA Microarray Experiment with Mitomycin C (MMC)
[0251] The E. coli strain MG1655 (rph-1) was used. Cultures grown
in LB at 37.degree. C. overnight were diluted 1 to 250 into fresh
LB and grown at 37.degree. C. with aeration. Each subculture was
split into two 100 ml cultures when the reading on a
Klett-Sammerson colorimeter with the red filter reads 20 Klett
units. MMC (Sigma, dissolved in ddH.sub.2O) at a final
concentration of 250 ng/ml, a sub-lethal dose, was added to one of
the split cultures. The other culture was a no addition control.
Incubation at 37.degree. C. continued for another 40 minutes, then
cells were collected for preparing total RNA. The MMC treated
culture and its control reached 60 and 55 Klett units, respectively
after 40 minute treatment.
[0252] Total RNA purification, first-strand cDNA labeling,
preparation of the E. coli whole genome high-density microarray
chips, hybridization and data analysis were done as previously
described (Wei et al. (2001) J. Bacteriol. 183: 545-556). Both cy3
and cy5 were used in probe labeling, and the hybridization
experiments were repeated by swapping the fluorescence cy dyes
between each pair of MMC treated sample and its blank control.
[0253] Ratios of expression in the mitomycin C treated samples vs.
controls were calculated for all genes in the DNA array. Ratios
greater than or equal to 2 were considered induced genes, while
those with ratios less than 2 fold were considered uninduced. The
known SOS genes (Kim et al., (1997) Proc Natl Acad Sci USA 1997 Dec
9;94(25):13792-7; Lomba et al., (1997) FEMS Microbiol Lett
156:119-122; Walker, G. C. (1996). In Escherichia coli and
Salmonella: Cellular and Molecular Biology. ASM Press pp.1400-1416)
fell into both the induced (Table 11) and uninduced classes (Table
12). In addition, 20 genes not previously known to be induced by
MMC were observed to be induced in the array experiment (Table
13).
11TABLE 11 Known SOS genes induced by MMC in array experiment Fold
induction/ Available Lux Induction Gene array experiment Fusion of
Fusion recN 8.3 NO recA 3.0 YES YES lexA 3.6 NO dinI 6.7 NO dinD
2.2 NO uvrA 2.3 YES YES uvrB 2.1 NO ruvA 2.0 YES YES sulA 5.8 NO
umuC 2.1 NO dinB (dinP) 2.0 YES YES b1848 (yebG) 5.8 NO
[0254]
12TABLE 12 Known SOS genes NOT induced by MMC in array experiment
Available Fold induction/ Lux Induction Gene array experiment
Fusion of Fusion uvrD 1.4 YES YES polB (dinA) 1.2 NO dinG 1.4 YES
YES dinF 1.8 YES YES himA 1.3 NO ruvB 1.5 NO umuD 1.2 NO
[0255]
13TABLE 13 Genes not previously known to be DNA damage-inducible
found induced by MMC in array experiment Available Fold induction/
Lux Induction of Gene array expt Fusion Fusion mioC 2.1 NO xseA 2.0
NO insB_2 2.2 NO insB_1 2.1 NO insA_4 2.1 NO secG 2.2 NO exbD 2.2
YES NO trkH 2.1 YES NO infA 2.3 NO hslS 6.7 NO hslT 4.0 NO cspA 2.9
NO dniR 2.1 YES NO b0531 3.3 NO b1847 (yebF) 3.3 YES YES (when
inverted) b1228 2.5 NO b2940 2.3 NO b0571 (ylcA) 2.2 YES NO b2559
2.0 NO b3199 2.0 NO
[0256] Verification of Expected Responses of MMC Induction of Known
SOS Genes
[0257] The Lux-A Collection of reporter gene fusions was examined
for presence of fusions to the genes in Table 11. Four were found
to be present in the collection. Each of these was tested for
induction by MMC at several doses over a time course of 100
minutes. The expected induction response of a lag period with no
change in gene expression followed by an induction of increased
bioluminescence was observed in all four cases at several doses of
MMC. Thus, this demonstrates that results from DNA array
experiments can be verified by using corresponding gene
fusions.
[0258] Questioning the Negative Result of Non-Induction of Known
SOS Genes
[0259] As expected, the expression of the known SOS genes was
elevated; however the expression of several was elevated less than
2-fold (Table 12) and as such were within a large group of 792
genes the expression of which was elevated by 20% or more. Most of
these are likely due to artifacts in the array data rather than to
actual biologically relevant responses. To test if strains carrying
luxCDABE gene fusions would yield the expected positive result, the
three gene fusions that were available in the Lux-A Collection of
reporter gene fusions were tested for mitomycin C responses. In all
three cases, mitomycin C induced increased bioluminescence. Thus,
this demonstrates that negative results from DNA arrays can be
questioned by contradictory positive results with corresponding
gene fusions.
[0260] Questioning or Confirmation of Induction of Previously
Unknown MMC Inducible Genes
[0261] The genes that were not previously known to be induced with
mitomycin C were further examined for correlation of DNA array and
gene fusion experimental data. For this class of genes, four
fusions were available in the Lux-A Collection of reporter gene
fusions.
[0262] The corresponding luxCDABE fusions to these four genes
provided no evidence of increased gene expression induced by
MMC.
[0263] An additional gene fusion in the Lux-A Collection of gene
fusions was found to have a genomic fragment that when inverted
would result in a fusion to yebF. In this case, a divergent
promoter to the purT gene was present in the chromosomal fragment
and strains containing the backward yebF fusion produced light.
Following isolation of plasmid DNA, XmaI digestion that releases
the insert DNA from the vector, religation and transformation, 10%
of the transformants produced light. Of these, 20% (or 2% of the
initial transformants) were found to be highly induced by nalidixic
acid, another DNA damaging agent. This result strongly suggested
that inversion of the insert DNA had occurred in these
nalidixic-acid inducible gene fusions. The orientation of the
inserted segment to yield a fusion of yebF to the luxCDABE operon
was confirmed by DNA sequence analysis. Furthermore, induction by
mitomycin C was demonstrated, as shown in FIG. 2.
Example 4
Functional Definition of Postulated Promoter Regions
[0264] The genes encoding production of type I extracellular
polysaccharide in E. coli are located in a cluster of 20 genes
(Stevenson et al. (1996) J. Bacteriol. 178: 4885-4893). An upstream
promoter for these genes has been identified (Stout, V. (1996) J.
Bacteriol. 178: 4273-4280) and its regulation has been
characterized (Gottesman, S. (1995) Two-component Signal
Transduction. American Society of Microbiology pp253-262; Wehland
and Bernhard (2000) J. Biol. Chem. 275:7013-7020). Nonetheless, the
transcriptional organization of this region has not been completely
defined. The annotated sequence for these genes (Blattner et al.
(1997) Science 277:1453-1462), which are transcribed from one
strand of the genome, suggests the existence of several putative
promoters and activator binding sites. Furthermore, a prediction of
operon structure in this region suggests that these genes may be
organized into several transcriptional units (Thieffry et al.
(1998) Bioinformatics 14:391-400). FIG. 8 summarizes the predicted
promoters in this region. Unexpectedly, an E. coli DNA
microarray-based experiment with strains 397C, containing a
truncated .beta.' subunit of RNA polymerase, and P90, an isogenic
rpoC.sup.+ control, suggested that RNA transcripts in this region
were affected by the rpoC mutation. Expression of genes b2043
through b2062 was coordinately upregulated in the rpoC mutant (FIG.
8). The elevated expression is most readily explained if a single
transcript starts before b2062 and ends between b2043 and b2042
(galF). If this is true, the region upstream of b2062 should
contain a promoter. That region was fused to the luxCDABE operon in
lux-a.pk033.g2. Transformants of P90 and 397C were grown at
30.degree. C. in LB medium containing 100 .mu.g/ml ampicillin.
Bioluminescence and turbidity, recorded with a Klett-Summerson
colorimeter (Van Dyk et al. (1995) J. Bacteriol. 177:6001-6004), of
actively growing cultures were used for calculation of light
production per 10.sup.9 cells. The unpaired t-test was used to
compare quadruplicate bioluminescence measurements of strains
carrying gene fusions to the control strain carrying the parental
plasmid. The bioluminescence produced when the lux-a.pk033.g2 gene
fusion was placed into strain P90 was weak (0.56+/-0.06
RLU/10.sup.9 CFU), but yet was significantly greater (P<0.0001)
than the bioluminescence produced by the parental plasmid in the
same host strain (0.028+/-0.010 RLU/10.sup.9 CFU). These data are
consistent with a promoter in the region upstream of b2062 that is
not very active in strain P90 growing in LB medium. The
bioluminescence of this gene fusion was elevated 1500-fold when
placed in strain 397C (FIG. 8). This strong promoter activity
driving luxCDABE gene expression is, therefore, dependent upon the
rpoC.sup.- mutation. In contrast, several other gene fusions to
chromosomal DNA segments in this region, whether they contained
predicted promoter regions or not, had very low levels of activity
in both the wild type and rpoC.sup.- mutant (FIG. 8). Thus, the
data from both the DNA microarray experiments and from gene fusions
are consistent with cotranscription of the twenty genes in this
region. The end of the operon was defined by a gene fusion to the
galF upstream region (in lux-a.pk07.d12) that strongly drove
luxCDABE transcription. The activity of this promoter region was
not dramatically effected by the rpoC mutation (FIG. 8), in
agreement with the DNA array data.
Example 5
Highly Parallel Transcription Analysis Using a High Density Array
of Cellular Reporters
[0265] Proof of principle with Luxarray 0.5
[0266] A solid phase assay system consisting of reporter clones
growing in the presence or absence of a perturbation, biological,
environmental or chemical was developed. This was accomplished by
growing the reporters on a porous membrane (Biodyne B, Nunc) seated
on top of solid growth media in a culture dish (OmniTray, Nunc).
Luminescence was measured either by a luminometer or by creating an
image of the entire culture and quantitating the pixel density.
Effects of perturbants can be determined by comparison of
luminescence generated by treated and control cultures. Growth on
the surface of the membrane allows the reporter assay to be moved
between conditions as required. Experimental protocols often
involve perturbations that prohibit long term exposure due to cell
death or other irreversible effects. The ability to move the entire
array to new growth conditions allows one a great variety of
experimental schemes such as pulsed or pulse/chase exposures,
reversibility, and short term kinetic studies.
[0267] Several important characteristics of the assay system needed
to be evaluated. In particular, growth density and conditions,
sensitivity, reproducibility, and the ability to perturb the
reporters and detect changes were major focus points. Development
of this assay system was initiated using a collection of 10 well
characterized clones, the parental plasmid clone, and media alone
(Table 14). The set of 10 clones represented a distribution of
signal strength as well as response to perturbation with DNA
damaging agents, in particular nalidixic acid. Initial experiments
were designed to benefit from the sensitivity and simplicity of
measuring luminescence with a 96-well luminometer (Dynex ML3000).
Printing was accomplished using a BioMek 2000 (Beckman Coulter)
equipped with a High Density Replication Tool (HDRT). Sterilization
in between transfers was accomplished by soaking the pins
successively in 0.2% SDS in water, sterile water, and 70% ethanol.
After sterilization, the pins are air-dried prior to the next
transfer.
14TABLE 14 Clone Biolumi- Strain No. Fusion to: nescence* Comment
DPD2083 N.A. N.A. none Parental plasmid without insert DPD2282
65.d4 lacZ moderate "Constitutive" expression in LB/ low expression
in LB + glucose DPD2247 85.a5 dinF low SOS regulon/ Nalidixic acid
and Mitomycin C inducible DPD2248 24.f5 dinG moderate SOS regulon/
Nalidixic acid and Mitomycin C inducible DPD2249 55.a3 dinP
moderate SOS regulon/ Nalidixic acid and Mitomycin C inducible
DPD2250 22.d4 recA high SOS regulon/ Nalidixic acid and Mitomycin C
inducible DPD2253 01.b6 uvrA moderate SOS regulon/ Nalidixic acid
and Mitomycin C inducible DPD2242 21.g11 lon moderate Heat shock
regulon DPD2243 54.c3 clpB moderate Heat shock regulon DPD2245
42.c8 rpoD low Heat shock regulon DPD2084 06.b4 yciG low
SigmaS-dependent stress responsive DPD2090 23.c7 osmY low
SigmaS-dependent stress responsive *Bioluminescence is measured in
the liquid culture by luminometer.
[0268] Strains were grown overnight at 37.degree. C. in 40 .mu.l LB
in 96-well dishes. These cultures were designated "printing plates"
and were used as the cell source to manufacture the arrays.
[0269] Membranes were sterilized with UV illumination for 10 min
then placed in contact with prewarmed media in a culture dish. The
clones and controls were printed onto an approximately 8.times.12
cm membrane mimicking the pattern of a 96-well plate (FIG. 3A).
Strains were printed to generate 8 repeating sections of the 10
strains and two controls and placed in a Dynex ML3000 luminometer
prewarmed to 37.degree. C. Luminescence data was collected for each
spot for 40 cycles of 20 min duration (overnight). FIG. 3B shows
the signal collected for each clone as a function of time. FIG. 3C
shows signal collected from the replicate spots of the same clone
(recA-luxCDABE). The data clearly indicated that the strains were
well behaved in the new solid phase system. Without exception, the
signal strength measured corroborated the data obtained in liquid
culture. Additionally, replicates varied minimally, again similar
to liquid culture measurements.
[0270] Next the system was evaluated for its ability to determine
responses to perturbation, in this case, DNA damage caused by
nalidixic acid. Parental clone and two nonresponding reporters,
osmY, and lacZYA, and three DNA damage responsive reporters, uvrA,
recA, dinG, were chosen. Strains were printed onto duplicate
membranes over LB agar from fresh overnight cultures as described
and incubated for 6 hr at 37.degree. C. to allow the cells to enter
an exponential growth phase. The membranes were moved to new,
pre-warmed plates containing varying amounts of nalidixic acid and
light readings collected with an ML3000 luminometer. Luxarray 0.5
clones (FIGS. 4A and B) were printed onto membranes and grown
initially on LB then moved to plates containing 0 .mu.g/ml
(diamonds), 1 .mu.g/ml (squares), or 5 .mu.g/ml nalidixic acid
(triangles). Luminescence was measured with a Dynex ML3000
luminometer every 30 min for 2 hrs. As shown in FIG. 4B, the
expected nalidixic acid mediated upregulation of genes in the SOS
regulon was detected as increased bioluminescence. In contrast,
strains containing fusions to non-DNA damage responsive genes, osmY
and lacZYA, were unaffected by nalidixic acid treatment (FIG. 4A).
The strain containing the parental plasmid, pDEW201, without a
promoter driving luxCDABE expression produces very low levels of
bioluminescence that are very close to the background measured on a
ML3000 luminometer for strains grown in 96-well microplates. The
apparent basal bioluminescence and upregulation of this strain
(FIG. 4A) is likely due to cross-talk from an adjacent strain
containing a DNA damage responsive gene fusion.
[0271] High Density Luxarray 0.5
[0272] The clone set of Luxarray 0.5 was used to develop a highly
parallel solid phase assay by printing the clones at high density
on the approximately 8.times.12 cm membrane. These arrays were used
to further develop the system.
[0273] Printing Density
[0274] In an effort to more closely approximate the final assay
system, the clones of Luxarray 0.5 were used to determine the
maximum density one could print the cells at and carry out useful
analyses. Arrays were printed as above at increasing density and
grown for 8 hr at 37.degree. C. Luminescence was imaged using an
EagleEye II (Stratagene, La Jolla, Calif.). From visual inspection
individual clonal areas of growth could be resolved at densities up
to 6144 individual spots per membrane. From a comparison of the
size of the growth areas from different densities, it appeared that
in the 8 hr growth period, there was a nutrient-limit effect that
resulted in an inversely proportional amount of growth as density
increases. At this density, there is over two-fold greater then the
estimated number of transcription units in the entire E. coli
genome. Thus a single 8.times.12 cm array could represent the
entire E. coli genome in duplicate with capacity left for controls.
Alternatively full genome coverage of two different strains or
different species could be printed on the same array.
[0275] High Density Array Image Collection
[0276] The essence of this assay is to collect an image of the
signal generated from reporter constructs such that the signal
intensity can be subsequently quantified. This requires not only
that the collection parameters (focal plane, magnification,
integration time, and algorithm) are constant but also that the
downstream image analysis software has the ability to process the
images generated. The most common application of this assay,
chemical perturbation, requires physically relocating a membrane
from one culture plate to another. This results in images with
minimal X-Y positional registration. Several commercially available
products can efficiently process these images. ArrayVision.TM.
(Imaging Research, Toronto, Canada) and ImageQuant (Molecular
Dynamics, Sunnyvale, Calif.) are two examples of appropriate
software packages.
[0277] High Density Luxarray 0.5 Nalidixic Acid Perturbation
[0278] The clones of Luxarray 0.5 were used to print arrays as
described above at a 4.times.4 density. That is to say that each
single spot as in FIG. 3 was replicated 16.times. in a 4.times.4
subarray. This resulted in each clone being printed 128 times on
different areas of the array. Arrays were printed in triplicate
from fresh overnight cultures onto membranes on plates containing
LB media. After 6 hr of growth at 37.degree. C. membranes were
moved to prewarmed plates containing either LB media or LB media
supplemented with 5 .mu.g/ml nalidixic acid (NA) previously
demonstrated to cause detectable induction of responsive promoters
for a wide range of promoter strength (FIG. 4). Cultures were
replaced at 37.degree. C. to continue growing. Images were
collected for each array every two hours from 0-8 hr after
relocation using a cooled CCD camera (FluorChem 8000: f.0.85 lens,
2 min exposure. AlphaInnotech). Spot intensity was determined using
ArrayVision.TM. (Imaging Research, Toronto, Canada). FIG. 5 shows
the results for selected strains containing reporter gene
fusions.
[0279] As found with the low density experiments quantified with
the luminometer, the expected responses for each clone were well
demonstrated. The five documented DNA damage-responsive reporter
constructs clearly show an upregulation of expression. In contrast,
light production from the strain carrying the lac promoter fusion
as well as several strains carrying other promoter fusions was
decreased. This decreased bioluminescence likely reflected the
decreased growth and metabolism of the nalidixic acid treated
strains and demonstrates the specificity of the upregulation of the
SOS-regulon gene fusions. Furthermore, the signal from the strain
containing the parental plasmid is of dramatically lower magnitude
than that of strains with promoter-lux fusions, thus, demonstrating
the advantage of the cooled CCD camera for data capture.
[0280] Lux Array 1.0, a Highly Parallel Promoter Activity
Assay.
[0281] Selection of a Maximal Non-Redundant Set of Lux Gene
Fusions.
[0282] The genome-registered Lux-A luxCDABE gene fusion collection
(described in Example 1) provided a list of 4988 plasmid-borne gene
fusions, each with boundary information relative to the E. coli
genome and the orientation relative to the lux operon. An operably
linked or a functional construct was defined as one consisting of a
genomic fragment encompassing a promoter adjacent to the
promoterless lux operon in an orientation that causes transcription
initiated at the promoter to proceed into and through the lux
operon. Therefore, to identify the functional subset of the
collection, criteria were computationally applied to filter the
list of clones first for functionality and second for redundancy. A
list of definitions of documented and predicted operons (Thieffry
et al. (1998) Bioinformatics 14:391-400) was used to define genomic
coordinates of the operons as the translational start codon
position of the first open reading frame (ORF) in the operon and
the translational stop codon position of the last ORF in the
operon. Additional information included the strand on which the
operon is coded (direction of transcription), and gene names
(common or "b" number). The lux gene fusions were filtered
computationally using the following assumptions; (i) a functional
transcriptional fusion would result from any genomic fragment
starting greater than 50 base pairs upstream of the start codon of
the first ORF and ending anywhere between the start codon of the
first ORF in the operon and the stop codon of the last ORF in the
operon, thereby eliminating the occurrence of a transcriptional
stop signal in the construct between the promoter and the lux
operon; and (ii) the promoter contained in the genomic fragment
must face in the correct orientation relative to the lux operon
(pictorially represented in FIG. 6). Finally, in cases when more
then one clone fit the criteria for a single operon, only the one
construct containing the genomic fragment representing the greatest
amount of upstream sequence was retained thereby eliminating
redundancy. The PERL code used and the resulting list of 689
selected gene fusions are found in Scheme 1 and Table 18 (following
Example 7). These fusions represent 27% of the 2584 known and
predicted transcriptional units in the E. coli genome. Individual
cultures of strains containing the identified gene fusions were
rearrayed from the ninety original culture plates to create a set
of sixteen 96-well microplates containing all the identified
fusions, duplicated with side by side symmetry, including
appropriately placed controls. These 16 plates represent were used
to generate the cellular arrays for use in subsequent analyses.
[0283] Preparation of Reporter Array.
[0284] The E. coli strains were grown overnight at 37.degree. C. in
40 .mu.l LB medium supplemented with 100 .mu.g/ml ampicillin in a
set of sixteen 96-well dishes. These cultures were designated
"printing plates" and were used as the cell source to manufacture
the arrays. Porous membranes (Biodyne B, Nunc) were sterilized with
UV illumination for 10min then placed in contact with pre-warmed of
solid LB growth media in a culture dish (OmniTray, Nunc). Printing
of 4.times.4 subarrays was accomplished using a BioMek 2000
(Beckman Coulter) equipped with a High Density Replication Tool
(HDRT). Sterilization in between transfers was accomplished by
soaking the pins successively in 0.2% SDS in water, sterile water,
and 70% ethanol. After sterilization, the pins are air-dried prior
to the next transfer. The E. coli strains in the LuxArray were
printed onto an approximately 8.times.12 cm membrane in two sets of
triplicates.
