U.S. patent application number 11/014621 was filed with the patent office on 2005-10-13 for quorum sensing signaling in bacteria.
This patent application is currently assigned to Aurora Biosciences Corporation. Invention is credited to Greenberg, E. Peter, Lee, Kimberly M., Muh, Ute, Whiteley, Marvin.
Application Number | 20050227345 11/014621 |
Document ID | / |
Family ID | 22545463 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227345 |
Kind Code |
A1 |
Whiteley, Marvin ; et
al. |
October 13, 2005 |
Quorum sensing signaling in bacteria
Abstract
The invention provides methods for identifying a modulator of
quorum sensing signaling in bacteria, and for identifying a quorum
sensing controlled gene in bacteria. In addition, the invention
provides quorum sensing controlled genetic loci in Pseudomas
aeruginosa. Novel indicator strains and vectors for engineering the
strains for use in the method of the invention are also
provided.
Inventors: |
Whiteley, Marvin;
(Coralville, IA) ; Lee, Kimberly M.; (Iowa City,
IA) ; Greenberg, E. Peter; (Iowa City, IA) ;
Muh, Ute; (Iowa City, IA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Aurora Biosciences
Corporation
11010 Torreyana Road
San Diego
CA
92121
UNIV OF IOWA RESEARCH FOUNDATION
214 Technology Innovation Center Oakdale Research Campus
Iowa City
IA
52242
|
Family ID: |
22545463 |
Appl. No.: |
11/014621 |
Filed: |
December 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11014621 |
Dec 15, 2004 |
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09653730 |
Sep 1, 2000 |
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6855513 |
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60153022 |
Sep 3, 1999 |
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Current U.S.
Class: |
435/252.34 ;
435/471; 536/23.7 |
Current CPC
Class: |
C07K 14/21 20130101 |
Class at
Publication: |
435/252.34 ;
435/471; 536/023.7 |
International
Class: |
C12N 001/21; C12N
015/74; C07H 021/04 |
Goverment Interests
[0002] This research was supported by grants and fellowships from
the National Institutes of Health (GM59026), and the National
Science Foundation (MCB9808308 and DBI9602247).
Claims
1.-45. (canceled)
46. An isolated nucleic acid molecule comprising a nucleotide
sequence, said nucleotide sequence comprising: a regulatory
sequence derived from the genome of Pseudomonas aeruginosa, wherein
said regulatory sequence regulates a quorum sensing controlled
genetic locus of the Pseudomonas aeruginosa chromosome, and wherein
said locus comprises a nucleotide sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36; and a
reporter gene operatively linked to said regulatory sequence.
47. An isolated nucleic acid molecule comprising a quorum sensing
controlled genetic locus derived from the genome of Pseudomonas
aeruginosa, wherein said locus comprises a nucleotide sequence
selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and
SEQ ID NO:36, operatively linked to a reporter gene.
48. An isolated nucleic acid molecule comprising a polynucleotide
having at least 80% identity to a quorum sensing controlled genetic
locus derived from the genome of Pseudomonas aeruginosa, wherein
said locus comprises a nucleotide sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, operatively
linked to a reporter gene.
49. An isolated nucleic acid molecule comprising a polynucleotide
that hybridizes under stringent conditions to the complement of a
nucleotide sequence comprising a quorum sensing controlled genetic
locus derived from the genome of Pseudomonas aeruginosa, wherein
said locus comprises a nucleotide sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ED NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, operatively
linked to a reporter gene.
50. The nucleic acid molecule of any one of claims 46, 47, 48 and
49, wherein said reporter gene is contained in a transposable
element.
51. A vector comprising the isolated nucleic acid molecule of any
one of claims 46, 47, 48 and 49.
52. A cell containing an isolated nucleic acid molecule of any one
of claims 46, 47, 48 and 49.
53.-74. (canceled)
75. The nucleic acid molecule of any one of claims 46, 47, 48 and
49, wherein said reporter gene is lacZ, GFP, or a variant thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/653,730, filed on Sep. 1, 2000, which
claims the benefit of U.S. Provisional Patent Application No.
60/153,022 filed on Sep. 3, 2000. The entire contents of each of
the aforementioned applications is hereby incorporated in its
entirety by reference.
BACKGROUND OF THE INVENTION
[0003] Bacteria communicate with each other to coordinate
expression of specific genes in a cell density dependent fashion.
This "bacterial signaling" is a phenomenon called quorum sensing
and response. Quorum sensing enables a bacterial species to sense
its own number and regulate gene expression according to population
density. In other words, quorum sensing is cell density-dependent
regulation of genes that involves a freely diffusible molecule
synthesized by the cell called an autoinducer (Fuqua, W. C. et al.
(1996) Annu. Rev. Microbiol. 50:727-751; Salmond, G. P. C. et al.
(1995) Mol. Microbiol. 16:615-624; Sitnikov, D. M. et al. (1995)
Mol. Microbiol. 17:801-812). Autoinducers are described, e.g., in
U.S. Pat. Nos. 5,591,872 and 5,593,827.
[0004] The paradigm system for quorum sensing is the lux system of
the luminescent marine bacterium, Vibrio fischeri. V. fischeri
exists at low cell densities in sea water and also at very high
cell densities within the light organs of various marine organisms,
such as the squid Euprymna scolopes (Pesci, E. C. et al. (1997)
Trends in Microbiol. 5(4): 132-135; Pesci, E. C. et al. (1997) J.
Bacteriol. 179:3127-3132; Ruby, E. G. (1996) Ann. Rev. Microbiol.
50:591-624