[0285] Growth in the Array.
[0286] The growth rate of individual colonies was evaluated because
initial experiments clearly demonstrated a large distribution of
growth rates for the reporter strains in the system. In order to
differentiate between clone-dependent or system-dependent sources
of this variability, bioluminescent cellular arrays were generated
in triplicate on different days using LB agar media containing 10
.mu.g/ml tetrazolium blue. The product generated when live cells
reduce tetrazolium blue is an insoluble blue precipitate. This
greatly increased contrast between the cells and the media
simplifying direct imaging of the cells by normal light. For each
of the triplicate experiments, each clone was visually scored to
determine the size of the growth generated during 8 hrs of
incubation at 37 C (data not shown). Variability was clearly
clone-specific and very consistent from day to day. As the majority
of proposed analyses are relative measurements, and inter-clone
comparisons are unlikely, this type of growth variability does not
effect the applicability or robustness of the overall assay system.
No further attempts were made to determine the source of the
variability, however it can be assumed that it is a result of the
plasmid constructs carried by the clones.
[0287] Bioluminescence of LuxArray 1.0.
[0288] Images of the bioluminescence were using a cooled CCD camera
(FluorChem 8000: f.0.85 lens, 2 min exposure, AlphaInnotech)
without additional light source. The bioluminescence from one such
array grown for 16 hours on rich media is shown in FIG. 7. The
array in FIG. 7 shows side by side replicate subarrays that include
control strains, a strain with lacZYA promoter fusion and a strain
containing the parental plasmid (pDEW201).
[0289] Identification of Nalidixic Acid Responsive Gene Fusions
Using LuxArray 1.0.
[0290] The antibiotic nalidixic acid, an inhibitor of DNA gyrase
known to be a effective inducer of the SOS DNA damage stress
response was used to demonstrate the utility of this array. Arrays
were printed from fresh overnight cultures onto membranes on plates
containing LB media. After 6 hr of growth at 37.degree. C.
membranes were moved to pre-warmed plates containing either LB
media or LB media supplemented with 5 .mu.g/ml nalidixic acid then
replaced at 37.degree. C. to continue growing. Images were
collected for each array every two hours from 0-8 hr after
relocation using a cooled CCD camera (FluorChem 8000: f.0.85 lens,
2 min exposure, AlphaInnotech).
[0291] Spot intensity of each image was determined using
ArrayVision.TM. (Imaging Research, Toronto, Canada) and the
resultant pixel density measurements imported into a template with
identifiers for each spot. The average signal for each of the
triplicate spots was calculated. The background signal, which
results from cross illumination of neighboring spots, was
calculated by finding the median of the 24 spots containing a
strain with the parental plasmid on each of the triplicate arrays
and the calculating the average of the three medians. This
background signal was calculated at each of the time points and
subtracted from each measurement at the corresponding time point.
All negative numbers were converted to zero.
[0292] Data normalization to account for inhibition of growth by
nalidixic acid was accomplished by finding the sum of the averaged
signals of each spot in the array for each treatment at each time
point. A normalization factor (NF) was calculated as follows:
NF=Total array signal (time zero, LB control)/Total array signal
(time x, condition y)
[0293] Each measurement was multiplied by NF to yield a normalized
signal. Ratios the nalidixic acid treated spot to the corresponding
control spot were calculated using the normalized data.
[0294] The ratios of the normalized data were compared for each of
the duplicate spots resulting from independent cultures in the
array. Putative nalidixic acid upregulated gene fusions were
selected as those for which the ratio in both duplicate spots was
at least 2 at both the 2 hour and 4 hour time points. Twelve gene
fusions were selected by these criteria (Table 15).
[0295] These twelve gene fusions include three well-characterized
members of the SOS regulon as well as several fusions to promoters
not previously known to be upregulated by nalidixic acid treatment.
The DNA sequence of plasmid DNA isolated from each of these twelve
cultures reconfirmed the identity of each inserted DNA. The
LuxArray contains a total of six gene fusions to SOS regulon
operons, all of which would be expected to be upregulated by
nalidixic acid. Three were, thus, scored falsely as negatives. An
examination of the data showed that two of these false negatives
were reproducibly upregulated but not at level of the selection
criteria; a fusion to uvrD was upregulated by 1.6 and 1.9 fold at 4
hours of nalidixic acid treatment, while one to ruvA was
upregulated 1.7 and 2.1 fold at that time point. The third did not
have consistent responses; the ratio of nalidixic acid treated to
untreated for the dinF-lux fusion was found to be 4.2 in one of
duplicate spots and 0.7 in the other.
[0296] Validation of Nalidixic Acid Upregulated Gene Fusions by
Retesting in Liquid Medium.
[0297] Each of the newly identified putative nalidixic acid
upregulated gene fusions and two of the known SOS gene fusions were
retested using exponentially growing cultures in liquid medium with
seven concentrations of nalidixic acid (80 .mu.g/ml in two fold
dilutions to 1.2 .mu.g/ml). Several concentrations of nalidixic
acid were used because the differences in responses between liquid
and solid growth were not known. Light production of 100 .mu.l
duplicate cultures at 37.degree. C. was quantitated using a 96 well
plate luminometer (Luminoskan Ascent, Labsystems). Table 15 shows
the results expressed as ratios of the signal from the nalidixic
acid treated cultures to the untreated control at 2 hours at the
concentration that yielded the maximal response. Also shown is the
number of concentrations of nalidixic acid tested that resulted in
response ratios of 1.5 or greater. It should be noted that the
liquid medium tests were not corrected for growth inhibition by
nalidixic acid. Using a standard of a maximal response ratio of at
least 1.8 and responses ratios that were >1.5 fold at 3 or more
concentrations, 7 of the 9 putative novel nalidixic acid
upregulated gene fusions were shown to be reproduced in liquid
medium.
[0298] Mitomycin C Responses and Effect of lexAind Mutation.
[0299] Mitomycin C, a DNA damaging compound with a different
mechanism of action from nalidixic acid, was used to determine if
these newly discovered nalidixic acid upregulated gene fusions were
generally responsive to DNA damage. In addition, the effect of a
lexAind mutation was tested to determine if any of these were part
of the SOS regulon. The expectation is that SOS regulon member will
be induced by both nalidixic acid and mitomycin C in a manner that
is dependent on lexA function (Walker, G. C., (1996) Escherichia
coli and Salmonella: Cellular and Molecular Biology ASM Press). As
shown in FIG. 9, a fusion of the promoter region of b1728 to
luxCDABE was clearly induced by both nalidixic acid and mitomycin C
in the lexA+ host, but was not induced by these chemicals in the
lexAind host strain. Thus, these results demonstrate that
upregulation by nalidixic acid as well as mitomycin C is controlled
by LexA. This is consistent with the observation of upregulation of
the b1728 mRNA transcript upon mitomycin C treatment in a LexA
dependent fashion (Fernndez de Henestrosa et al. (2000). Mol.
Microbiol. 35:1560-1572).--Likewise, two gene fusions, those to
oraA and yigN were identified as new members of the SOS regulon
(Table 16). Interestingly, four of the nalidixic acid responsive
gene fusions were not upregulated by mitomycin C in the lexA+ host
suggesting that they are not generally DNA damage responsive, but
rather are more specifically responsive to nalidixic acid.
Negative, non-DNA damage responsive gene fusions were also included
(Table 16).
[0300] Thus, the LuxArray assay was useful to identify novel
nalidixic acid upregulated genes in E. coli. Likewise, this robust
assay can be used generally in a fashion parallel to hybridization
assays to monitor transcriptional changes. The multiple whole
genome scale capacity and relative simplicity of manufacture allow
for significant throughput. This assay is an important addition to
efforts making functional assignment of promoter activity.
15TABLE 15 Nalidixic acid responses on solid and in liquid medium
Solid Solid Nal conc Liquid Solid expt expt expt Solid expt of max
expt. # of conc Fusion to gene or NA/LB NA/LB, NA/LB, NA/LB,
induction NA/LB with > 1.5 operon: Lux ID t = 2 hr t = 4 hr t =
6 hr t = 8 hr in liquid t = 2 hr fold up Known SOS regulon members:
dinG ybiB lux-a.pk0024.f5 3.4 4.2 3.1 2.6 5 ug/ml 4.18 6
lux-a.pk0024.f5 2.5 3.7 3.5 2.9 dinP lux-a.pk055.a3 3.1 5.1 4.2 3.1
5 ug/ml 7.60 7 lux-a.pk055.a3 5.5 11.0 8.1 6.3 uvrA lux-a.pk0001.b6
2.5 2.3 1.6 1.0 nd nd nd lux-a.pk0001.b6 2.9 2.4 1.1 0.6 Nalidixic
acid upregulated on both the solid LuxArray and in liquid culture
b1169 lux-a.pk0015.d6 2.3 6.4 2.7 0.6 20 ug/ml 2.71 6
lux-a.pk0015.d6 2.2 4.1 1.9 0.8 b1728 lux-a.pk033.c5 3.7 4.3 3.2
2.6 5 ug/ml 3.67 7 lux-a.pk033.c5 2.1 3.1 3.3 2.5 b1936
lux-a.pk0019.gl 2.5 7.7 9.7 7.1 10 ug/ml 2.34 6 lux-a.pk0019.gl 4.4
8.2 15.5 10.2 lpxA lpxB mhB dnaE lux-a.pk061.c3 3.5 3.1 2.8 1.7 2.5
ug/ml 1.82 3 lux-a.pk061.c3 2.7 3.5 3.0 1.9 oraA lux-a.pk058.f5 5.1
7.9 5.9 3.9 10 ug/ml 7.19 7 lux-a.pk058.f5 4.7 7.7 5.6 3.8 yaaF
lux-a.pk031.e7 2.1 2.0 1.3 1.0 2.5 ug/ml 1.85 3 lux-a.pk031.e7 2.0
2.1 1.8 1.5 yigN lux-a.pk046.f11 2.1 2.5 0.3 0.1 1.2 ug/ml 2.54 5
lux-a.pk046.f11 7.1 4.3 2.5 2.2 Nalidixic acid upregulation not
reproduced in liquid culture frvR frvX frvB frvA lux-a.pk046.e6 2.8
2.3 0.8 0.5 20 ug/ml 1.46 0 lux-a.pk046.e6 2.1 2.3 2.0 1.7 yfhJ fdx
hscA yfhE lux-a.pk0019.g2 2.1 2.0 1.7 1.4 1.2 ug/ml 1.02 0
lux-a.pk0019.g2 2.1 2.2 1.5 1.3
[0301]
16TABLE 16 Mitomycin C and Nalidixic acid Responses in lexA+ and
lexAind hosts NA MitC Fusion to Ratio, Ratio, gene or Host 2 hr 2
hr operon: Lux ID Strain 10 ug/ml 250 ng/ml Known SOS regulon
member: dinG ybiB lux- lexA.sup.+ 4.03 5.01 a.pk0024.f5
lexA.sup.ind 0.97 1.15 Nalidixic acid upregulated on both the solid
LuxArray and in liquid culture New SOS regulon members b1728
lux-a.pk033.c5 lexA.sup.+ 5.17 8.97 lux-a.pk033.c5 lexA.sup.ind
0.40 0.90 oraA lux-a.pk058.f5 lexA.sup.+ 11.06 15.27 lux-a.pk058.f5
lexA.sup.ind 1.06 1.21 yigN lux- lexA.sup.+ 5.74 9.84 a.pk046.f11
lux- lexA.sup.ind 0:59 0.93 a.pk046.f11 Nalidixic acid upregulated,
not generally DNA damage inducible b1169 lux- lexA.sup.+ 3.22 1.12
a.pk0015.d6 lux- lexA.sup.ind 2.29 1.73 a.pk0015.d6 b1936 lux-
lexA.sup.+ 3.25 1.22 a.pk0019.g1 lux- lexA.sup.ind 1.55 2.46
a.pk0019.g1 lpxA lpxB lux-a.pk061.c3 lexA.sup.+ 2.87 1.06 rnhB dnaE
lux-a.pk061.c3 lexA.sup.ind 2.39 1.29 yaaF lux-a.pk031.e7
lexA.sup.+ 1.53 1.02 lux-a.pk031.e7 lexA.sup.ind 1.19 0.99 Negative
controls yciG lux-a.pk006.b4 lexA.sup.+ 0.32 0.56 lexA.sup.ind 0.67
0.61 lacZ 65.d4 lexA.sup.+ 0.81 0.80 lexA.sup.ind 1.05 1.24
Example 6
Additions to Collections and Alternative Methods of Generating
Collections of Bacterial Genomic Fragments Fused to a Reporter
[0302] There are several methods for making fusions of bacterial
promoter regions to reporter genes to add to a genome-registered
collection of randomly generated gene fusions. Some of these
methods are also alternative methods for building a large
collection of gene fusions.
[0303] Firstly, DNA sequence data from more random gene fusions
will result in identification of additional useful members to a
collection that is not completely saturated. These additional
sequences can be generated from members of the same originally
sequenced library of genetic fusions, LuxA for instance, or an
independently generated library. The steps outlined in previous
examples allow the genome registration of the newly sequenced
fusions.
[0304] DNA sequencing of randomly generated fusions will lead to
identification of fusions that would be useful except that the
orientation of the chromosomal DNA is inverted, such that the
promoter regions of interest are not operably linked to the
reporter genes. Selecting such fusions and inverting the
orientation of the insert DNA can significantly enhance the utility
of a sequenced collection by adding many more operable linked
fusion to the collections. A simple way to do this is to digest the
plasmid DNA with a restriction enzyme that cuts just outside the
cloned region and religate the pieces. Although a mixture of
plasmids results from this procedure, in many cases the correctly
oriented plasmid can be found because cells containing it, but not
other possible products, will produce light. This method has the
advantage of avoiding use of the polymerase chain reaction and thus
avoiding possible changes in the DNA sequence from
amplification.
[0305] This approach was demonstrated by identifying a non-operably
linked clpB fusion in the Lux-A Collection, inverting the
chromosomal DNA segment relative to the vector, and comparing an E.
coli strain carrying the resultant plasmid to one in the collection
with a proper orientation of the clpB promoter region. One isolate
from the Lux-A Collection, lux-a.pk043.d3 contains the region of
the E. coli chromosome with the clpB promoter, but is oriented in
the opposite direction than required to operably link the promoter
and reporter genes. Very low levels of light production of this
strain were measured. In contrast, the strain, lux-a.pk054.c3, that
carries the plasmid with the clpB promoter region in the proper
orientation to operably link it to the luxCDABE reporters gene had
a high level of light production. Plasmid DNA was isolated from
lux-a.pk043.d3, digested with restriction enzyme XmaI, and ligated
with T4 DNA ligase. The ligation reaction was used to transform E.
coli strain DPD1675 by electroporation. Transformants were selected
by ampicillin resistance. Three percent of the resultant
transformed colonies produced bioluminescence as detected by
exposure of X-ray film. The response to ethanol treatment of two of
these light producing transformants that putatively contain
inverted chromosomal inserts, of lux-a.pk054.c3 (DPD2243) that
contains a plasmid with the clpB promoter operably linked to the
luxCDABE reporter and of the original lux-a.pk043.d3 were compared.
Actively growing cultures at 37.degree. C. in LB medium were
treated with 4% ethanol and light production was quantitated at
37.degree. C. using a microplate luminometer (Dynex ML3000). Table
17 summarizes the average bioluminescence of duplicate samples at
38 min after treatment.
17TABLE 17 Light production by strains containing a clpB-luxCDABE
gene fusion resulting from inversion of the chromosomal insert, and
controls Light Light production Culture production in LB + 4%
Response Strain turbidity.sup.1 in LB.sup.2 ethanol.sup.2 Ratio
lux-a.pk043.d3 11 0.0015 0.0025 N/A Inverted 14 1.25 8.17 6.5
lux-a.pk043.d3 Inverted 18 5.34 29.63 5.5 lux-a.pk043.d3
lux-a.pk054.c3 12 1.37 13.00 9.4 (DPD2243) .sup.1At time zero;
Klett units .sup.2RLU
[0306] Thus, the presence of a promoter that drives transcription
of the luxCDABE operon in the inverted isolates was shown by the
light production in LB medium. Furthermore, the expected biological
response of induction of up-regulation by ethanol treatment for
this member of the heat shock regulon was also demonstrated.
[0307] This inverting method was implemented in a parallel manner
to add gene fusions to the Lux-A Collection. First, a list of about
400 gene fusions from the Lux-A Collection that contained the
correct genomic fragment but in the wrong orientation relative to
the lux operon, and were not present in the current collection was
derived. Each of the strains containing these fusions was picked
out of the original collection of 8000 fusions and regrown. The
bioluminescence of each was quantitated because, in some cases, the
presence of a divergent promoter in the cloned DNA fragment
resulted in light production even though the DNA was inverted with
respect to the promoter of interest. About half of the selected
strains produced >0.1 RLU of light following 3 hours of
incubations at 37.degree. C. after inoculation of 100 .mu.l LB
medium with 10 .mu.l of culture from a working plate. This
suggested that the set of strains with light production of <0.1
RLU would be easier to pursue initially. Thus, following isolation
of plasmid DNA in a 96 well format, XmaI digestion, and religation,
the ligation reaction mixes originating from strains with light
production of less than 0.1 RLU were sorted into separate plates
from those that originated from strains with light production
greater than or equal to 0.1 RLU. E. coli DPD1675 was made
competent by calcium chloride treatment and transformed with the
religated plasmid DNA selecting for ampicillin resistance. Placing
the petri plate with the transformant colonies on X-ray film and
developing the exposed film at various time points of contact
identified light producing transformants. The light producing
colonies were purified by isolating single colonies in solidified
LB medium containing ampicillin and light production was verified
by luminometry. This process was completed for 36 plasmids isolated
from strains with light production of less than 0.1 RLU. Plasmid
DNA from each of the strains containing putative inverted
chromosomal segments was isolated and DNA sequence was determined
to assess if the inverted product had been obtained. Thirteen of
the 36 plasmids yielded DNA sequence information that had a high
scoring hit when compared with the E. coli genomic DNA sequence
using BLASTN (default setting). Of these, five had DNA in the
inverted orientation from the original clone.
[0308] There are several potential reasons for this relatively low
success rate. For example, some promoters that are annotated may
not be functional in the conditions used or may not be biologically
relevant promoters. Thus, selection by light production of the
inverted clone would not be successful. In contrast, divergent
promoters that were not active in the liquid culture test for
bioluminescence used for sorting may have been active on the
solidified medium used to screen for light producing clones. This
may have lead to selection of a colony containing the initial
plasmid. Furthermore, there may have been poor plasmid DNA yield,
poor cutting, and/or poor religations when done in parallel in
microplates. This may have lead to DNA concentrations in the
ligations such that intermolecular ligations were minimized or such
that a low number of transformant colonies resulted and rare
transformants with the inverted insert DNA were not represented.
Thus, this approach of restriction digestion, religation,
transformation and selection of light producing transformants has
been demonstrated to work. However, further optimization for
efficient parallel implementation is required.
[0309] To test inverting of a chromosomal segment in a plasmid
containing divergent promoters, the backward fusion to yebF was
chosen. Obtaining an operably linked reporter gene fusion to yebF
was of particular interest because the yebF gene is an otherwise
unknown open reading frame that was found in a DNA microarray
experiment to be highly induced by mitomycin C, a DNA damaging
agent. In this case, a divergent promoter to the purT gene was
present in the chromosomal fragment and strains containing the
backward yebF fusion produced light. Following XmaI digestion,
religation and transformation, 10% of the transformants produced
light. Of these, 20% (or 2% of the initial transformants) were
found to be highly induced by nalidixic acid, another DNA damaging
agent, This result strongly suggesting that the desired inversion
of the chromosomal segment had occurred. This was verified by DNA
sequence analysis. Thus, inverting chromosomal segments containing
divergent promoters is possible and is facilitated if the two
promoters can be distinguished by their biological activity.
[0310] An alternative inverting procedure uses plasmid DNA from
clones requiring reorientation as templates for the polymerase
chain reaction (PCR). Universal PCR primers were designed which
hybridize specifically with the pDEW201 plasmid flanking the
genomic fragment cloning site (Bam HI). Use of the first primer,
pDEWE2S (SEQ ID NO:3, 5'-GGAATTGGGGATCGGAGCTCCCGGG-3'), an EcoRI
site (GAATTC) is converted to a SacI site (GAGCTC) via an internal
AT to GC mismatch. Use of the second primer, pDEWS2E (SEQ ID NO:4,
5'-GAATGGCGCGAATTCGGTACCCGGG-3'), results in the conversion of the
SacI site to an EcoRI site via an internal GC to AT conversion.
Thus the resultant PCR products from any pDEW201 clone can be
digested with EcoRI and SacI and ligated into EcoRI- and
SacI-digested pDEW201. The resultant plasmid contains the original
chromosomal segment in the opposite orientation of the original
clone, relative to the lux operon. As the primers are specific to
the vector they can be used for all clones and are amenable to high
throughput (96-well plate) approaches.
[0311] Construction, Sequencing and Registering an Additional
Random Library of E. coli Genomic Fragments Fused to a luxCDABE
Reporter.
[0312] An independent random library of E. coli genomic fragments
in plasmid pDEW201 was constructed and called the LuxZ library.
Chromosomal DNA isolated from E. coli strain W3110 (Ernsting et
al., (1992) J. Bacteriol. 174:1109-1118) was partially digested
with the restriction enzyme Sau3A1, size fractionated by agarose
gel electrophoreses, and a fraction with an average size of
approximately 0.7 KB isolated. This fraction was ligated to pDEW201
that had previously been digested with BamHI and treated with calf
intestinal alkaline phosphatase. The ligation products were used to
transform ultracompetent E. coli XL2Blue cells (Stratagene) to
ampicillin resistance using the protocol provided by Stratagene.
Preliminary characterization of individual XL2Blue transformants
that were picked in random indicated that a large percentage (16 of
16) contained insert DNA with sizes ranging from 0.1 to 1.5 KB.
Approximately 150,000 of these transformants were pooled and used
as a source of heterogeneous LuxZ library plasmid DNA isolated
using Qiagen tip20 columns (Qiagen Corp). This plasmid DNA pool was
diluted 100-fold then used to transform E. coli DPD1675 (Van Dyk
and LaRossa (1998) Methods in Molecular Biology: Bioluminescence
Methods and Protocols, Humana Press inc. vol. 102:85-95) by
electroporation. Electrocompetent DPD1675 cells were prepared
starting with 1 liter of LB culture at approximately
2.4.times.10.sup.8 cells/ml. Following chilling on ice, the cells
from this culture were collected by centrifugation at an average
relative centrifugal force of 6,555 for 5 minutes. The cells were
washed twice ice-cold, sterile distilled water (1 liter for the
first wash and 500 ml for the second wash) and centrifuged. Then
the cells were washed with 10 ml of ice-cold, sterile 10% glycerol
in distilled water and centrifuged. The final resuspension of the
cell pellet was done with ice-cold, sterile 10% glycerol in
distilled water to yield a final volume of 2.1 ml. 55 .mu.l
aliquots were prepared, quick frozen in dry ice and ethanol, then
stored at -80.degree. C. until used. One such aliquot was thawed on
ice, 1.0 .mu.l of the diluted LuxZ library plasmid DNA was added,
and the cells and plasmid DNA were transferred to an
electroporation cuvette (Biorad, Gene Pulser.RTM./E. coli
Pulser.TM.) on ice. The capped cuvette was electroporated with 1.85
volts at a capacitance of 25 .mu.F then 0.5 ml of SOC medium (per
liter: 20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 2.5 mM KCl,
2.5 mM MgCl.sub.2, 20 mM glucose, pH 7.0) was added. A 30 minute
phenotypic expression time at 37.degree. C. was used to minimize
the presence of siblings. Following this incubation, 500 .mu.l of
24% sterile glycerol in water was added and 100 .mu.l aliquots were
prepared, frozen on dry ice, and stored at -80.degree. C. until
used. These aliquots were thawed, plated on LB medium containing
ampicillin, and 4608 single colonies were picked for use as
inoculum for cultures grown in 80 .mu.l 1.times. freezing medium
(LB with 100 .mu.g/ml ampicillin, 5.54% glycerol, 36 mM
K.sub.2HPO.sub.4, 13.2 mM KH.sub.2PO.sub.4, 1.7 mM
NaC.sub.6H.sub.5O.sub.7:2H.sub.2O, 40 mM MgSO.sub.4, 6.8 mM
NH.sub.4SO.sub.4) in the wells of 384 well microplates. These
plates were stored at -80.degree. C. until used for inoculation and
growth to saturation in Terrific Broth (Gibco-BRL, Inc) containing
100 .mu.g/ml ampicillin in 96 well deep-well plates. Plasmid DNA
was extracted from the cultures using the Qiagen R.E.A.L..TM. prep
method with the following modification: after lysis of cells, the
plates were placed in a boiling water bath for 5 minutes and then
rapidly chilled in an ice-water bath before precipitation with
Buffer R3. This modification prevented degradation of the plasmid
DNA by the nucleases present in the non-endA host strain, DPD1675.
DNA sequencing reactions were performed with approximately 1 .mu.g
of plasmid DNA under standard ABI Prism.TM. dRhodamine
DyeTerminator Ready Reaction conditions with the primers
pDEW201.forward (SEQ ID NO:1, 5'-GGATCGGAATTCCCGGGGAT-3') and
pDEW201.reverse (SEQ ID NO:2, 5'-CTGGCCGTTAATAATGAATG-3') to obtain
sequence information from each end of the insert. DNA sequences
were determined on ABI377.TM.-XL 96-lane upgraded Sequencers under
4.times. run conditions on 5% PAG (polyacrylamide gel)
LongRanger.TM. (FMC, Inc.) gels and analyzed with ABI software. DNA
sequences were transferred to a UNIX based utility for further
analysis. The homology search for the sequence from the beginning
and end of each Lux-Z clone (in both orientations) and registration
to the E. coli genome was done as described in Example 1 resulting
in 1799 additional genome-registered gene fusions.
Example 7
Lux Array 1.04, a Genome-Wide, Promoter Activities Assay in Liquid
Medium, and its Use to Discover a Limonene Sensor
[0313] Selection of Additional, Non-Redundant Lux Gene Fusions to
Expand LuxArray 1.0.
[0314] Operably linked gene fusions were selected from the
genome-registered LuxZ gene fusion collection described in Example
6 using the computational filter described in Example 5. This list
was then compared with the current list of promoters contained in
Lux Array 1.0 to eliminate redundancy. This process resulted in
identification of 149 gene fusions useful to expand the
LuxArray.
[0315] In addition, the five gene fusions generated by inversion of
previously sequenced gene fusions as described in Example 6 and six
gene fusions from other sources were added. Table 19 lists these
additional 160 gene fusions that together with the gene fusions in
LuxArray 1.0 yielded LuxArray 1.04. The 849 gene fusions in Lux
Array 1.04 thus represent 33% of the 2584 known and predicted
transcriptional units in the E. coli genome. Individual cultures of
strains containing the newly identified gene fusions were added to
empty wells of the existing microplate number 15 and new
microplates numbered 17 to 19 were made with duplicated side by
side symmetry. The former microplate 16 was eliminated because it
contained only strains with the parental plasmid and sterile
controls. Thus, the Lux Array 1.04 is contained in 18 microplates
representing the cellular array. These were stored at -80.degree.
C. in freezing medium.
[0316] Limonene Stress of the Reporter Array in Liquid Culture.
[0317] Microplates from -80.degree. C. containing the LuxArray 1.04
E. coli strains were thawed and used to inoculate 100 .mu.l LB
medium supplemented with 100 .mu.g/ml ampicillin in Costar#3595
flat bottomed 96-well microplates. Following overnight growth at
37.degree. C., the plates were visually examined and any wells with
little or no turbidity were recorded. Then these cultures were
diluted by transfer of 15 .mu.l to 135 .mu.l LB medium in Costar
#3595, flat bottom 96-well microplates and were moderately swirl
shaken on an IKA Schuttler MTS 4 shaker at setting 400 for two hrs
at 37.degree. C. in a humidified box. Twenty .mu.l of each these
actively growing cultures were transferred to the corresponding
well in sets of three microplates, one containing 80 .mu.l of LB
medium, another containing 80 .mu.l of LB medium saturated with
limonene at room temperature, and the third containing 40 .mu.l of
LB medium saturated with limonene and 40 .mu.l of LB medium. An
initial (zero time) reading was taken using an MLX microplate
luminometer (Dynex) pre-warmed to 37.degree. C. Then the plates
were placed at 37.degree. C. without shaking. Additional
bioluminescent measurements were taken at 45, 90, and 135 minutes.
To limit the number of microplates handled each day, LuxArray 1.04
microplates 1 to 5, microplates 6 to 10, microplates 11 to 15, and
microplates 17 to 19 were used on each of four different days.
[0318] Data Analysis of Limonene Induced Responses.
[0319] The data were normalized to correct for growth inhibition
caused by limonene on the basis of each individual day that data
was collected. The bioluminescence (RLU) of each individual culture
for each day of experimentation under each of the three conditions,
were added to determine the daily array total RLU. These were used
to find a daily normalization factor (DNF) as follows:
DNF=Daily total array signal (time zero, LB control)/Daily total
array signal (time x, condition y)
[0320] Each measurement from the corresponding day, time point, and
condition was multiplied by DNF to yield a normalized signal. The
data from wells that were scored to have little or no growth were
not used in further analysis. The normalized data were analyzed
with GeneSpring (Silicon Genetics) software, which averaged the
signal from each of the duplicate cultures for each reporter gene
fusion.
[0321] Overall, there was little to no upregulation of gene
expression under the condition of 40% saturated limonene in LB
medium. Thus, patterns of gene expression from the 80% saturated
limonene in LB medium were further examined. Lists were made of
gene fusions that were upregulated by 2 fold or more at each time
point. These lists were examined and gene fusions with very low
normalized RLU values (<0.012 RLU) at all conditions were
eliminated. The upregulated gene fusions are listed in Table 20,
which uses the name of the gene in each operon with the smallest b#
to identify each gene fusion. The designation of ">2.times." is
given for upregulation while "*" is listed if the expression was
less than 2 fold increased. At 45 minutes, the expression of
fourteen gene fusions was increased; none of these was increased 3
fold or higher. At 90 minutes, 37 gene fusions were induced; these
included all but one of the gene fusions observed at 45 minutes.
However, the most highly upregulated gene fusions at 90 minutes
were not previously observed to be upregulated at 45 minutes. These
were fusions of the luxCDABE reporter to the promoter regions of
uhpT (3.8.times.), nirB (3.7.times.) and narK (3.6.times.). At 135
minutes, 39 gene fusions were induced, which included 13 fusions
not observed at 45 or 90 minutes. At this time point, the most
highly upregulated gene fusions were those to the promoter regions
of uhpT (7.7.times.), nirB (4.1.times.) and katG (4.1.times.).
Thus, the gene fusion to the promoter region of uhpT was the most
highly upregulated at both the 90 and 135 minute time points.
[0322] Verification of uhpT-Lux Upregulation by Limonene.
[0323] E. coli strain DPD3228 is identical to lux-a.pk034.b9 the
strain containing the uhpT-luxCDABE gene fusion. This strain was
grown overnight at 37.degree. C. in LB medium containing 100
.mu.g/ml ampicillin and diluted the following day into LB medium
and grown to log phase at 37.degree. C. 20 .mu.l aliquots of this
culture were added to 80 .mu.l of LB medium saturated with
limonene, to 80 .mu.l of a series of two fold dilutions into LB
medium of LB saturated with limonene, and to 80 .mu.l of LB medium.
Light production was measured in a Luminoskan Ascent (Labsystems)
microplate luminometer at 37.degree. C. FIG. 10 shows the result.
Similar to the initial LuxArray observations, a late response to
the presence of limonene was observed. This response was maximal at
the highest concentration of limonene tested, but was also detected
when limonene was present at 40% or 20% of saturating amounts in LB
medium. Thus, the upregulation of bioluminescence from this strain
by limonene was confirmed, demonstrating its utility as a sensor
for limonene.
[0324] Scheme 1: PERL Script Used to Filter the Lux Clone
Collection for Functional Reporter Constructs
18 #Filename:luxfilter2 #This perl script is designed to compare an
operon list #containing genome coordinates and directionality #and
clone ID to a list of clones containing the same and output #their
intersect to a new file open (OPERONS, "< operons_gg.txt")
.parallel. die "can't open operon list: $!"; open (OUTPUT, ">
full_list2.txt") .parallel. die "can't open output file: $!"; open
(CLONES, "< luxclones.txt") .parallel. die "can't open clone
list: $!"; @clones = <CLONES>; close CLONES; while ($line =
<OPERONS>){ print OUTPUT $line; print $line; if ($line
=.about. /complement/) { ($start) = $line =.about.
/.backslash.d+.backslash...backslash.. (.backslash.d+)/; ($op_end)
= $line =.about. /(.backslash.d+).backslash...backslash...ba-
ckslash.d+/; $promoter = $start+50; foreach $clone (@clones) { if
($clone =.about. /.backslash.t-.backslash.t/) { ($clone_start) =
$clone =.about.
/.backslash.d+.backslash.t(.backslash.d+).backslash.t.backslash.d+/;
($clone_end) = $clone =.about.
/(.backslash.d+).backslash.t.backslash.d+.backslash.t.backslash.d+/;
if (($clone_start > $promoter) && 10 ($clone_end >
$op_end) && ($clone_end < $start)) { print OUTPUT
$clone; print $clone; } } } } else { ($start) = $line =.about.
/(.backslash.d+).backslash...backslash...backslash.d+/; ($op_end) =
$line =.about. /.backslash.d+.backslash...backslash..(.back-
slash.d+)/; $promoter = $start-50; foreach $clone (@clones) { if
($clone =.about. /.backslash.+/) { ($clone_start) = $clone =.about.
/(.backslash.d+).backslash.-
t.backslash.d+.backslash.t.backslash.d+/; ($clone_end) = $clone
=.about. /.backslash.d+.backslash.t(.backslash.d+-
).backslash.t.backslash.d+/; if (($promoter > $clone_start)
&& ($clone_end < $op_end) && ($clone_end >
$start)) { print OUTPUT $clone; print $clone; } } } } } close
OUTPUT; close OPERONS;
[0325]
19TABLE 18 Luxarray 1.0 Clone Collection Coding Lux ID strand
Operon lux-lacZ - complement(360473 . . . 365529) lux-a.pk007.b9 -
Operon complement(3399029 . . . 3400969) /note = "predicted operon"
/note = "ordered genes contained in the operon: yhdA "
lux-a.pk0017.h6 + Operon (510865 . . . 513092) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0485 b0486
" lux-a.pk0022.c6 - Operon complement(2325387 . . . 2334712) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b2225 b2226 b2227 b2228 b2229 yfaA " lux-a.pk0026.g3 -
Operon complement(120178 . . . 121551) /note = "documented aroP
operon" lux-a.pk0032.g2 + Operon (4161218 . . . 4171626) /note =
"predicted operon" /note = "ordered genes contained in the operon:
btuB murI murB birA " lux- - Operon complement(1067734 . . .
1073234) /note = "predicted a.pk0037.d11 operon" /note = "ordered
genes contained in the operon: b1006 b1007 b1008 b1009 b1010 b1011
b1012 " lux- + Operon (342108 . . . 343157) /note = "predicted
operon" /note a.pk0041.f10 = "ordered genes contained in the
operon: b0325 " lux-a.pk047.d10 Operon complement(4592507 . . .
4593313) /note = "predicted operon" /note = "ordered genes
contained in the operon: yjjM " lux-a.pk052.f3 + Operon (3246594 .
. . 3248016) /note = "predicted operon" /note = "ordered genes
contained in the operon: yqjC yqjD yqjE b3100 " lux-a.pk058.f5 -
Operon complement(2820162 . . . 2820662) /note = "predicted operon"
/note = "ordered genes contained in the operon: oraA"
lux-a.pk066.a3 - Operon complement(1998496 . . . 2001629) /note =
"predicted operon" /note = "ordered genes contained in the operon:
fliZ fliA fliC " lux-a.pk072.e2 + Operon (2543793 . . . 2547426)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2428 b2429 b2430 " lux-a.pk078.c5 - Operon
complement(3117613 . . . 3119295) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2975 " lux-a.pk086.e2 -
Operon complement(4311389 . . . 4322743) /note = "documented
phnCDE-b4103-phnFGHIJKLMNOPQ operon" lux- - Operon
complement(1734145 . . . 1735314) /note = "predicted a.pk0001.a11
operon" /note = "ordered genes contained in the operon: b1657 "
lux-a.pk007.c1 - Operon complement(2255449 . . . 2257316) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yeiN yeiC " lux-a.pk0018.a4 + Operon (3714927 . . . 3715913) /note
= "predicted operon" /note = "ordered genes contained in the
operon: yiaE" lux-a.pk0022.d3 + Operon (801110 . . . 802543) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b0770 " lux-a.pk026.e11 + Operon (84191 . . . 87848) /note
= "documented leuO-ilvIH operon" lux-a.pk032.g4 + Operon (214291 .
. . 215979) /note = "predicted operon" /note = "ordered genes
contained in the operon: yaeQ yaeJ cutF " lux-a.pk037.d4 + Operon
(89634 . . . 103153) /note = "predicted operon" /note = "ordered
genes contained in the operon: yabB yabC ftsL ftsI murE murF mraY
murD ftsW murG murC ddlB " lux-a.pk041.f12 + Operon (3542470 . . .
3543201) /note = "predicted operon" /note = "ordered genes
contained in the operon: yhgH " lux-a.pk047.d4 - Operon
complement(3334196 . . . 3334459) /note = "predicted operon" /note
= "ordered genes contained in the operon: yrbA" lux-a.pk052.g10 +
Operon (4233811 . . . 4237309) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjbF yjbG yjbH"
lux-a.pk058.h10 - Operon complement(578407 . . . 578859) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0558 " lux-a.pk066.b10 - Operon complement(4297143 . . . 4300516)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yjcPyjcQ " lux-a.pk072.e3 + Operon (797809 . . . 7988040
/note = "predicted operon" /note = "ordered genes contained in the
operon: ybhE " lux-a.pk078.d2 - Operon complement(1336594 . . .
1337184) /note = "predicted operon" /note = "ordered genes
contained in the operon: ribA" lux-a.pk086.h2 - Operon
complement(3390094 . . . 3393895) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhdP yhdR " lux-lacZ
complement(360473 . . . 365529) lux-a.pk007.d12 - Operon
complement(2111456 . . . 2112349) /note = "predicted operon" /note
= "ordered genes contained in the operon: galF" lux-a.pk0018.a6 +
Operon (4140109 . . . 4141523) /note = "predicted operon" /note =
"ordered genes contained in the operon: frwC frwB " lux- + Operon
(2693959 . . . 2695377) /note = "predicted operon" a.pk0022.g11
/note = "ordered genes contained in the operon: yfhD "
lux-a.pk027.b11 - Operon complement(3598659 . . . 3601874) /note =
"documented ftsYEX operon" lux-a.pk032.h3 + Operon (274525 . . .
276871) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0260 b0261 " lux-a.pk037.d6 + Operon (4401964 . . .
4402161) /note = "predicted operon" /note = "ordered genes
contained in the operon: b4176 " lux-a.pk041.g11 - Operon
complement(2904665 . . . 2905963) /note = "predicted operon" /note
= "ordered genes contained in the operon: eno" lux-a.pk047.e10 +
Operon (4152580 . . . 4155802) /note = "documented argCBH operon"
lux-a.pk052.g3 - Operon complement(3852741 . . . 3853934) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yidF yidG yidH " lux-a.pk059.a8 + Operon (621523 . . . 622773)
/note = "predicted operon" /note = "ordered genes contained in the
operon: ybdA " lux-a.pk066.b12 + Operon (284619 . . . 287623) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b0270 b0271 " lux-a.pk072.f11 - Operon complement(2061410 .
. . 2063786) /note = "documented cobUST operon" lux-a.pk078.d7 -
Operon complement(3092119 . . . 3093144) /note = "predicted operon"
/note = "ordered genes contained in the operon: b2950 "
lux-a.pk087.b5 - Operon complement(1952602 . . . 1956156) /note =
"predicted operon" /note = "ordered genes contained in the operon:
bisZ b1873 " lux-a.pk0001.a2 - Operon complement(3767870 . . .
3769371) /note = "predicted operon" /note = "ordered genes
contained in the operon: yibH yibI " lux-a.pk007.d2 - Operon
complement(2506481 . . . 2507446) /note = "predicted operon" /note
= "ordered genes contained in the operon: glk " lux-a.pk0018.b1 +
Operon (4048927 . . . 4049436) /note = "predicted operon" /note =
"ordered genes contained in the operon: yihI " lux-a.pk0022.g7 -
Operon complement(783105 . . . 784046) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0752 "
lux-a.pk027.c11 + Operon (1779419 . . . 1782701) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1699 b1700
ydiD " lux-a.pk033.a3 - Operon complement( 1978212 . . . 1980411)
/note = "documented otsAB operon" lux-a.pk037.e3 - Operon
complement(3290116 . . . 3290976) /note = "predicted operon" /note
= "ordered genes contained in the operon: yraL" lux-a.pk041.g5 -
Operon complement(1551996 . . . 1553720) /note = "predicted operon"
/note = "ordered genes contained in the operon: sfcAft
lux-a.pk047.e11 + Operon (2932257 . . . 2938121) /note =
"documented fucPIKUR operon" lux-a.pk052.g6 - Operon
complement(317900 . . . 319252) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0304 " lux-a.pk059.b4 -
Operon complement(3302214 . . . 3303458) /note = "documented mtr
operon" lux-a.pk066.c7 + Operon (87860 . . . 89032) /note =
"predicted operon" /note = "ordered genes contained in the operon:
fruL fruR " lux-a.pk072.f2 - Operon complement(3553466 . . .
3555711) /note = "predicted operon" /note = "ordered genes
contained in the operon: yhgj yhgK yhgL " lux-a.pk078.d9 + Operon
(4257900 . . . 4258885) /note = "predicted operon" /note = "ordered
genes contained in the operon: yjbL yjbM " lux-a.pk087.b7 + Operon
(594823 . . . 596196) /note = "predicted operon" /note = "ordered
genes contained in the operon: b0572 " lux-lacZ complement(360473 .
. . 365529) lux-a.pk007.d3 + Operon (794312 . . . 796835) /note =
"documented modABC operon" lux- + Operon (1635056 . . . 1635481)
/note = "predicted operon" a.pk0018.b10 /note = "ordered genes
contained in the operon: b1549 " lux-a.pk0022.h4 + Operon (816267 .
. . 818970) /note = "documented moaABCDE operon" lux-a.pk027.d12 +
Operon (1879936 . . . 1881021) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1800 " lux-a.pk033.a4 +
Operon (4213057 . . . 4217911) /note = "documented aceBAK operon"
lux-a.pk037.e6 + Operon (2232053 . . . 2234520) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2146 yeiA
" lux-a.pk041.h8 + Operon (2493599 . . . 2494585) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2378 " lux-a.pk047.e8 - Operon complement(2239830 . . . 2241672)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yeiB folE " lux-a.pk052.h2 - Operon complement(3748758 . .
. 3749498) /note = "predicted operon" /note = "ordered genes
contained in the operon: yiaT " lux-a.pk059.c12 - Operon
complement(3944752 . . . 3945590) /note = "predicted operon" /note
= "ordered genes contained in the operon: yifA pssR "
lux-a.pk066.c9 + Operon (3500404 . . . 3502421) /note = "predicted
operon" /note = "ordered genes contained in the operon: yhfP yhfQ
yhfR" lux-a.pk072.f7 - Operon complement(1577657 . . . 1580581)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1497 b1498" lux-a.pk078.e3 + Operon (2680877 . . .
2682076) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2550 " lux-a.pk087.c1 - Operon
complement(1234932 . . . 1236464) /note = "predicted operon" /note
= "ordered genes contained in the operon: ycgB" lux-a.pk0001.b6 -
Operon complement(4268628 . . . 4271450) /note = "documented uvrA
operon" lux-a.pk007.e12 + Operon (4248534 . . . 4249862) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjbI " lux-a.pk0018.c7 Operon complement(2245083 . . . 2246552)
/note = "predicted operon" /note = "ordered genes contained in the
operon: lysP " lux-a.pk0022.h5 + Operon (2363915 . . . 2371298)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2253 b2254 b2255 b2256 b2257 b2258 " lux-a.pk027.e11 -
Operon complement( 1762958 . . . 1766709) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1685 b1686
b1687 " lux-a.pk033.a6 + Operon (3676830 . . . 3677978) /note =
"predicted operon" /note = "ordered genes contained in the operon:
kdgK " lux-a.pk037.e9 Operon complement(2097884 . . . 2099290)
/note = "predicted operon" /note = "ordered genes contained in the
operon: gnd " lux-a.pk042.a6 - Operon complement(4228938 . . .
4229210) /note = "predicted operon" /note = "ordered genes
contained in the operon: yjbD" lux-a.pk047.f2 - Operon
complement(280053 . . . 281207) /note = "predicted operon" /note =
"ordered genes contained in the operon: yagA " lux-a.pk052.h9 -
Operon complement(2769861 . . . 2770706) /note = "predicted operon"
/note = "ordered genes contained in the operon: b2638 b2639"
lux-a.pk060.a10 + Operon (3432844 . . . 3435531) /note = "predicted
operon" /note = "ordered genes contained in the operon: fmu trkA "
lux-a.pk066.d11 + Operon (4411838 . . . 44134780 /note = "predicted
operon" /note = "ordered genes contained in the operon: aidB "
lux-a.pk072.g1 + Operon (3483757 . . . 3484389) /note = "documented
crp operon" lux-a.pk078.f11 + Operon (3491648 . . . 3496838) /note
= "documented nirBDC- cysG operon" lux-a.pk087.c4 - Operon
complement(1232399 . . . 1233940) /note = "predicted operon" /note
= "ordered genes contained in the operon: nhaB" lux-a.pk0001.c2 -
Operon complement(1694486 . . . 1695076) /note = "predicted operon"
/note = "ordered genes contained in the operon: gusR"
lux-a.pk007.e7 + Operon (2837547 . . . 2840437) /note = "documented
ascFB operon" lux-a.pk0018.d2 - Operon complement(605488 . . .
606606) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0581 " lux- + Operon (2088214 . . . 2095247) /note
= "documented a.pk0023.c11 hisGDCBHAFI operon" lux-a.pk027.e3 -
Operon complement(344890 . . . 345561) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0328 "
lux-a.pk033.c5 + Operon (1808223 . . . 1808825) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1728 "
lux-a.pk037.f1 - Operon complement(2474714 . . . 2475649) /note =
"documented dsdC operon" lux-a.pk042.b3 - Operon complement(1838807
. . . 1839433) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1758" lux-a.pk047.f7 + Operon (2861616 .
. . 2863035) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2738 b2739" lux-a.pk053.b5 - Operon
complement(464836 . . . 466536) /note = "predicted operon" /note =
"ordered genes contained in the operon: ybaE " lux-a.pk060.a6 +
Operon (2775136 . . . 2775803) /note = "predicted operon" /note =
"ordered genes contained in the operon: b2645 b2646 "
lux-a.pk066.f11 - Operon complement(820765 . . . 823720) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0788 b0789 b0790 " lux-a.pk072.g5 + Operon (1418389 . . . 1421668)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1357 b1358 b1359 b1360 b1361 b1362" lux-a.pk079.b10 -
Operon complement(177001 . . . 179153) /note = "predicted operon"
/note = "ordered genes contained in the operon: yadS yadT pfs "
lux-a.pk087.c6 - Operon complement(2992482 . . . 2993114) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2855 b2856" lux-a.pk0001.c7 + Operon (2857783 . . . 2858439) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b2734 " lux-a.pk007.f11 - Operon complement(4429669 . . .
4430643) /note = "predicted operon" /note = "ordered genes
contained in the operon: ytfF" lux-a.pk0018.d7 - Operon
complement(3077663 . . . 3079654) /note = "predicted operon" /note
= "ordered genes contained in the operon: tktA " lux-a.pk023.c3 +
Operon (3632372.3633523) /note = "predicted operon" /note =
"ordered genes contained in the operon: yhiM " lux-a.pk027.e7 +
Operon (997713 . . . 1003880) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0939 b0940 b0941 b0942
b0943 ycbF " lux-a.pk033.d5 + Operon (4100373 . . . 4101077) /note
= "predicted operon" /note = "ordered genes contained in the
operon: yiiM " lux-a.pk037.f5 - Operon complement(2908778 . . .
2909361) /note = "predicted operon" /note = "ordered genes
contained in the operon: chpA chpR " lux-a.pk042.b7 + Operon
(3237584 . . . 3238828) /note = "predicted operon" /note = "ordered
genes contained in the operon: ygjU " lux-a.pk047.g9 - Operon
complement(2083726 . . . 2085090) /note = "predicted operon" /note
= "ordered genes contained in the operon: yeeF " lux-a.pk053.b8 +
Operon (3841591 . . . 3843357) /note = "predicted operon" /note =
"ordered genes contained in the operon: yicP " lux-a.pk060.b12 -
Operon complement(1325791 . . . 1327136) /note = "predicted operon"
/note = "ordered genes contained in the operon: btuR yciK "
lux-a.pk066.g9 -
Operon complement(1889349 . . . 1891259) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1808"
lux-a.pk073.a11 - Operon complement(439426 . . . 440567) /note =
"documented xseB-ispA operon" lux-a.pk079.e5 - Operon
complement(346081 . . . 347667) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0330 " lux-a.pk087.d4 +
Operon (252005 . . . 253161) /note = "predicted operon" /note =
"ordered genes contained in the operon: yafN yafO yafP " lux- -
Operon complement(2591092 . . . 2594757) /note = "predicted
a.pk0001.d10 operon" /note = "ordered genes contained in the
operon: b2473 b2474 b2475 " lux-a.pk008.c11 - Operon
complement(2420669 . . . 2421559) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2305 " lux-a.pk0018.f1 +
Operon (980270 . . . 982117) /note = "predicted operon" /note =
"ordered genes contained in the operon: ycbB " lux-a.pk0023.c6 +
Operon (774376 . . . 778255) /note = "documented tolQRAB operon"
lux-a.pk027.f6 - Operon complement(691097 . . . 692640) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0659 b0660 " lux-a.pk033.e12 - Operon complement(2112524 . . .
2116426) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2043 b2044 b2045 " lux-a.pk037.h4 -
Operon complement(4343258 . . . 4344904) /note = "documented fumB
operon" lux-a.pk042.c7 + Operon (3416786 . . . 3418859) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yhdW yhdX " lux-a.pk048.c7 + Operon (384399 . . . 387870) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0365 b0366 b0367 b0368 " lux-a.pk053.d10 + Operon (4010643 . . .
4012904) /note = "documented metE operon" lux-a.pk060.b6 + Operon
(4349421 . . . 4349935) /note = "predicted operon" /note = "ordered
genes contained in the operon: yjdI yjdJ " lux-a.pk067.b10 - Operon
complement(2254105 . . . 2255355) /note = "predicted operon" /note
= "ordered genes contained in the operon: yeiM " lux-a.pk073.d7 -
Operon complement( 1990897 . . . 1992663) /note = "predicted
operon" /note = "ordered genes contained in the operon: uvrC "
lux-a.pk079.f10 - Operon complement(4452185 . . . 4453183) /note =
"predicted operon" /note = "ordered genes contained in the operon:
fbp" lux-a.pk087.e2 + Operon (2278652 . . . 2280412) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yejH " lux- - Operon complement(59687 . . . 63264) /note =
"predicted a.pk0001.f10 operon" /note = "ordered genes contained in
the operon: yabO hepA " lux-a.pk008.e12 + Operon (71351 . . .
72115) /note = "predicted operon" /note = "ordered genes contained
in the operon: yabI " lux- + Operon (2890237 . . . 2892794) /note =
"predicted operon" a.pk0018.f11 /note = "ordered genes contained in
the operon: b2765 b2766 b2767 b2768" lux-a.pk0023.d2 + Operon
(15445 . . . 16557) /note = "predicted operon" /note = "ordered
genes contained in the operon: yi81_1 " lux-a.pk027.h4 - Operon
complement(4590931 . . . 4592292) /note = "predicted operon" /note
= "ordered genes contained in the operon: yjiZ " lux-a.pk033.e2 +
Operon (471822 . . . 473476) /note = "predicted operon" /note =
"ordered genes contained in the operon: glnK amtB " lux-a.pk037.h9
- Operon complement(904963 . . . 906012) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0868 "
lux-a.pk042.c8 + Operon (3208422 . . . 3212529) /note = "documented
rpsU- dnaG-rpoD operon" lux-a.pk048.d11 - Operon complement(1844989
. . . 1846032) /note = "documented selD operon" lux-a.pk053.d5 +
Operon (4414530 . . . 4415279) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjfP " lux-a.pk060.e5 +
Operon (3344219 . . . 3345879) /note = "predicted operon" /note =
"ordered genes contained in the operon: ptsN b3205 ptsO "
lux-a.pk067.d11 - Operon complement(2715511 . . . 2716548) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yfiFIf lux-a.pk073.f10 + Operon (3637741 . . . 3638175) /note =
"predicted operon" /note = "ordered genes contained in the operon:
uspA " lux-a.pk079.f4 + Operon (234798 . . . 235538) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0213 " lux-a.pk087.f10 - Operon complement(4308686 . . . 4309621)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yjcXII lux-a.pk0001.f2 - Operon complement(2426077 . . .
2429677) /note = "documented dedD-cvpA-purF-ubiX operon"
lux-a.pk009.a4 + Operon (3579494 . . . 3581811) /note = "predicted
operon" /note = "ordered genes contained in the operon: yhhZ b3443
insA_5 insB_5" lux-a.pk0018.g6 + Operon (2415080 . . . 2416621)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2298 " lux-a.pk0023.e3 + Operon (3223875 . . . 3225308)
/note = "predicted operon" /note = "ordered genes contained in the
operon: ygjI " lux-a.pk028.g6 - Operon complement(3663810 . . .
3665210) /note = "predicted operon" /note = "ordered genes
contained in the operon: gadA" lux-a.pk033.e6 - Operon
complement(3825087 . . . 3826292) /note = "predicted operon" /note
= "ordered genes contained in the operon: gltS" lux-a.pk038.a4 -
Operon complement(3623310 . . . 3628232) /note = "predicted operon"
/note = "ordered genes contained in the operon: yhhJ yhiG yhiI "
lux-a.pk042.d9 + Operon (1864932 . . . 1866866) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1783 "
lux-a.pk048.g4 + Operon (3029318 . . . 3030835) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2888 "
lux-a.pk053.d8 - Operon complement(2523950 . . . 2524876) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2409 " lux-a.pk060.e8 - Operon complement(740298 . . . 741779)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0709" lux-a.pk067.f8 - Operon complement(1921389 . . .
1922993) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1839 b1840 b1841 " lux-a.pk073.f3 -
Operon complement(1515906 . . . 1516870) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1447 b1448"
lux-a.pk079.g6 - Operon complement(988377 . . . 989579) /note =
"documented pncB operon" lux-a.pk087.g3 - Operon complement(3475544
. . . 3476134) /note = "predicted operon" /note = "ordered genes
contained in the operon: slyD " lux- + Operon (484985 . . . 485632)
/note = "predicted operon" /note a.pk0002.b12 = "ordered genes
contained in the operon: acrR " lux-a.pk009.a9 - Operon
complement(232597 . . . 233955)/note = "predicted operon" /note =
"ordered genes contained in the operon: dniR " lux-a.pk0018.h2 +
Operon (3782887 . . . 3785724) /note = "predicted operon" /note =
"ordered genes contained in the operon: yibO yibP " lux-a.pk023.f2
- Operon complement(2030406 . . . 2031524) /note = "predicted
operon" /note = "ordered genes contained in the operon: yedJ b1963
" lux-a.pk029.b2 + Operon (3972208 . . . 3978309) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yifH yifI yifJ rffT rffM " lux-a.pk033.f3 + Operon (945094 . . .
947882) /note = "predicted operon" /note = "ordered genes contained
in the operon: ycaD b0899 " lux-a.pk038.b10 + Operon (1036963 . . .
1041138) /note = "documented appCBA operon" lux-a.pk042.e10 -
Operon complement(3464797 . . . 3467490) /note = "predicted operon"
/note = "ordered genes contained in the operon: yheB "
lux-a.pk049.a4 - Operon complement( 1946774 . . . 1948546) /note =
"predicted operon" /note = "ordered genes contained in the operon:
aspS" lux-a.pk053.e9 - Operon complement(2077555 . . . 2078613)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yeeA " lux-a.pk060.g1 - Operon complement(1823979 . . .
1830006) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1744 b1745 b1746 b1747 b1748 "
lux-a.pk068.a3 - Operon complement(926697 . . . 930185) /note =
"documented cydCD operon" lux-a.pk073.h2 + Operon (3646158 . . .
3648292) /note = "documented arsRBC operon" lux-a.pk079.h9 - Operon
complement(1532989 . . . 1533882) /note = "predicted operon" /note
= "ordered genes contained in the operon: yddE " lux-a.pk088.c3 -
Operon complement(264844 . . . 266191) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0250 b0251 "
lux-lacZ complement(360473 . . . 365529) lux-a.pk009.b2 - Operon
complement(1397745 . . . 1402604) /note = "predicted operon" /note
= "ordered genes contained in the operon: ogt ydaH b1337 b1338"
lux- - Operon complement(3065360 . . . 3066100) /note = "predicted
a.pk0019.a12 operon" /note = "ordered genes contained in the
operon: yggE " lux-a.pk0023.h6 - Operon complement(1797417 . . .
1800594) /note = "documented thrS-infC-rpmI-rplT operon"
lux-a.pk029.b8 - Operon complement(3487903 . . . 3489257) /note =
"predicted operon" /note = "ordered genes contained in the operon:
pabA fic yhfG " lux-a.pk033.f6 + Operon (2380733 . . . 2381944)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2269 " lux-a.pk038.b6 + Operon (601182 . . . 602558) /note
= "predicted operon" /note = "ordered genes contained in the
operon: pheP " lux-a.pk042.g1 + Operon (2898614 . . . 2901396)
/note = "predicted operon" /note = "ordered genes contained in the
operon: + ygcE " lux-a.pk049.a8 + Operon (2556791 . . . 2558086)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2442 " lux-a.pk053.g7 - Operon complement(2180055 . . .
2180801) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2101 " lux-a.pk061.a5 - Operon
complement(3838176 . . . 3841494)/note = "predicted operon"/note =
"ordered genes contained in the operon: yicM yicN yicO "
lux-a.pk068.b3 - Operon complement(114407 . . . 117549) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0105 hofC hofB ppdD" lux-a.pk074.c11 + Operon (3555900 . . .
3557498) /note = "predicted operon" /note = "ordered genes
contained in the operon: yhgB " lux-a.pk080.d1 + Operon (209679 . .
. 212266) /note = "predicted operon" /note = "ordered genes
contained in the operon: IdcC b0187 " lux-a.pk088.c3 - Operon
complement(264844 . . . 266191) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0250 b0251 "
lux-a.pk0002.b7 - Operon complement(273325 . . . 274341) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yi52_1 " lux-a.pk009.c11 - Operon complement(3080896 . . . 3081816)
/note = "predicted operon"/note = "ordered genes contained in the
operon: speB " lux-a.pk0019.a8 + Operon (1896421 . . . 1898049)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1815 " lux-a.pk0024.a3 + Operon (1244383 . . . 1244823)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1196 " lux-a.pk029.c10 - Operon complement(1550422 . . .
1550784) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1477 " lux-a.pk033.g10 - Operon
complement(1566978 . . . 1568513) /note = "predicted operon" /note
= "ordered genes contained in the operon: xasA " lux-a.pk038.c2 +
Operon (1599514 . . . 1605313) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1513 b1514 b1515 b1516
b1517 b1518" lux-a.pk043.c1 - Operon complement(2929887 . . .
2931710) /note = "documented fucAO operon" lux-a.pk049.d8 - Operon
complement(5683 . . . 6459) /note = "predicted operon" /note =
"ordered genes contained in the operon: yaaA " lux-a.pk054.a6 -
Operon complement(4350778 . . . 4352295) /note = "documented lysU
operon" lux-a.pk061.b6 - Operon complement(1815172 . . . 1819643)
/note = "documented celABCDF operon" lux-a.pk068.b8 + Operon
(1473162 . . . 1475474) /note = "predicted operon" /note = "ordered
genes contained in the operon: ydbD " lux-a.pk074.d9 + Operon
(1903658 . . . 1904278) /note = "predicted operon" /note = "ordered
genes contained in the operon: b1821 " lux-a.pk080.e12 + Operon
(243543 . . . 244121) /note = "predicted operon" /note = "ordered
genes contained in the operon: gmhA " lux-a.pk088.d5 - Operon
complement(3105038 . . . 3107233) /note = "documented speC operon"
lux-lacZ complement(360473 . . . 365529) lux-a.pk009.d6 - Operon
complement(642780 . . . 643190) /note = "predicted operon" /note =
"ordered genes contained in the operon: rnk " lux-a.pk0019.a9 +
Operon (3484440 . . . 3486530) /note = "predicted operon" /note =
"ordered genes contained in the operon: yhfK " lux-a.pk0024.b6 +
Operon (122092 . . . 129336) /note = "documented pdhR- aceEF-lpdA
operon" lux-a.pk029.c11 + Operon (4506526 . . . 4507122) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b4285 " lux-a.pk033.g11 + Operon (1289465 . . . 1290478) /note =
"predicted operon" /note = "ordered genes contained in the operon:
hnr " lux-a.pk038.c7 + Operon (2609941 . . . 2612802) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2491 b2492 " lux-a.pk043.e11 - Operon complement(2885601 . . .
2889921) /note = "documented cysJIH operon" lux-a.pk049.g7 - Operon
complement(1886085 . . . 1887770) /note = "predicted operon" /note
= "ordered genes contained in the operon: fadD " lux-a.pk054.c10 -
Operon complement(3317629 . . . 3319272) /note = "predicted operon"
/note = "ordered genes contained in the operon: yhbX "
lux-a.pk061.b7 + Operon (180884 . . . 182308 /note = "predicted
operon" /note = "ordered genes contained in the operon: htrA "
lux-a.pk068.b9 + Operon (467520 . . . 471641 /note = "predicted
operon" /note = "ordered genes contained in the operon: b0447 mdlA
mdlB " lux-a.pk074.e3 - Operon complement(4594719 . . . 4596971)
/note = "predicted operon" /note = "ordered genes contained in the
operon: mdoB " lux-a.pk080.g3 + Operon (655780 . . . 656340) /note
= "predicted operon" /note = "ordered genes contained in the
operon: ybeG " lux-a.pk088.e1 - Operon complement(1932863 . . .
1934338) /note = "documented zwf operon" lux-a.pk0002.e4 + Operon
(1735868 . . . 1736893) /note = "documented purR operon"
lux-a.pk009.d11 - Operon complement(4363050 . . . 4364351) /note =
"predicted operon" /note = "ordered genes contained in the operon:
dcuA " lux- + Operon (2621064 . . . 2623130) /note = "documented
ppk a.pk0019.c11 operon" lux-a.pk0024.c7 - Operon complement(592551
. . . 594666) /note = "predicted operon" /note = "ordered genes
contained in the operon: b0570 b0571 " lux-a.pk029.c5 - Operon
complement(266408 . . . 267244) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0252 " lux-a.pk033.g2 -
Operon complement(2131512 . . . 2135.265) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2060 b2061
b2062" lux-a.pk038.d9 - Operon complement(65855 . . . 70048) /note
= "documented araBAD operon" lux-a.pk043.f11 + Operon (4200898 . .
. 4202223) /note = "predicted operon" /note = "ordered genes
contained in the operon: hydG " lux-a.pk049.h9 + Operon
(3352267.3359575) /note = "documented gltBDF operon" lux-a.pk054.c3
- Operon complement(2729620 . . . 2732193) /note = "predicted
operon" /note = "ordered genes contained in the operon: clpB"
lux-a.pk061.c3 + Operon (202560 . . . 208608) /note = "predicted
operon"
/note = "ordered genes contained in the operon: lpxA lpxB rnhB dnaE
" lux-a.pk068.h2 + Operon (103155 . . . 106456) /note = "documented
ftsQAZ operon" lux-a.pk074.f1 + Operon (2550372 . . . 2552144)
/note = "predicted operon" /note = "ordered genes contained in the
operon: amiA hemF" lux-a.pk081.b5 + Operon (3728760 . . . 3733786)
/note = "documented xylFGHR operon" lux-a.pk088.g2 + Operon (882896
. . . 884128) /note = "predicted operon" /note = "ordered genes
contained in the operon: b0842 " lux-lacZ complement(360473 . . .
365529) lux-a.pk009.f3 - f176; This 176 aa ORF is 45 pct identical
(2 gaps) to 172 residues of an approx. 184 aa protein FIMF_ECOLI
SW: P08189 lux- - Operon complement(4061182 . . . 4066856) /note =
"predicted a.pk0019.d12 operon" /note = "ordered genes contained in
the operon: yihO yihO yihP yihQ " lux-a.pk0024.e1 - Operon
complement(2854476 . . . 2854829) /note = "predicted operon" /note
= "ordered genes contained in the operon: ygbA " lux-a.pk029.c5 +
Operon (2766686 . . . 2767507) /note = "predicted operon" /note =
"ordered genes contained in the operon: b2633 " lux-a.pk033.g7 +
Operon (2001895 . . . 2004101) /note = "documented fliDST operon"
lux-a.pk038.e11 + Operon (3785854 . . . 3786687) /note = "predicted
operon" /note = "ordered genes contained in the operon: yibQ "
lux-a.pk043.f6 - Operon complement(3040509 . . . 3041168) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2899 " lux-a.pk050.a11 - Operon complement(4437449 . . . 4438792)
/note = "predicted operon" /note = "ordered genes contained in the
operon: ytfL " lux-a.pk054.e11 - Operon complementer(3711690 . . .
3713909) /note = "predicted operon" /note = "ordered genes
contained in the operon: bisCII lux-a.pk061.d8 - Operon
complement(3148833 . . . 3149999) /note = "documented exbDB operon"
lux-a.pk068.h4 + Operon (3180566 . . . 3181339) /note = "predicted
operon" /note = "ordered genes contained in the operon: ygiE "
lux-a.pk074.g9 - Operon complement(4000900 . . . 4002326) /note =
"predicted operon" /note = "ordered genes contained in the operon:
rarDyigI " lux-a.pk081.b6 + Operon (879950 . . . 881152) /note =
"predicted operon" /note = "ordered genes contained in the operon:
dacC " lux-a.pk088.h3 + Operon (122092 . . . 129336) /note =
"documented pdhR-aceEF- pdA operon" lux-a.pk0002.g5 + Operon
(2342885 . . . 2346534) /note = "documented nrdAB operon"
lux-a.pk009.g3 - Operon complement(2181736 . . . 2183321) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2103 b2104" lux-a.pk0019.e3 - Operon complement(4012721 . . .
4013719) /note = "predicted operon" /note = "ordered genes
contained in the operon: " lux-a.pk0024.e6 + Operon (893007 . . .
897152) /note = "documented potFGHI operon" lux-a.pk029.c9 + Operon
(89634 . . . 103153) /note = "predicted operon" /note = "ordered
genes contained in the operon: yabB yabC ftsL ftsI murE murF mraY
murD ftsW murG murC ddlB " lux-a.pk034.a11 - Operon
complement(2116702 . . . 2119576) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2046 b2047"
lux-a.pk038.f1 + Operon (1830452 . . . 1831258) /note = "predicted
operon" /note = "ordered genes contained in the operon: xthA "
lux-a.pk043.g5 - Operon complement(1957304 . . . 1957876) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1875 " lux-a.pk050.a12 - Operon complement(4338298 . . . 4339206)
/note = "documented melR operon" lux-a.pk054.e5 - Operon
complement(3858976 . . . 3861491)/note = "predicted operon"/note =
"ordered genes contained in the operon: glvG glvB glvC"
lux-a.pk061.e9 - Operon complement(2980519 . . . 2982146) /note =
"predicted operon" /note = "ordered genes contained in the operon:
kduD kduI " lux-a.pk069.a4 - Operon complement(3026544 . . .
3028966) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2886 b2887" lux-a.pk074.h10 + Operon
(4109895 . . . 4110335) /note = "predicted operon" /note = "ordered
genes contained in the operon: yiiR " lux-a.pk081.c2 - Operon
complement(4457474 . . . 4457938) /note = "predicted operon" /note
= "ordered genes contained in the operon: nrdG" lux-a.pk089.a11 +
Operon (1094746 . . . 1096052) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1028 b1029"
lux-a.pk0003.a2 + Operon (349236 . . . 353816) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0333 b0334
b0335 " lux-a.pk009.g7 - FecR protein lux-a.pk0019.e4 + Operon
(1303788 . . . 1304792) /note = "predicted operon" /note = "ordered
genes contained in the operon: oppF " lux-a.pk0024.f5 + Operon
(832293 . . . 835433) /note = "predicted operon" /note = "ordered
genes contained in the operon: dinG ybiB " lux-a.pk029.e7 - Operon
complement(1852120 . . . 1852878) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1770 " lux-a.pk034.b8 +
Operon (4126252 . . . 4129847) /note = "documented metBL operon"
lux-a.pk038.f3 - Operon complement(303719 . . . 309250) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0289 b0290 b0291 b0292 " lux-a.pk043.h1 + Operon (2383874 . . .
2384851) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2271 " lux-a.pk050.b9 - Operon
complement(360473 . . . 365529) /note = "documented lacAYZ operon"
lux-a.pk054.f7 + Operon (1057307 . . . 1061621) /note = "documented
torCAD operon" lux-a.pk061.f10 - Operon complement(2612840 . . .
2613901) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2493 " lux-a.pk069.a6 - Operon
complement(4372207 . . . 4373235) /note = "predicted operon" /note
= "ordered genes contained in the operon: yjeKft lux-a.pk075.a10 +
Operon (4609980 . . . 4611053) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjjU " lux-a.pk081.e3 -
Operon complement(2024345 . . . 2026054) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1956 "
lux-a.pk089.c4 + Operon (434858 . . . 436331) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0417 b0418
" lux-a.pk0003.c1 + Operon (1216509 . . . 1218074) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1168 " lux- + Operon (738224 . . . 740148) /note = "predicted
operon" /note = a.pk0010.a12 "ordered genes contained in the
operon: ybgA phrB " lux-a.pk0019.f8 + Operon (970975 . . . 971868)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0919 " lux-a.pk0024.f7 - Operon complement(4555923 . . .
4558261) /note = "predicted operon" /note = "ordered genes
contained in the operon: iadA yjiG yjiH " lux-a.pk029.g2 - Operon
complement(50380 . . . 51222) /note = "predicted operon" /note =
"ordered genes contained in the operon: apaH " lux-a.pk034.b9 -
Operon complement(3843403 . . . 3844794) /note = "documented uhpT
operon" lux-a.pk038.g11 + Operon (2212886 . . . 2213617) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yehV " lux-a.pk044.b6 - Operon complement(3451145 . . . 3453035)
/note = "predicted operon" /note = "ordered genes contained in the
operon: pinO yheD " lux-a.pk050.d9 - Operon complement(2759372 . .
. 2763174) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2627 b2628" lux-a.pk054.h8 + Operon
(195785 . . . 200360)/note = "predicted operon" /note = "ordered
genes contained in the operon: cdsA yaeL b0177 " lux-a.pk061.f4 +
Operon (4382971 . . . 4383596) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjeN yjeO " lux-a.pk069.a8
- Operon complement(3673920 . . . 3675995) /note = "predicted
operon" /note = "ordered genes contained in the operon: yhjF "
lux-a.pk075.c5 + Operon (2192320 . . . 2194353) /note = "predicted
operon" /note = "ordered genes contained in the operon: metG "
lux-a.pk081.f12 + Operon (3031085 . . . 3031633) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2889 "
lux-a.pk089.c6 - Operon complement(4091029 . . . 4095029) /note =
"documented rhaBAD operon" lux-a.pk0003.d1 - Operon
complement(1246919 . . . 1250091) /note = "predicted operon" /note
= "ordered genes contained in the operon: ycgC b1199 b1200 "
lux-a.pk0010.b4 + Operon (1923464 . . . 1924806) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1843
b1844" lux-a.pk0019.g1 + Operon 2010524 . . . 2010802 /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1936 " lux-a.pk0024.g2 - Operon complement(394354 . . . 395511)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yaiH " lux-a.pk029.g4 - Operon complement(4112149 . . .
4113159) /note = "predicted operon" /note = "ordered genes
contained in the operon: glpX " lux-a.pk034.d1 - Operon
complement(4519695 . . . 4523371) /note = "predicted operon" /note
= "ordered genes contained in the operon: yjhG yjhH yjhI "
lux-a.pk038.h1 - Operon complement( 1654771 . . . 1655517) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1585 " lux-a.pk044.b7 - Operon complement(4097072 . . . 4098106)
/note = "documented rhaT operon" lux-a.pk050.e1 - Operon
complement(2906051 . . . 2907688) /note = "predicted operon" /note
= "ordered genes contained in the operon: pyrG " lux-a.pk055.a12 -
Operon complement(3275497 . . . 3276306) /note = "predicted operon"
/note = "ordered genes contained in the operon: agaR "
lux-a.pk061.f5 - Operon complement(3924173 . . . 3924631) /note =
"documented asnC operon" lux-a.pk069.d3 - Operon complement(4125658
. . . 4125975) /note = "documented metJ operon" lux-a.pk075.d9 +
Operon 3220238 . . . 3223812 /note = "documented ebgAC operon"
lux-a.pk081.f3 - Operon complement(2570177 . . . 2573897) /note =
"predicted operon" /note = "ordered genes contained in the operon:
cchA eutI b2459 b2460 b2461 b2462 " lux-a.pk089.e2 - Operon
complement(2624715 . . . 2626958) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2503 " lux- + Operon
(4022578 . . . 4024818) /note = "predicted operon" a.pk0003.e12
/note = "ordered genes contained in the operon: yigC ubiB "
lux-a.pk0010.c7 - Operon complement(3750593 . . . 3752058)/note =
"predicted operon"/note = "ordered genes contained in the operon:
yiaV yiaW" lux-a.pk0019.g2 - Operon complement(2654556 . . .
2657487) /note = "predicted operon" /note = "ordered genes
contained in the operon: yfhJ fdx hscA yfhE " lux-a.pk0024.g3 +
Operon (2032043 . . . 2032777) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1964 b1965" lux-a.pk030.b3
- Operon complement(4272339 . . . 4272689) /note = "predicted
operon" /note = "ordered genes contained in the operon: yjcB "
lux-a.pk034.d3 - Operon complement(2858490 . . . 2859287) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2735 " lux-a.pk038.h6 - Operon complement(2441911 . . . 2446793)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2325 b2326 yfcA mepA aroC yfcB " lux-a.pk044.d10 - Operon
complement(3668922 . . . 3669524) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhjB" lux-a.pk050.e10 +
Operon (4439959 . . . 4445812) /note = "predicted operon" /note =
"ordered genes contained in the operon: ytfM ytfN ytfP "
lux-a.pk055.a3 + Operon (250898 . . . 251953) /note = "predicted
operon" /note = "ordered genes contained in the operon: "
lux-a.pk061.h3 - Operon complement(489334 . . . 490036) /note =
"predicted operon" /note = "ordered genes contained in the operon:
ybaM priC " lux-a.pk069.d8 + Operon (424235 . . . 429700) /note =
"documented queA-tgt- yajC-secD-secF operon" lux-a.pk075.f11 -
Operon complement(2478658 . . . 2481359) /note = "predicted operon"
/note = "ordered genes contained in the operon: emrY emrK "
lux-a.pk081.g10 + Operon (1773611 . . . 1776371) /note = "predicted
operon" /note = "ordered genes contained in the operon: ydiF b1695
" lux-a.pk089.e4 - Operon complement(2563501 . . . 2570070) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2451 eutH eutG eutJ eutE cchB " lux-a.pk0003.f1 - Operon
complement(1252308 . . . 1255175) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1202 " lux-a.pk0010.d6 -
Operon complement(2181736 . . . 2183321) /note = "predicted
operon"/note = "ordered genes contained in the operon: b2103 b2104
" lux- - Operon complement(805221 . . . 806504) /note = "predicted
a.pk0020.b12 operon" /note = "ordered genes contained in the
operon: ybhC " lux-a.pk0024.g8 - Operon complement(3463886 . . .
3464362) /note = "predicted operon" /note = "ordered genes
contained in the operon: bfr " lux-a.pk030.b8 + Operon (208621 . .
. 209580) /note = "documented accA operon" lux-a.pk034.d6 + Operon
(4501566 . . . 4503973) /note = "predicted operon" /note = "ordered
genes contained in the operon: yjhB yjhC " lux-a.pk039.a5 - Operon
complement(437539 . . . 439401) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0420 " lux-a.pk044.f8 -
Operon complement(4620670 . . . 4622358) /note = "predicted operon"
/note = "ordered genes contained in the operon: lplA smp "
lux-a.pk050.e4 + Operon (1958086 . . . 1959819) /note = "predicted
operon" /note = "ordered genes contained in the operon: argS "
lux-a.pk055.a4 - Operon complement(899067 . . . 902957) /note =
"documented artPIQMJ operon" lux-a.pk062.a12 - Operon
complement(1431108 . . . 1431698) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1374" lux-a.pk069.f11 -
Operon complement(3393963 . . . 3396015) /note = "predicted operon"
/note = "ordered genes contained in the operon: cafA yhdE "
lux-a.pk075.f4 + Operon (47246 . . . 49631) /note = "predicted
operon" /note = "ordered genes contained in the operon: yabF kefC "
lux-a.pk081.h2 + Operon (2852361 . . . 2854439) /note = "predicted
operon" /note = "ordered genes contained in the operon: fhlA "
lux-a.pk089.e7 + Operon (3208422 . . . 3212529) /note = "documented
rpsU- dnaG-rpoD operon" lux-a.pk0004.a2 - Operon complement(2493070
. . . 2493312) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2377 " lux- - Operon complement(411831 .
. . 416176) /note = "predicted a.pk0010.f10 operon" /note =
"ordered genes contained in the operon: sbcC sbcD " lux-a.pk0020.b4
+ Operon (4105132 . . . 4106094) /note = "predicted operon" /note =
"ordered genes contained in the operon: pfkA " lux-a.pk0024.h2 +
Operon (2808791 . . . 2809321) /note = "predicted operon" /note =
"ordered genes contained in the operon: emrR " lux-a.pk030.d3 +
Operon (2042885 . . . 2050036) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1978 " lux-a.pk034.e1 +
Operon (253467 . . . 254202) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0235 prfH " lux-a.pk039.a6
- Operon complement(4553059 . . . 4553889) /note = "predicted
operon" /note = "ordered genes contained in the operon: yjiC"
lux-a.pk044.f9 + Operon (4339489 . . . 4342368) /note = "documented
melAB operon" lux-a.pk050.e9 - Operon complement(910405 . . .
913043) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0872 b0873 " lux-a.pk055.b2 -
Operon complement(930308 . . . 931273) /note = "predicted operon"
/note = "ordered genes contained in the operon: trxB "
lux-a.pk062.b4 - Operon complement(2513663 . . . 2515969) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yfeA " lux-a.pk069.f4 + Operon (3866983 . . . 3868068) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yidS " lux-a.pk075.f5 - Operon complement(2447248 . . . 2453021)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2332 b2333 b2334 b2335 b2336 b2337 b2338 " lux-a.pk081.h3
- Operon complement(4577638 . . . 4580618) /note = "documented
hsdMS operon" lux-a.pk089.f1 - Operon complement(3144871 . . .
3145706) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2999 b3000" lux-lacZ complement(360473 .
. . 365529) lux- - Operon complement(3261327 . . . 3264706) /note =
a.pk0010.g10 "documented tdcABC operon" lux-a.pk0020.c2 + Operon
(320832 . . . 323677) /note = "predicted operon" /note = "ordered
genes contained in the operon: b0306 b0307 b0308 " lux-a.pk0024.h3
+ Operon (2990116 . . . 2991492) /note = "predicted operon" /note =
"ordered genes contained in the operon: b2852 " lux-a.pk030.f10 -
Operon complement(4067055 . . . 4067981) /note = "predicted operon"
/note = "ordered genes contained in the operon: yihR "
lux-a.pk034.f5 - Operon complement(1417789 . . . 1418265) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1356" lux-a.pk039.b6 + Operon (547838 . . . 550555) /note =
"predicted operon" /note= "ordered genes contained in the operon:
b0519 b0520 ybcF " lux-a.pk044.g3 + Operon (1421806 . . . 1424004)
/note = "predicted operon" /note = "ordered genes contained in the
operon: trkG b1364 b1365 b1366" lux-a.pk050.f6 - Operon
complement(2643033 . . . 2650307) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2519 b2520"
lux-a.pk055.b3 + Operon (179237 . . . 180754) /note = "predicted
operon" /note = "ordered genes contained in the operon: dgt "
lux-a.pk062.c6 - Operon complement(602639 . . . 603886) /note =
"predicted operon" /note = "ordered genes contained in the operon:
ybdG " lux-a.pk069.f6 + Operon (4324713 . . . 4327816) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjdA yjcZ " lux-a.pk075.g1 - Operon complement(2519613 . . .
2522898) /note = "predicted operon" /note = "ordered genes
contained in the operon: xapR xapB xapA " lux-a.pk081.h7 - Operon
complement(2224529 . . . 2225290) /note = "predicted operon" /note
= "ordered genes contained in the operon: yohF" lux-a.pk089.g1 +
Operon (3776681 . . . 3778644) /note - "predicted operon" /note =
"ordered genes contained in the operon: lctR lctD " lux-a.pk0004.c1
+ Operon (1848884 . . . 1849900) /note = "documented ansA operon"
lux- + Operon (3225442 . . . 3228880) /note = "predicted operon"
a.pk0010.g12 /note = "ordered genes contained in the operon: ygjJ
ygjK " lux-a.pk0020.c3 - Operon complement(2638706 . . . 2640864)
/note = "predicted operon" /note = "ordered genes contained in the
operon: gcpE yfgA " lux- - Operon complement(3132887 . . . 3134386)
/note = "predicted a.pk0025.c11 operon" /note = "ordered genes
contained in the operon: pitB" lux-a.pk030.f2 - Operon
complement(1061773 . . . 1062998) /note = "predicted operon" /note
= "ordered genes contained in the operon: yccD cbpA "
lux-a.pk034.g4 + Operon (106557 . . . 107474) /note = "predicted
operon" /note = "ordered genes contained in the operon: lpxC "
lux-a.pk039.d1 + Operon (339389 . . . 341731) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0323 b0324
" lux-a.pk044.h4 + Operon (3535122 . . . 3537344) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yhgF " lux-a.pk050.g1 + Operon (3150251 . . . 3151438) /note =
"documented metC operon" lux-a.pk055.d10 - Operon
complement(3363337 . . . 3364353) /note = "predicted operon" /note
= "ordered genes contained in the operon: yi52_1 " lux-a.pk062.e11
+ Operon (4055987 . . . 4057762) /note = "predicted operon" /note =
"ordered genes contained in the operon: yihK " lux-a.pk069.g7 +
Operon (190 . . . 5020) /note = "documented thrLABC operon"
lux-a.pk075.h9 - Operon cpmplement(858436 . . . 859251) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0822 " lux-a.pk081.h8 - Operon complement(1145234 . . . 1145857)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yceFft lux-a.pk090.a2 + Operon (1312044 . . . 1312682)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yciD " lux-lacZ complement(360473 . . . 365529)
lux-a.pk0011.a2 + Operon (539783 . . . 542257) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0513 b0514
" lux- - Operon complement(296605 . . . 301797) /note = "predicted
a.pk0020.d10 operon" /note = "ordered genes contained in the
operon: b0282 b0283 b0284 b0285 b0286 " lux-a.pk0025.c9 - Operon
complement(2238648 . . . 2239688) /note = "documented galS operon"
lux-a.pk030.f8 - Operon complement(4535227 . . . 4537078) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjhT yjhA " lux-a.pk034.h2 - Operon complement(720279 . . . 723637)
/note = "documented kdpDE operon" lux-a.pk039.d10 - Operon
complement(1411555 . . . 1415410) /note = "predicted operon" /note
= "ordered genes contained in the operon: ydaC lar recT recE "
lux-a.pk045.c12 - Operon complement(3068185 . . . 3071711) /note =
"predicted operon" /note = "ordered genes contained in the operon:
fba pgk epd " lux-a.pk050.g10 - Operon complement(2227458 . . .
2228405) /note = "predicted operon" /note = "ordered genes
contained in the operon: yohI" lux-a.pk055.e12 - Operon
complement(1447100 . . . 1449373) /note = "predicted operon" /note
= "ordered genes contained in the operon: tynA" lux-a.pk062.e7 -
Operon complement(3171520 . . . 3175926) /note = "predicted operon"
/note = "ordered genes contained in the operon: parE yqiA icc yqiB
b3034 " lux-a.pk070.a4 - Operon complement(3904481 . . . 3909153)
/note = "documented pstSCAB-phoU operon" lux-a.pk076.c10 + Operon
(22391 . . . 27227) /note = "documented ileS-lspA-lytB operon"
lux-a.pk082.a8 + Operon (972760 . . . 980009) /note = "predicted
operon" /note = "ordered genes contained in the operon: smtA mukF
mukE mukB " lux-a.pk090.g5 - Operon complement(3043178 . . .
3043921) /note = "predicted operon" /note = "ordered genes
contained in the operon: ygfF" lux- - Operon complement(1492172 . .
. 1493236) /note = "predicted a.pk0004.e11 operon" /note = "ordered
genes contained in the operon: b1422 " lux-a.pk0011.a6 + Operon
(4619338 . . . 4620669) /note = "predicted operon" /note = "ordered
genes contained in the operon: yjjJ " lux- + Operon (5234 . . .
5530) /note = "predicted operon" /note = a.pk0020.d11 "ordered
genes contained in the operon: b0005 " lux- - Operon
complement(2409459 . . . 2410632) /note = "predicted a.pk0025.d10
operon" /note = "ordered genes contained in the operon: b2293
b2294" lux-a.pk030.g7 - Operon complement(10643 . . . 11356) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b0011 " lux-a.pk034.h6 + Operon (2160898 . . . 2163020)
/note = "documented baeSR operon" lux-a.pk039.e8 + Operon (4008666
. . . 4009565) /note = "predicted operon" /note = "ordered genes
contained in the operon: yigM " lux-a.pk045.c2 - Operon
complement(2699761 . . . 2702083) /note = "documented rnc-era-recO
operon" lux-a.pk050.g2 - Operon complement(550750 . . . 552323)
/note = "documented purEK operon" lux-a.pk055.g12 - Operon
complement(1877427 . . . 1877972) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1796 b1797"
lux-a.pk062.f10 - Operon complement(4294798 . . . 4296945) /note =
"documented fdhF operon" lux-a.pk070.b7 - Operon complement(2223064
. . . 2223675) /note = "predicted operon" /note = "ordered genes
contained in the operon: yohC " lux-a.pk076.c5 - Operon
complement(3795866 . . . 3805725) /note = "documented rfaQGPSBIJYZK
operon" lux-a.pk082.b4 - Operon complement(3396024 . . . 3398784)
/note = "documented mreBCD operon" lux-a.pk089.g1 + Operon (3776681
. . . 3778644) /note = "predicted operon" /note = "ordered genes
contained in the operon: lctR lctD " lux-lacZ complement(360473 . .
. 365529) lux-a.pk0011.d6 - Operon complement(542485 . . . 545587)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0515 b0516 b0517 " lux- - Operon complement(3981965 . . .
3983620) /note = "predicted a.pk0020.g10 operon" /note = "ordered
genes contained in the operon: aslA " lux- - Operon
complement(1274402 . . . 1276841) /note = a.pk0025.e12 "documented
narXL operon" lux-a.pk030.h2 + Operon (3234934 . . . 3235938) /note
= "predicted operon" /note = "ordered genes contained in the
operon: ygjR " lux-a.pk035.a10 - Operon complement(747144 . . .
751401) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0716 b0717 b0718 " lux-a.pk039.f11 + Operon (269466
. . . 270978) /note = "predicted operon" /note = "ordered genes
contained in the operon: b0255 tra8_1 " lux-a.pk045.d11 - Operon
complement(3508698 . . . 3509763) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhfY yhfZ "
lux-a.pk050.g3 - Operon complement(276980 . . . 279099) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yagC b0263 insB_1 insA_2 " lux-a.pk055.g3 - Operon
complement(149715 . . . 152854) /note = "predicted operon" /note =
"ordered genes contained in the operon: yadC yadK yadL yadM "
lux-a.pk062.h4 + Operon (632809 . . . 633969) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0600 "
lux-a.pk070.c1 + Operon (440773 . . . 442221) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0423 "
lux-a.pk076.c8 + Operon (319451 . . . 320305) /note = "predicted
operon" /note = "ordered genes contained in the operon: b0305 "
lux-a.pk082.b5 - Operon complement(3868065 . . . 3869402) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yidT" lux-a.pk090.a2 + Operon (1312044 . . . 1312682) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yciD " lux-a.pk0004.f7 - Operon complement(2034816 . . . 2036893)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1968 b1969" lux-a.pk0011.d7 + Operon (70387 . . . 71265)
/note = "documented araC operon" lux-a.pk0020.g3 + Operon (770678 .
. . 773404) /note = "documented cydAB operon" lux-a.pk0025.e2 +
Operon (4569935 . . . 4571500) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjiT " lux-a.pk031.a12 +
Operon (4266993 . . . 4267706) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjbP " lux-a.pk035.b7 -
Operon complement(3348330 . . . 3350660) /note = "predicted operon"
/note = "ordered genes contained in the operon: arcB "
lux-a.pk039.g11 + Operon (2923370 . . . 2924218) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2794 "
lux-a.pk045.d6 + Operon (1143671 . . . 1144045) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1085 "
lux-a.pk050.g7 + Operon (1475639 . . . 1480225) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1408 b1409
b1410 b1411" lux-a.pk055.h3 + Operon (3089126 . . . 3091935) /note
= "predicted operon" /note = "ordered genes contained in the
operon: yggJ gshB b2948 b2949 " lux-a.pk063.d5 - Operon
complement(3545619 . . . 3550106) /note = "documented malPQ operon"
lux-a.pk070.c11 - Operon complement(1041253 . . . 1048555) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yccC b0982 b0983 b0984 b0985 b0986 " lux-a.pk076.e8 - Operon
complement(3056686 . . . 3057345) /note = "predicted operon" /note
= "ordered genes contained in the operon: rpiA " lux-a.pk082.c8 +
Operon (3698192 . . . 3699463) /note = "predicted operon" /note =
"ordered genes contained in the operon: yhjV " lux-a.pk090.g5 -
Operon complement(3043178 . . . 3043921) /note = "predicted operon"
/note = "ordered genes contained in the operon: ygfF"
lux-a.pk005.b11 - Operon complement(2752029 . . . 2752785) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2618 b2619" lux-a.pk0011.h8 - Operon (420210 . . . 421583) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0402 " lux-a.pk0020.g6 - Operon complement(587205 . . . 592401)
/note = "documented nfrBA operon" lux-a.pk0025.f1 + Operon (1892829
. . . 1894772) /note = "predicted operon" /note = "ordered genes
contained in the operon: pabB yeaB " lux-a.pk031.a3 + Operon
(3405238 . . . 3407588) /note = "documented panF- prmA operon"
lux-a.pk035.c7 - Operon complement(3383856 . . . 3387041) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yhcP yhcQ b3242 " lux-a.pk039.h3 - Operon complement(2868278 . . .
2870843) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2745 ygbB b2747 b2748 " lux-a.pk045.f3 +
Operon (1463416 . . . 1465974) /note = "predicted operon" /note =
"ordered genes contained in the operon: ydbA_1 " lux-a.pk050.h3 +
Operon (4483786 . . . 4485968) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjgP yjgQ " lux-a.pk055.h9
+ Operon (784856 . . . 785908) /note = "documented aroG operon"
lux-a.pk063.d9 + Operon (705316 . . . 706980) /note = "predicted
operon" /note = "ordered genes contained in the operon: glnS "
lux-a.pk070.d2 - Operon complement(859397 . . . 862761) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0823 b0824 " lux-a.pk076.f2 + Operon (1777641 . . . 1779363) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b1697 b1698" lux-a.pk082.d4 + Operon (4552145 . . .
4552918) /note = "predicted operon" /note = "ordered genes
contained in the operon: uxuR " lux-a.pk005.b6 + Operon (2151891 .
. . 2160901) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2074 b2075 b2076 b2077" lux-a.pk0012.c3 +
Operon (3478926 . . . 3482073) /note = "predicted operon" /note =
"ordered genes contained in the operon: yheS yheT yheU " lux- -
Operon complement(2500010 . . . 2506262) /note = "predicted
a.pk0020.h11 operon" /note = "ordered genes contained in the
operon: b2383 b2384 b2385 b2386 b2387 " lux-a.pk0025.f3 + Operon
(2194494 . . . 2201931) /note = "predicted operon" /note = "ordered
genes contained in the operon: molR molR molR yehI "
lux-a.pk031.c11 - Operon complement(3146992 . . . 3147486) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b3002 " lux-a.pk035.g11 + Operon (3101031 . . . 3102386) /note =
"predicted operon" /note = "ordered genes contained in the operon:
mutY b2962" lux-a.pk039.h5 - Operon complement(2469097 . . .
2471266) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2354 b2355 b2356 b2357 b2358 "
lux-a.pk045.g1 - Operon complement(4346893 . . . 4349240) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjdG yjdH " lux-a.pk051.b1 - Operon
complement(1158585 . . . 1160774) /note = "predicted operon" /note
= "ordered genes contained in the operon: fhuE" lux-a.pk056.a11 +
Operon (571689 . . . 572956) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0547 b0548 b0549 b0550 "
lux-a.pk063.e1 - Operon complement(1181006 . . . 1184817) /note =
"documented potABCD operon" lux-a.pk070.e2 + Operon (624108 . . .
628520) /note = "documented entCEBA operon" lux-a.pk076.f8 - Operon
complement(3640010 . . . 3642812) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhiQ prlC "
lux-a.pk082.d5 - Operon complement(2706774 . . . 2708032) /note =
"documented rpoE-rseA operon" lux-a.pk005.e12 + Operon (4435285 . .
. 4435785) /note = "predicted operon" /note = "ordered genes
contained in the operon: b4215 " lux-a.pk0012.c7 - Operon
complement(3472315 . . . 3474089) /note = "predicted operon" /note
= "ordered genes contained in the operon: yheL b3344 yheN b3346 "
lux-a.pk0020.h5 - Operon complement(1841855 . . . 1844984) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1762 topB " lux-a.pk0025.f5 + Operon (164730 . . . 167264) /note =
"predicted operon" /note = "ordered genes contained in the operon:
mrcB " lux-a.pk031.c7 - Operon complement(3031677 . . . 3034226)
/note = "documented prfB-lysS operon" lux-a.pk035.h9 - Operon
complement(1863750 . . . 1864496) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1782 " lux-a.pk040.a11 +
Operon (1710793 . . . 1712295) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1634 " lux-a.pk045.g4 -
Operon complement(2833196 . . . 2835448) /note = "predicted operon"
/note = "ordered genes contained in the operon: hypF"
lux-a.pk051.b11 - Operon complement(3112567 . . . 3117128) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2973 b2974" lux-a.pk056.b3 - Operon complement(2948657 . . .
2956906) /note = "predicted operon" /note = "ordered genes
contained in the operon: recD recB ptr" lux-a.pk063.e8 + Operon
(1807404 . . . 1808072) /note = "predicted operon" /note = "ordered
genes contained in the operon: b1727 " lux-a.pk070.e4 + Operon
(2589267 . . . 2590754) /note = "predicted operon" /note = "ordered
genes contained in the operon: yffB dapE " lux-a.pk077.a11 - Operon
complement(608682 . . . 611717) /note = "documented fepA-entD
operon" lux-a.pk082.e8 + Operon ( 4028751 . . . 4032033) /note =
"predicted operon" /note = "ordered genes contained in the operon:
pepQ yigZ trkH " lux-a.pk005.f6 + Operon (3602024 . . . 3602879)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yhhF b3466 " lux-a.pk0012.e3 + Operon (3540803 . . .
3541681) /note = "predicted operon" /note = "ordered genes
contained in the operon: yhgA " lux- + Operon (3285731 . . .
3290073) /note = "predicted operon" a.pk0021.a10 /note = "ordered
genes contained in the operon: yraI yraJ yraK " lux- - Operon
complement(4159749 . . . 4160849) /note = "predicted a.pk0025.g11
operon" /note = "ordered genes contained in the operon: trmA "
lux-a.pk031.d1 - Operon complement(1396798 . . . 1397550) /note =
"documented fnr operon" lux-a.pk036.a5 - Operon complement(4586446
. . . 4588864) /note = "predicted operon" /note = "ordered genes
contained in the operon: yjiX yjiY " lux-a.pk040.b5 - Operon
complement(3913181 . . . 3920080) /note = "documented atpIBEFHAGDC
operon" lux-a.pk046.a5 + Operon (1846861 . . . 1848717) /note =
"predicted operon" /note = "ordered genes contained in the operon:
sppA " lux-a.pk051.c11 - Operon complement(1607253 . . . 1608704)
/note = "documented uxaB operon" lux-a.pk056.b4 - Operon
complement(1588878 . . . 1590466) /note = "documented hipBA operon"
lux-a.pk063.g7 + Operon (1741481 . . . 1742854) /note = "predicted
operon" /note = "ordered genes contained in the operon: ydhE "
lux-a.pk070.f11 + Operon (3834580 . . . 3835764) /note = "predicted
operon" /note = "ordered genes contained in the operon: yicK "
lux-a.pk077.a3 - Operon complement(3130469 . . . 3131972) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2984 b2985" lux-a.pk082.g4 + Operon (108279 . . . 110984) /note =
"predicted operon" /note = "ordered genes contained in the operon:
secA " lux-a.pk005.g10 + Operon (1486256 . . . 1487695) /note =
"predicted operon" /note = "ordered genes contained in the operon:
aldA " lux- + Operon (45807 . . . 47138) /note = "predicted operon"
/note = a.pk0012.f11 "ordered genes contained in the operon: yaaU "
lux-a.pk0021.b6 - Operon complement(3427403 . . . 3429566) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yrdB aroE yrdC yrdD " lux-a.pk0025.g5 + Operon (738224 . . .
740148) /note = "predicted operon" /note = "ordered genes contained
in the operon: ybgA phrB" lux-a.pk031.e7 + Operon (27293 . . .
28207) /note = "predicted operon" /note = "ordered genes contained
in the operon: yaaF " lux-a.pk036.b6 - Operon complement(4428899 .
. . 4429561) /note = "predicted operon" /note = "ordered genes
contained in the operon: b4209 " lux-a.pk040.b7 - Operon
complement(1582231 . . . 1584510) /note = "predicted operon" /note
= "ordered genes contained in the operon: b1501 " lux-a.pk046.b5 -
Operon complement(3044188 . . . 3048687) /note = "documented gcvTHP
operon" lux-a.pk051.d10 - Operon complement(4156969 . . . 4158303)
/note = "predicted operon" /note = "ordered genes contained in the
operon: udhA " lux-a.pk056.c3 - Operon complement(332725 . . .
333657) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0316" lux-a.pk064.c10 - Operon complement(2945779 .
. . 2947122) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2817 " lux-a.pk070.g5 + Operon (4328080 .
. . 4329582) /note = "documented proP operon" lux-a.pk077.a9 -
Operon complement(229967 . . . 230881) /note = "predicted operon"
/note = "ordered genes contained in the operon: yafC lux-a.pk082.h2
+ Operon (1753722 . . . 1755134) /note = "predicted operon" /note =
"ordered genes contained in the operon: pykF " lux-a.pk005.g2 +
Operon (4049619 . . . 4050998) /note = "predicted operon" /note =
"ordered genes contained in the operon: hemN " lux-a.pk0015.d6 +
Operon (1218824 . . . 1220344) /note = "predicted operon" /note =
"ordered genes contained in the operon: b1169 " lux-a.pk0021.b8 +
Operon (3928943 . . . 3930502) /note = "predicted operon" /note =
"ordered genes contained in the operon: kup " lux-a.pk0025.h1 +
Operon (1785469 . . . 1786302) /note = "predicted operon" /note =
"ordered genes contained in the operon: ydiA " lux-a.pk031.e9 +
Operon (533140 . . . 535710) /note = "predicted operon" /note =
"ordered genes contained in the operon: gcl gip " lux-a.pk036.b9 +
Operon (938651 . . . 939943) /note = "predicted operon" /note =
"ordered genes contained in the operon: serS " lux-a.pk040.c9 -
Operon complement(583903 . . . 586131) /note = "documented
envY-ompT operon" lux-a.pk046.c1 + Operon (535810 . . . 538311)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0509 b0510 b0511 " lux-a.pk051.d5 + Operon (638168 . . .
640541) /note = "documented ahpCF operon" lux-a.pk056.d6 + Operon
(1360767 . . . 1364839) /note = "predicted operon" /note = "ordered
genes contained in the operon: aldH ordL goaG " lux-a.pk064.c7 -
Operon complement(1853015 . . . 1859356) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1771 b1772 b1773
b1774 b1775 b1776 " lux-a.pk070.g8 - Operon complement(408332 . . .
409243) /note = "predicted operon" /note = "ordered genes contained
in the operon: yaiD" lux-a.pk077.b1 - Operon complement(751452 . .
. 752018) /note = "predicted operon" /note = "ordered genes
contained in the operon: ybgD " lux-a.pk083.a4 - Operon
complement(505827 . . . 506306) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0481 " lux-lacZ
complement(360473 . . . 365529) lux-a.pk0016.c4 + Operon (819107 .
. . 819811) /note = "predicted operon" /note = "ordered genes
contained in the operon: b0786 " lux-a.pk0021.c6 - Operon
complement(1244902 . . . 1246599) /note = "predicted operon" /note
= "ordered genes contained in the operon: treA" lux-a.pk0025.h5 -
Operon complement(764376 . . . 765098) /note = "predicted operon"
/note = "ordered genes contained in the operon: farR"
lux-a.pk031.f10 + Operon (2261883 . . . 2263064) /note = "predicted
operon" /note = "ordered genes contained in the operon: yeiO "
lux-a.pk036.d10 + Operon (4156069 . . . 4156986) /note =
"documented oxyR operon" lux-a.pk040.d7 - Operon complement(2671836
. . . 2677387) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2544 b2545 b2546 b2547 b2548 "
lux-a.pk046.c12 + Operon (2282149 . . . 2284156) /note = "predicted
operon" /note = "ordered genes contained in the operon: yejL yejM "
lux-a.pk051.g11 - Operon complement(1722760 . . . 1724082) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1647 b1648" lux-a.pk056.f2 + Operon (1631646 . . . 1632236) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b1545 " lux-a.pk064.f4 + Operon (3274643.3275442) /note =
"predicted operon" /note = "ordered genes contained in the operon:
sohA yhaV " lux-a.pk071.a11 - Operon complement(906075 . . .
910272) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0869 b0870 poxB " lux-a.pk077.c5 + Operon (3884457
. . . 3885821) /note = "predicted operon" /note = "ordered genes
contained in the operon: thdF " lux-a.pk083.d3 - Operon
complement(2436962 . . . 2438140) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2322 " lux-a.pk005.g3 -
Operon complement(841019 . . . 841423) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0806 " lux- -
Operon complement(4333272 . . . 4334609) /note = "predicted
a.pk0016.a11 operon" /note = "ordered genes contained in the
operon: yjdD " lux-a.pk0021.d4 + Operon (4133655 . . . 4134593)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yijE " lux- + Operon (4448633 . . . 4452152) /note =
"predicted operon" a.pk0026.a12 /note = "ordered genes contained in
the operon: ytfR ytfS ytfT yjfF " lux-a.pk031.f7 + Operon (3735126
. . . 3737156) /note = "documented malS operon" lux-a.pk036.d6 -
Operon complement(837753 . . . 840754) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0804 b0805 "
lux-a.pk040.e4 + Operon (3791614 . . . 3795834) /note = "documented
rfaDFCL operon" lux-a.pk046.c3 + Operon (2765725 . . . 2766594)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b2632 " lux-a.pk051.h4 - Operon complement(2943058 . . .
2943864) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2812 " lux-a.pk056.g9 - Operon
complement(717485 . . . 719683) /note = "documented speF operon"
lux-a.pk064.f8 + Operon (3058870 . . . 3064300) /note = "predicted
operon" /note = "ordered genes contained in the operon: sbm ygfD
b2919 b2920 " lux-a.pk071.a4 - Operon complement(4051449 . . .
4055614) /note = "documented glnALG operon" lux-a.pk077.c7 + Operon
(3416027 . . . 3416248) /note = "predicted operon" /note = "ordered
genes contained in the operon: yhdV " lux-a.pk083.e7 - Operon
complement(1120784 . . . 1121830) /note = "documented pyrC operon"
lux-lacZ complement(360473 . . . 365529) lux-a.pk0016.g4 + Operon
(2848670 . . . 2852287) /note = "documented hypABCDE operon"
lux-a.pk0021.e5 + Operon (331595 . . . 332683) /note = "predicted
operon" /note = "ordered genes contained in the operon: yahA "
lux-a.pk0026.b5 + Operon (3229306 . . . 3231324) /note = "predicted
operon" /note = "ordered genes contained in the operon: ygjL "
lux-a.pk031.g10 + Operon (1097070 . . . 1098047) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1033 "
lux-a.pk036.d7 - Operon complement(1665368 . . . 1666588) /note =
"predicted operon" /note = "ordered genes contained in the operon:
mlc" lux-a.pk040.e5 + Operon (4422696 . . . 4424135) /note =
"documented rpsF- priB-rpsR-rplI operon" lux-a.pk046.e6 - Operon
complement(4085688 . . . 4090404) /note = "predicted operon" /note
= "ordered genes contained in the operon: frvR frvX frvB frvA "
lux-a.pk052.a4 - Operon complement(476291 . . . 477847) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0457 " lux-a.pk056.h3 + Operon (1725861 . . . 1726268) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1651 " lux-a.pk064.g8 - Operon complement(2225343 . . . 2226859)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yohG yohH " lux-a.pk071.e1 + Operon (3244277 . . . 3245068)
/note = "predicted operon" /note = "ordered genes contained in the
operon: exuR " lux-a.pk077.d11 + Operon (3291041 . . . 3294625)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yraM yraN yraO yraP " lux-a.pk083.g7 + Operon (1532048 . .
. 1532893) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1463 " lux-a.pk006.b2 - Operon
complement(2417861 . . . 2418505) /note = "predicted operon" /note
= "ordered genes contained in the operon: b2301 " lux-a.pk0016.h6 +
Operon (8238 . . . 9191) /note = "predicted operon" /note =
"ordered genes contained in the operon: talB " lux-a.pk0021.e6 +
Operon (1944176 . . . 1944877) /note = "predicted operon" /note =
"ordered genes contained in the operon: yebB " lux- - Operon
complement(3875333 . . . 3877747) /note = "predicted a.pk0026.c12
operon" /note = "ordered genes contained in the operon: gyrB "
lux-a.pk031.g7 - Operon complement(1020953 . . . 1023571) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0960 yccF " lux-a.pk036.d9 - Operon complement(3250958 . . .
3251854) /note = "predicted operon" /note = "ordered genes
contained in the operon: yhaJ " lux-a.pk040.g11 + Operon (1321244 .
. . 1324665) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1266 yciO yciQ " lux-a.pk046.e9 - Operon
complement(184257 . . . 188650) /note = "predicted operon" /note =
"ordered genes contained in the operon: yaeI b0165 dapD glnD "
lux-a.pk052.a9 - Operon complement(4280832 . . . 4282792) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjcG yjcH " lux-a.pk057.f5 - Operon complement(4458096 . . .
4460234) /note = "predicted operon" /note = "ordered genes
contained in the operon: nrdD " lux-a.pk064.h7 - Operon
complement(2748136 . . . 2748729) /note = "predicted operon" /note
= "ordered genes contained in the operon: grpE " lux-a.pk071.f7 -
Operon complement(2547666 . . . 2548592) /note = "predicted operon"
/note = "ordered genes contained in the operon: b2431 "
lux-a.pk077.d9 + Operon (4628275 . . . 4630239) /note = "predicted
operon" /note = "ordered genes contained in the operon: slt "
lux-a.pk083.h2 - Operon complement(1169741 . . . 1173187) /note =
"predicted operon" /note = "ordered genes contained in the operon:
mfd " lux-lacZ complement(360473 . . . 365529) lux-a.pk0017.a4 +
Operon (142779 . . . 144472) /note = "predicted operon" /note =
"ordered genes contained in the operon: yadG yadH "
lux-a.pk0021.e8 - Operon complement(4612249 . . . 4614634) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjjW yjjI " lux-a.pk0026.c7 + Operon (2318063 . . . 2321271) /note
= "predicted operon" /note = "ordered genes contained in the
operon: atoS atoC " lux-a.pk031.g8 - Operon complement(3129356 . .
. 3130333) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2983 " lux-a.pk036.e6 - Operon
complement(3836802 . . . 3837620) /note = "predicted operon" /note
= "ordered genes contained in the operon: nlpA " lux-a.pk040.g3 -
Operon complement(3927224 . . . 3928744) /note = "predicted operon"
/note = "ordered genes contained in the operon: yieN "
lux-a.pk046.f11 + Operon (4014920 . . . 4016347) /note = "predicted
operon" /note = "ordered genes contained in the operon: yigN "
lux-a.pk052.b6 + Operon (3995596 . . . 3997758) /note = "documented
uvrD operon" lux-a.pk058.a11 - Operon complement(947883 . . .
948791) /note = "predicted operon" /note = "ordered genes contained
in the operon: b0900 " lux-a.pk064.h8 - Operon complement(1550852 .
. . 1551892) /note = "predicted operon" /note = "ordered genes
contained in the operon: b1478 " lux-a.pk071.g2 - Operon
complement(4585479 . . . 4586333) /note = "predicted operon" /note
= "ordered genes contained in the operon: yjiA m lux-a.pk077.e4 -
Operon complement(1596641 . . . 1598233) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1511 "
lux-a.pk085.a11 + Operon (2435970 . . . 2436965) /note = "predicted
operon" /note = "ordered genes contained in the operon: div "
lux-a.pk006.b4 - Operon complement(1313880 . . . 1314116) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yciG" lux- - Operon complement(4413595 . . . 4413897) /note =
"predicted a.pk0017.c10 operon" /note = "ordered genes contained in
the operon: yjfN " lux-a.pk0021.f6 - Operon complement(3215197 . .
. 3216717) /note = "predicted operon" /note = "ordered genes
contained in the operon: air " lux-a.pk0026.c8 + Operon (2496691 .
. . 2500007) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2380 b2381 b2382 " lux-a.pk031.g9 +
Operon (358023 . . . 360370) /note = "documented cynTSX operon"
lux-a.pk036.e8 - Operon complement(2455035 . . . 2458489) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2341 b2342" lux-a.pk040.g4 - Operon complement(3137731 . . .
3143155) /note = "documented hybGFEDCBA operon" lux-a.pk046.g7 -
Operon complement(1355826 . . . 1357265) /note = "predicted operon"
/note = "ordered genes contained in the operon: b1296 "
lux-a.pk052.b7 - Operon complement(1884888 . . . 1886015) /note =
"predicted operon" /note = "ordered genes contained in the operon:
rnd" lux-a.pk058.a9 + Operon (3963322 . . . 3963705) /note =
"predicted operon" /note = "ordered genes contained in the operon:
trxA " lux-a.pk065.d9 - Operon complement(1878910 . . . 1879854)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b1799" lux-a.pk071.g4 - Operon complement(3160760 . . .
3161497) /note = "predicted operon" /note = "ordered genes
contained in the operon: plsC" lux-a.pk077.f10 + Operon (2991660 .
. . 2991878) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2853 " lux-a.pk085.a5 + Operon (4254694 .
. . 4256700) /note = "documented lexA- dinF operon" lux-a.pk006.c9
+ Operon (4381375 . . . 4382919) /note = "predicted operon" /note =
"ordered genes contained in the operon: yjeM " lux-a.pk0017.c7 +
Operon (504138 . . . 505790) /note = "predicted operon" /note =
"ordered genes contained in the operon: ushA " lux- + Operon
(458112 . . . 460466) /note = "predicted operon" /note a.pk0021.g11
= "ordered genes contained in the operon: lon " lux-a.pk0026.d1 +
Operon (113444 . . . 114487) /note = "predicted operon" /note =
"ordered genes contained in the operon: guaC " lux-a.pk032.b10 +
Operon (1262937 . . . 1268242) /note = "predicted operon" /note =
"ordered genes contained in the operon: hemA prfA hemK b1213 ychA
kdsA " lux-a.pk036.h7 + Operon (182445 . . . 183620) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yaeG " lux-a.pk040.g6 - Operon complement(2434735 . . . 2435871)
/note = "predicted operon" /note = "ordered genes contained in the
operon: pdxB " lux-a.pk047.a10 + Operon (2011251 . . . 2017535)
/note = "documented fliFGHIJK operon" lux-a.pk052.c11 + Operon
(1124785 . . . 1126952) /note = "predicted operon" /note = "ordered
genes contained in the operon: rimJ yceH " lux-a.pk058.c1 - Operon
complement(3253729 . . . 3255597) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhaN yhaO "
lux-a.pk065.e4 + Operon (3272923 . . . 3274494) /note = "predicted
operon" /note = "ordered genes contained in the operon: yhaG "
lux-a.pk071.g6 - Operon complement(4454888 . . . 4455439) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yjgA " lux-a.pk077.f12 - Operon complement(2841059 . . . 2848458)
/note = "documented hycABCDEFGH operon" lux-a.pk085.c1 - Operon
complement(944154 . . . 944780) /note = "predicted operon" /note =
"ordered genes contained in the operon: ycaC " lux-a.pk006.e10 -
Operon complement(2122545 . . . 2131419) /note = "predicted operon"
/note = "ordered genes contained in the operon: yefD yefC yefB yefA
b2054 b2055 b2056 b2057 wcaB b2059 " lux-a.pk0017.c9 + Operon
(3958292 . . . 3960313) /note = "predicted operon" /note = "ordered
genes contained in the operon: rep " lux-a.pk0022.a2 - Operon
complement(2536692 . . . 2541548) /note = "documented cysPUWAM
operon" lux- + Operon (3610600 . . . 3611187) /note = "predicted
operon" a.pk0026.e10 /note = "ordered genes contained in the
operon: yhhU " lux-a.pk032.b2 + Operon (3281811 . . . 3284666)
/note = "predicted operon" /note = "ordered genes contained in the
operon: agaB agaC agaD agaI " lux-a.pk036.h9 + Operon (830095 . . .
831459) /note = "predicted operon" /note = "ordered genes contained
in the operon: rhlE " lux-a.pk041.a10 - Operon complement(3780269 .
. . 3781755) /note = "predicted operon" /note = "ordered genes
contained in the operon: gpsA secB " lux-a.pk047.a11 + Operon
(4016442 . . . 4021926) /note = "predicted operon" /note = "ordered
genes contained in the operon: yigO yigP yigQ yigTa yigTb yigTc
yigU yigW yigW " lux-a.pk052.c5 - Operon complement(2574118 . . .
2576397) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2463 " lux-a.pk058.c11 - Operon
complement(1980578 . . . 1984151) /note = "documented
araFG-b1899-araH operon" lux-a.pk065.f11 - Operon
complement(3376505 . . . 3377632) /note - "predicted operon" /note
= "ordered genes contained in the operon: yhcM " lux-a.pk072.a10 -
Operon complement(149715 . . . 152854) /note = "predicted operon"
/note = "ordered genes contained in the operon: yadC yadK yadL yadM
" lux-a.pk077.f3 + Operon (3965531 . . . 3972101) /note =
"predicted operon" /note = "ordered genes contained in the operon:
rfe b3785 rffE rffD rffG rffH " lux-a.pk085.f1 - Operon
complement(54755 . . . 57109) /note = "predicted operon" /note =
"ordered genes contained in the operon: imp" lux-a.pk006.f7 -
Operon complement(3865689 . . . 3866939) /note = "predicted operon"
/note = "ordered genes contained in the operon: yidR"
lux-a.pk0017.d3 + Operon (4476036 . . . 4476452) /note = "predicted
operon" /note = "ordered genes contained in the operon: yjgD "
lux-a.pk0022.b1 - Operon complement(4025199 . . . 4028561) /note =
"documented fadBA operon" lux- - Operon complement(78848 . . .
83708) /note = "documented a.pk0026.f11 leuLABCD operon"
lux-a.pk032.c3 - Operon complement(2744454 . . . 2745815) /note =
"predicted operon" /note = "ordered genes contained in the operon:
ffh " lux-a.pk037.a3 + Operon (3857880 . . . 3858803) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b3680" lux-a.pk041.a6 - Operon complement(640662 . . . 641090)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0607 " lux-a.pk047.a6 - Operon complement(661975 . . .
663186) /note = "predicted operon" /note = "ordered genes contained
in the operon: dacA " lux-a.pk052.d6 - Operon complement(3892901 .
. . 3894238) /note = "predicted operon" /note = "ordered genes
contained in the operon: yieG" lux-a.pk058.c9 - Operon
complement(1187539 . . . 1189670) /note = "documented phoQP operon"
lux-a.pk065.f9 + Operon (2135858 . . . 2137507) /note = "predicted
operon" /note = "ordered genes contained in the operon: b2063 "
lux-a.pk072.b1 - Operon complement(1846149 . . . 1846700) /note =
"predicted operon" /note = "ordered genes contained in the operon:
ydjA " lux-a.pk077.h3 - Operon complement(4534182 . . . 4535162)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yjhS " lux-a.pk085.g10 - Operon complement(1015762 . . .
1017522) /note = "predicted operon" /note = "ordered genes
contained in the operon: b0955 " lux-a.pk006.h1 + Operon (989845 .
. . 992457) /note = "predicted operon" /note = "ordered genes
contained in the operon: pepN " lux-a.pk0017.e3 - Operon
complement(2334813 . . . 2337440) /note = "documented gyrA operon"
lux-a.pk0022.b3 - Operon complement(4188313 . . . 4193677) /note =
"documented thiCEFGH operon" lux-a.pk0026.f3 + Operon (4276058 . .
. 4277407) /note = "predicted operon" /note = "ordered genes
contained in the operon: yjcD " lux-a.pk032.d2 + Operon (2461032 .
. . 2462090) /note = "predicted operon" /note = "ordered genes
contained in the operon: b2345 " lux-a.pk037.c11 - Operon
complement(824225 . . . 829878) /note = "predicted operon" /note =
"ordered genes contained in the operon: b0792 b0793 b0794 b0795
ybiH " lux-a.pk041.b3 - Operon complement(3334604 . . . 3337706)
/note = "predicted operon" /note = "ordered genes contained in the
operon: yrbB yrbC b3193 yrbE b3195 " lux-a.pk047.b4 + Operon
(2735619 . . . 2736925) /note = "documented pheLA operon"
lux-a.pk052.d7 + Operon (1732459 . . . 1733274) /note = "predicted
operon" /note = "ordered genes contained in the operon: b1655 "
lux-a.pk058.e10 + Operon (1928905 . . . 1930083) /note = "predicted
operon" /note = "ordered genes contained in the operon: purT "
lux-a.pk065.g3 - Operon complement(2246757 . . . 2247638) /note =
"predicted operon" /note = "ordered genes contained in the operon:
yeiE" lux-a.pk072.b6 + Operon (538371 . . . 539732) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0512 " lux-a.pk078.b3 - Operon complement(383283 . . . 383693)
/note = "predicted operon" /note = "ordered genes contained in the
operon: b0364 " lux-a.pk085.h9 + Operon (532235 . . . 533050) /note
= "predicted operon" /note = "ordered genes contained in the
operon: b0506 " lux-a.pk006.h2 - Operon complement(2595851 . . .
2597780) /note = "documented dapA-nlpB operon" lux-a.pk0017.e7 +
Operon (3656862 . . . 3661157) /note = "predicted operon" /note =
"ordered genes contained in the operon: yhiU yhiV " lux-a.pk0022.b5
- Operon complement(4148026 . . . 4150677) /note = "predicted
operon" /note = "ordered genes contained in the operon: ppc" lux- +
Operon (940269 . . . 944119) /note = "documented dmsABC
a.pk0026.g10 operon" lux-a.pk032.d7 + Operon (432226 . . . 434780)
/note = "predicted operon" /note = "ordered genes contained in the
operon: ybaD ribD ribH nusB " lux-a.pk037.c3 - Operon
complement(658474 . . . 661435) /note = "predicted operon" /note =
"ordered genes contained in the operon: lipA ybeF lipB"
lux-a.pk041.d4 + Operon (1737935 . . . 1739146) /note = "predicted
operon" /note = "ordered genes contained in the operon: ydhC "
lux-a.pk047.c5 - Operon complement(3322642 . . . 3325305) /note =
"documented ftsJ-hflB operon" lux-a.pk052.e11 - Operon
complement(2712459 . . . 2713385) /note = "predicted operon" /note
= "ordered genes contained in the operon: yfiE " lux-a.pk058.f2 -
Operon complement(1942370 . . . 1944000) /note = "documented ruvBA
operon" lux-a.pk065.g6 + Operon (3155664 . . . 3156593) /note =
"predicted operon" /note = "ordered genes contained in the operon:
+ " lux-a.pk072.b8 + Operon (3018561 . . . 3022207) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b2880 b2881 " lux-a.pk078.c2 - Operon complement(288525 . . .
289529) /note = "documented argF operon" lux-a.pk086.a5 - Operon
complement(4302191 . . . 4304188) /note = "predicted operon" /note
= "ordered genes contained in the operon: yjcS " lux-a.pk007.a10 -
Operon complement(2338437 . . . 2342189) /note = "predicted operon"
/note = "ordered genes contained in the operon: yfaL" lux- - Operon
complement(2633619 . . . 2635415) /note = "predicted a.pk0017.f11
operon" /note = "ordered genes contained in the operon: b2510
b2511" lux-a.pk0022.c1 + Operon (334504 . . . 339313) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b0318 b0319 b0320 b0321 b0322 " lux-a.pk0026.g2 - Operon
complement(786066 . . . 786818) /note = "predicted operon" /note =
"ordered genes contained in the operon: gpmA " lux-a.pk032.e4 +
Operon (2141288 . . . 2144605) /note = "predicted operon" /note =
"ordered genes contained in the operon: yegE " lux-a.pk037.c8 -
Operon complement(262552 . . . 263231) /note = "predicted operon"
/note = "ordered genes contained in the operon: b0245 b0246 "
lux-a.pk041.d8 - Operon complement(622777 . . . 623733) /note =
"documented fepB operon" lux-a.pk047.c6 + Operon (3154754 . . .
3155464) /note = "predicted operon" /note = "ordered genes
contained in the operon: b3012 " lux-a.pk052.f1 - Operon
complement(3665421 . . . 3666818) /note = "predicted operon" /note
= "ordered genes contained in the operon: yhjA " lux-a.pk058.f3 -
Operon complement(3371333 . . . 3372124) /note = "predicted operon"
/note = "ordered genes contained in the operon: b3226 "
lux-a.pk065.h2 - Operon complement(2009370 . . . 2009891) /note =
"predicted operon" /note = "ordered genes contained in the operon:
b1933 b1934" lux-a.pk072.d1 - Operon complement(4300657 . . .
4301688) /note = "predicted operon" /note = "ordered genes
contained in the operon: yjcR" lux-a.pk078.c2 - Operon
complement(4474870 . . . 4475874) /note = "documented argI operon"
lux-a.pk086.b7 + Operon (1481142 . . . 1484987) /note = "predicted
operon" /note = "ordered genes contained in the operon: hrpA " The
numbers in parenthesis represent each end of the nucleotide
sequence designation in the E. coli genomic sequence. The sequence
designation is based on Blattner et al. ((1997) Science
277:1453-1462)
[0326]
20TABLE 19 lux-z.pk013.a15 - Operon complement(3366649 . . .
3370239) note = "predicted operon" note = "ordered genes contained
in the operon: b3221 yhcI b3223 nanT" lux-z.pk013.g17 - Operon
complement(4488774 . . . 4490093) note = "predicted operon" note =
"ordered genes contained in the operon: yjgT" lux-z.pk013.i9 +
Operon 3128193 . . . 3129209 note = "predicted operon" note =
"ordered genes contained in the operon: yi52_9" lux-z.pk013.k1 -
Operon complement(2978786 . . . 2980204) note = "documented araE
operon" lux-z.pk013.k11 + Operon 2027561 . . . 2028481 note =
"predicted operon" note = "ordered genes contained in the operon:
yedA" lux-z.pk013.k5 + Operon 2418641 . . . 2419288 note =
"predicted operon" note = "ordered genes contained in the operon:
b2302" lux-z.pk013.o5 - Operon complement(1347004 . . . 1348209)
note = "predicted operon" note = "ordered genes contained in the
operon: b1287" lux-z.pk014.a14 + Operon 1063259 . . . 1064515 note
= "predicted operon" note = "ordered genes contained in the operon:
yccE" lux-z.pk014.a22 + Operon 557402 . . . 557977 note =
"predicted operon" note = "ordered genes contained in the operon:
b0530" lux-z.pk014.c16 + Operon 607288 . . . 608400 note =
"predicted operon" note = "ordered genes contained in the operon:
yi81_2" lux-z.pk014.c5 - Operon complement(1561358 . . . 1565164)
note = "predicted operon" note = "ordered genes contained in the
operon: b1489 b1490" lux-z.pk014.g11 - Operon complement(3055198 .
. . 3056430) note = "documented serA operon" lux-z.pk014.h9 +
Operon 948891 . . . 949481 note = "predicted operon" note =
"ordered genes contained in the operon: ycaK" lux-z.pk014.k13 +
Operon 3769908 . . . 3773786 note = "documented mtlADR operon"
lux-z.pk014.l1 + Operon 3132146 . . . 3132838 note = "predicted
operon" note = "ordered genes contained in the operon: b2986"
lux-z.pk014.m1 + Operon 2752917 . . . 2753399 note = "predicted
operon" note = "ordered genes contained in the operon: smpB"
lux-z.pk014.n19 - Operon complement(4439115 . . . 4439753) note =
"predicted operon" note = "ordered genes contained in the operon:
msrA" lux-z.pk014.o9 - Operon complement(2486043 . . . 2488206)
note = "predicted operon" note = "ordered genes contained in the
operon: b2371 b2372" lux-z.pk014.p3 + Operon 4077307 . . . 4077549
note = "predicted operon" note = "ordered genes contained in the
operon: yiiF" lux-z.pk014.p4 - Operon complement(1930139 . . .
1932628) note = "predicted operon" note = "ordered genes contained
in the operon: eda edd" lux-z.pk014.p8 - Operon complement 1122630
. . . 1123277) note = "predicted operon" note = "ordered genes
contained in the operon: grxB" lux-z.pk015.a7 + Operon 3408908 . .
. 3409204 note = "documented fis operon" lux-z.pk015.b23 + Operon
4059826 . . . 4061091 note = "predicted operon" note = "ordered
genes contained in the operon: yihN" lux-z.pk015.c7 + Operon
2758568 . . . 2759194 note = "predicted operon" note = "ordered
genes contained in the operon: b2626" lux-z.pk015.d10 + Operon
2745907 . . . 2748081 note = "predicted operon" note = "ordered
genes contained in the operon: b2611 b2612 yfjD" lux-z.pk015.e17 -
Operon complement(2794358 . . . 2794807) note = "predicted operon"
note = "ordered genes contained in the operon: b2665"
lux-z.pk015.f1 + Operon 2321467 . . . 2325313 note = "documented
atoDAB operon" lux-z.pk015.g1 + Operon 1509678 . . . 1515026 note =
"predicted operon" note = "ordered genes contained in the operon:
b1440 b1441 b1442 b1443 b1444" lux-z.pk015.g13 + Operon 3214420 . .
. 3215043 note = "predicted operon" note = "ordered genes contained
in the operon: b3071" lux-z.pk015.g23 - Operon complement(2828798 .
. . 2830387) note = "predicted operon" note = "ordered genes
contained in the operon: ygaA" lux-z.pk015.i5 - Operon
complement(376759 . . . 377592) note = "predicted operon" note =
"ordered genes contained in the operon: b0355" lux-z.pk015.n1 -
Operon complement(586314 . . . 587204) note = "predicted operon"
note = "ordered genes contained in the operon: ybcH"
lux-z.pk015.n24 + Operon 2576686 . . . 2579659 note = "predicted
operon" note = "ordered genes contained in the operon: b2464 tktB"
lux-z.pk015.n4 - Operon complement(4145045 . . . 4145896) note =
"predicted operon" note = "ordered genes contained in the operon:
yijO" lux-z.pk015.p2 - Operon complement(3111560 . . . 3112492)
note = "predicted operon" note = "ordered genes contained in the
operon: b2972" lux-z.pk015.p6 + Operon 1768612 . . . 1768995 note =
"predicted operon" note = "ordered genes contained in the operon:
b1689" lux-z.pk016.a4 - Operon complement(4580819 . . . 4584385)
note = "predicted operon" note = "ordered genes contained in the
operon: hsdR" lux-z.pk016.c22 + Operon 4210813 . . . 4211196 note =
"predicted operon" note = "ordered genes contained in the operon:
yjaA" lux-z.pk016.c9 + Operon 1073465 . . . 1074103 note =
"predicted operon" note = "ordered genes contained in the operon:
b1013" lux-z.pk016.e11 - Operon complement(2812905 . . . 2814461)
note = "predicted operon" note = "ordered genes contained in the
operon: gshA" lux-z.pk016.f16 + Operon 2405581 . . . 2406798 note =
"predicted operon" note = "ordered genes contained in the operon:
b2290" lux-z.pk016.f6 + Operon 1234161 . . . 1234880 note =
"predicted operon" note = "ordered genes contained in the operon:
fadR" lux-z.pk016.h16 + Operon 3054261 . . . 3054809 note =
"predicted operon" note = "ordered genes contained in the operon:
ygfA" lux-z.pk016.i17 - Operon complement(2817403 . . . 2820033)
note = "predicted operon" note = "ordered genes contained in the
operon: alaS" lux-z.pk016.i2 + Operon 4041737 . . . 4043209 note =
"predicted operon" note = "ordered genes contained in the operon:
yihF" lux-z.pk016.i24 + Operon 1055484 . . . 1056512 note =
"predicted operon" note = "ordered genes contained in the operon:
torT" lux-z.pk016.n14 - Operon complement(1228706 . . . 1229623)
note = "predicted operon" note = "ordered genes contained in the
operon: b1182" lux-z.pk016.o11 - Operon complement(4108320 . . .
4109087) note = "predicted operon" note = "ordered genes contained
in the operon: tpiA" lux-z.pk016.o17 + Operon 2475867 . . . 2478550
note = "documented dsdXA operon" lux-z.pk017.c2 + Operon 3550718 .
. . 3553423 note = "documented malT operon" lux-z.pk017.g19 +
Operon 420210 . . . 421583 note = "predicted operon" note =
"ordered genes contained in the operon: b0402" lux-z.pk017.g23 -
Operon complement(4468560 . . . 4469969) note = "documented pyrBI
operon" lux-z.pk017.i21 - Operon complement(3633838 . . . 3635040)
note = "predicted operon" note = "ordered genes contained in the
operon: yhiN" lux-z.pk017.i4 + Operon 3004356 . . . 3005447 note =
"predicted operon" note = "ordered genes contained in the operon:
b2870" lux-z.pk017.k15 - Operon complement(2657583 . . . 2659575)
note = "predicted operon" note = "ordered genes contained in the
operon: yfhF b2529 b2530" lux-z.pk017.m18 + Operon 2270378 . . .
2275909 note = "predicted operon" note = "ordered genes contained
in the operon: yejA yejB yejE yejF" lux-z.pk018.a16 - Operon
complement(3738738 . . . 3739211) note = "predicted operon" note =
"ordered genes contained in the operon: not-yiaI" lux-z.pk018.e4 +
Operon 4256816 . . . 4257025 note = "predicted operon" note =
"ordered genes contained in the operon: b4045" lux-z.pk018.g18 -
Operon complement(2488276 . . . 2489970) note = "predicted operon"
note = "ordered genes contained in the operon: b2373"
lux-z.pk018.i2 + Operon 2037500 . . . 2039140 note = "predicted
operon" note = "ordered genes contained in the operon: b1971 b1972"
lux-z.pk018.o10 + Operon 959463 . . . 960251 note = "predicted
operon" note = "ordered genes contained in the operon: b0909"
lux-z.pk019.a18 - Operon complement(2242798 . . . 2244789) note =
"documented cirA operon" lux-z.pk019.a24 - Operon
complement(4559066 . . . 4560244) note = "predicted operon" note =
"ordered genes contained in the operon: yjiJ" lux-z.pk019.b15 +
Operon 4611194 . . . 4611829 note = "predicted operon" note =
"ordered genes contained in the operon: yjjV" lux-z.pk019.b21 -
Operon complement(3309474 . . . 3310819) note = "predicted operon"
note = "ordered genes contained in the operon: truB rbfA"
lux-z.pk019.c6 - Operon complement(4388035 . . . 4389048) note =
"predicted operon" note = "ordered genes contained in the operon:
yjeQ" lux-z.pk019.e12 + Operon 4263361 . . . 4264440 note =
"predicted operon" note = "ordered genes contained in the operon:
alr" lux-z.pk019.e2 + Operon 1225823 . . . 1226191 note =
"predicted operon" note = "ordered genes contained in the operon:
b1177" lux-z.pk019.g1 - Operon complement(663325 . . . 668151) note
= "predicted operon" note = "ordered genes contained in the operon:
rlpA mrdB mrdA ybeA ybeB" lux-z.pk019.h5 - Operon
complement(1555136 . . . 1561100) note = "predicted operon" note =
"ordered genes contained in the operon: b1483 b1484 b1485 b1486
b1487 b1488" lux-z.pk019.j11 + Operon 2662383 . . . 2663264 note =
"predicted operon" note = "ordered genes contained in the operon:
b2534" lux-z.pk019.j15 + Operon 1498597 . . . 1500179 note =
"documented tehAB operon" lux-z.pk019.o21 - Operon
complement(3961980 . . . 3963245) note = "predicted operon" note =
"ordered genes contained in the operon: rhlB" lux-z.pk020.a23 +
Operon 3572704 . . . 3573297 note = "predicted operon" note =
"ordered genes contained in the operon: b3434" lux-z.pk020.c16 -
Operon complement(3809518 . . . 3810192) note = "predicted operon"
note = "ordered genes contained in the operon: radC" lux-z.pk020.c8
+ Operon 4292060 . . . 4293373 note = "predicted operon" note =
"ordered genes contained in the operon: gltP" lux-z.pk020.e3 +
Operon 167484 . . . 173444 note = "documented fhuACDB operon"
lux-z.pk020.h21 + Operon 4007918 . . . 4008433 note = "predicted
operon" note = "ordered genes contained in the operon: yigL"
lux-z.pk020.j20 - Operon complement(379293 . . . 380511) note =
"predicted operon" note = "ordered genes contained in the operon:
b0358 b0359" lux-z.pk020.j9 + Operon 2322776 . . . 2324098 note =
"predicted operon" note = "ordered genes contained in the operon:
atoE" lux-z.pk020.l21 + Operon 1329072 . . . 1331669 note =
"predicted operon" note = "ordered genes contained in the operon:
topA" lux-z.pk020.n12 - Operon complement(216179 . . . 218775) note
= "predicted operon" note = "ordered genes contained in the operon:
yaeF proS" lux-z.pk021.a14 + Operon 4277559 . . . 4279208 note =
"predicted operon" note = "ordered genes contained in the operon:
yjcE" lux-z.pk021.b3 - Operon complement(2962383 . . . 2964059)
note = "documented lgt-thyA operon" lux-z.pk021.c10 + Operon
3407917 . . . 3408882 note = "predicted operon" note = "ordered
genes contained in the operon: yhdG" lux-z.pk021.d1 + Operon
1687876 . . . 1689384 note = "predicted operon" note = "ordered
genes contained in the operon: b1614" lux-z.pk021.d22 - Operon
complement(4523674 . . . 4525548) note = "predicted operon" note =
"ordered genes contained in the operon: yjhJ yjhK yjhL"
lux-z.pk021.e10 - Operon complement(2287085 . . . 2288101) note =
"predicted operon" note = "ordered genes contained in the operon:
yi52_8" lux-z.pk021.g16 - Operon complement(2421756 . . . 2423936)
note = "predicted operon" note = "ordered genes contained in the
operon: hisP hisM hisQ" lux-z.pk021.h14 - Operon complement(3096577
. . . 3097584) note = "predicted operon" note = "ordered genes
contained in the operon: yggM" lux-z.pk021.h3 + Operon 1805820 . .
. 1806680 note = "predicted operon" note = "ordered genes contained
in the operon: b1725" lux-z.pk021.i24 + Operon 3814303 . . .
3815166 note = "predicted operon" note = "ordered genes contained
in the operon: yicC" lux-z.pk021.k6 - Operon complement(4375389 . .
. 4376522) note = "predicted operon" note = "ordered genes
contained in the operon: ampC" lux-z.pk021.l19 - Operon
complement(2660603 . . . 2661343) note = "predicted operon" note =
"ordered genes contained in the operon: b2532" lux-z.pk021.n20 +
Operon 3225442 . . . 3228880 note = "predicted operon" note =
"ordered genes contained in the operon: ygjJ ygjK" lux-z.pk021.n20
+ Operon 3225442 . . . 3228880 note = "predicted operon" note =
"ordered genes contained in the operon: ygjj ygjK" lux-z.pk021.o16
- Operon complement(2710047 . . . 2710904) note = "predicted
operon" note = "ordered genes contained in the operon: yfiC"
lux-z.pk021.o4 + Operon 3490205 . . . 3491386 note = "predicted
operon" note = "ordered genes contained in the operon: yhfC"
lux-z.pk021.p19 + Operon 4199504 . . . 4200901 note = "documented
hydH operon" lux-z.pk022.b12 - Operon complement(635939 . . .
636841) note = "predicted operon" note = "ordered genes contained
in the operon: b0603" lux-z.pk022.b20 - Operon complement(156299 .
. . 156883) note = "predicted operon" note = "ordered genes
contained in the operon: yadN" lux-z.pk022.c5 - Operon
complement(2432102 . . . 2432761) note = "predicted operon" note =
"ordered genes contained in the operon: dedA" lux-z.pk022.f12 +
Operon 2264265 . . . 2265731 note = "predicted operon" note =
"ordered genes contained in the operon: yeiQ" lux-z.pk022.g13 -
Operon complement(2221958 . . . 2222899) note = "predicted operon"
note = "ordered genes contained in the operon: pbpG" lux-z.pk022.g8
- Operon complement(3960360 . . . 3961844) note = "predicted
operon" note = "ordered genes contained in the operon: gppA"
lux-z.pk022.h21 + Operon 2163690 . . . 2165051 note = "predicted
operon" note = "ordered genes contained in the operon: b2081"
lux-z.pk022.i1 - Operon complement(2940940 . . . 2941170) note =
"predicted operon" note = "ordered genes contained in the operon:
b2809" lux-z.pk022.j21 + Operon 2597860 . . . 2598968 note =
"predicted operon" note = "ordered genes contained in the operon:
gcvR bcp" lux-z.pk022.k8 - Operon complement(4486129 . . . 4487631)
note = "predicted operon" note = "ordered genes contained in the
operon: yjgR" lux-z.pk022.n7 + Operon 4002473 . . . 4003342 note =
"predicted operon" note = "ordered genes contained in the operon:
pldA" lux-z.pk022.p18 + Operon 1277180 . . . 1278571 note =
"documented narK operon" lux-z.pk023.a11 - Operon
complement(4282992 . . . 4284950) note = "predicted operon" note =
"ordered genes contained in the operon: acs"
lux-z.pk023.c18 + Operon 1194346 . . . 1195596 note = "documented
icdA operon" lux-z.pk023.c21 + Operon 3387155 . . . 3388084 note =
"predicted operon" note = "ordered genes contained in the operon:
b3243" lux-z.pk023.e17 + Operon 3418958 . . . 3420830 note =
"predicted operon" note = "ordered genes contained in the operon:
yhdY yhdZ" lux-z.pk023.f12 + Operon 855186 . . . 856778 note =
"predicted operon" note = "ordered genes contained in the operon:
b0820" lux-z.pk023.g8 - Operon complement(2957082 . . . 2962199)
note = "predicted operon" note = "ordered genes contained in the
operon: recC ppdC ygdB ppdB ppdA" lux-z.pk023.j2 - Operon
complement(3436342 . . . 3437146) note = "predicted operon" note =
"ordered genes contained in the operon: yhdM yhdN" lux-z.pk023.k11
- Operon complement(3239467 . . . 3242381) note = "documented uxaCA
operon" lux-z.pk023.k5 - Operon complement(1690914 . . . 1694095)
note = "predicted operon" note = "ordered genes contained in the
operon: uidB uidA" lux-z.pk023.l18 - Operon complement(1928481 . .
. 1928771) note = "predicted operon" note = "ordered genes
contained in the operon: yebG" lux-z.pk023.m21 + Operon 3963846 . .
. 3965291 note = "predicted operon" note = "ordered genes contained
in the operon: rhoL rho" lux-z.pk023.m22 + Operon 1426547 . . .
1427008 note = "predicted operon" note = "ordered genes contained
in the operon: b1371" lux-z.pk023.m22 - Operon complement(3127058 .
. . 3128230) note = "predicted operon" note = "ordered genes
contained in the operon: b2981" lux-z.pk023.o7 + Operon 4380191 . .
. 4381198 note = "predicted operon" note = "ordered genes contained
in the operon: yjeA" lux-z.pk023.o8 + Operon 2263215 . . . 2264042
note = "predicted operon" note = "ordered genes contained in the
operon: yeiP" lux-z.pk025.b18 - Operon complement(1186342 . . .
1187472) note = "predicted operon" note = "ordered genes contained
in the operon: ycfD" lux-z.pk025.b8 - Operon complement(1349852 . .
. 1355134) note = "predicted operon" note = "ordered genes
contained in the operon: sapF sapD sapC sapB sapA" lux-z.pk025.c11
+ Operon 1146017 . . . 1146538 note = "predicted operon" note =
"ordered genes contained in the operon: yceD" lux-z.pk025.d13 -
Operon complement(2549297 . . . 2550269) note = "predicted operon"
note = "ordered genes contained in the operon: b2433 b2434"
lux-z.pk025.e10 + Operon 1391230 . . . 1392864 note = "predicted
operon" note = "ordered genes contained in the operon: b1329"
lux-z.pk025.f5 - Operon complement(4487709 . . . 4488707) note =
"predicted operon" note = "ordered genes contained in the operon:
yjgS" lux-z.pk025.h13 - Operon complement(1731778 . . . 1732125)
note = "predicted operon" note = "ordered genes contained in the
operon: ydhD" lux-z.pk025.h22 + Operon 4098391 . . . 4099011 note =
"documented sodA operon" lux-z.pk025.i24 - Operon
complement(1261249 . . . 1262723) note = "predicted operon" note =
"ordered genes contained in the operon: ychB hemM" lux-z.pk025.j11
- Operon complement(529356 . . . 530450) note = "predicted operon"
note = "ordered genes contained in the operon: ybbB"
lux-z.pk025.j16 + Operon 1712401 . . . 1713006 note = "predicted
operon" note = "ordered genes contained in the operon: gst"
lux-z.pk025.j4 + Operon 3578769 . . . 3S79257 note = "predicted
operon" note = "ordered genes contained in the operon: b3441"
lux-z.pk025.m6 - Operon complement(1581786 . . . 1581983) note =
"predicted operon" note = "ordered genes contained in the operon:
b1500" lux-z.pk025.m7 + Operon 3775026 . . . 3776681 note =
"documented lldP operon" lux-z.pk025.o12 + Operon 3057773 . . .
3058666 note = "predicted operon" note = "ordered genes contained
in the operon: iciA" lux-z.pk025.o8 + Operon 402927 . . . 404042
note = "predicted operon" note = "ordered genes contained in the
operon: yaiC" lux-z.pk018.i18 - surA lux-z.pk013.g19 + yacK
lux-z.pk014.h24 - map lux-z.pk014.l8 + betT lux-z.pk020.g15 + yaiB
lux-z.pk017.o4 - proC lux-z.pk023.g7 - bioA lux-t.pk001.a1 osmY
lux-t.pk001.a2 inaA lux-t.pk001.a3 hisP1 lux-a.pk068.c1 hisP3
lux-t.pk001.a4 katG lux-t.pk001.a5 yebF lux-t.pk001.a6 flhB
lux-t.pk001.a7 ppa lux-t.pk001.a8 sgcR(yjhJ) lux-t.pk001.a9 flhC
lux-a.pk0022.d4 recA
[0327]
21TABLE 20 1st gene of the operon 45 minutes 90 minutes 135 minutes
b0116 (lpdA) >2.times. >2.times. * b0168 (map) >2.times.
>2.times. * b0767 (ybhE) >2.times. >2.times. >2.times.
b0842 >2.times. >2.times. >2.times. b1186 (nhaB)
>2.times. >2.times. >2.times. b1413 (hrpA) >2.times.
>2.times. >2.times. b1676 (pykF) >2.times. >2.times.
>2.times. b1993 (cobU) >2.times. >2.times. >2.times.
b2081 >2.times. >2.times. * b2999 >2.times. * * b3012
>2.times. >2.times. >2.times. b3904 (rhaB) >2.times.
>2.times. >2.times. b4106 (phnC) >2.times. >2.times.
>2.times. b4392 (slt) >2.times. >2.times. * b0314 (betT) *
>2.times. >2.times. b0417 * >2.times. >2.times. b0422
(xseB) * >2.times. * b0572 * >2.times. >2.times. b0593
(entC) * >2.times. >2.times. b0839 (dacC) * >2.times.
>2.times. b1188 (ycgB) * >2.times. * b1223 (narK) *
>2.times. >2.times. b1872 (bisZ) * >2.times. >2.times.
b2237 (inaA) * >2.times. >2.times. b2322 * >2.times.
>2.times. b2367 (emrY) * >2.times. * b2451 * >2.times.
>2.times. b2550 * >2.times. * b2699 (recA) * >2.times.
>2.times. b3245 * >2.times. >2.times. b3267 (yhdV) *
>2.times. >2.times. b3336 (bfr) * >2.times. * b3365 (nirB)
* >2.times. >2.times. b3419 (yhgJ) * >2.times.
>2.times. b3666 (uhpT) * >2.times. >2.times. b3942 (katG)
* >2.times. >2.times. b4043 (lexA) * >2.times. * b4264
(yjgS) * >2.times. b0005 * >2.times. b0123 (yacK) *
>2.times. b0386 (proC) * * >2.times. b0450 (glnK) * *
>2.times. b0774 (bioA) * * >2.times. b1847 (yebF) * *
>2.times. b1852 (zwf) * * >2.times. b2019 (hisG) * *
>2.times. b2114 (metG) * * >2.times. b2428 * * >2.times.
b3573 * * >2.times. b3779 (gppA) * * >2.times. b4226 (ppa) *
* >2.times.
[0328]
Sequence CWU 1
1
4 1 20 DNA Artificial Sequence Description of Artificial Sequence
primer 1 ggatcggaat tcccggggat 20 2 20 DNA Artificial Sequence
Description of Artificial Sequence primer 2 ctggccgtta ataatgaatg
20 3 25 DNA Artificial Sequence Description of Artificial Sequence
primer 3 ggaattgggg atcggagctc ccggg 25 4 25 DNA Artificial
Sequence Description of Artificial Sequence primer 4 gaatggcgcg
aattcggtac ccggg 25
* * * * *
References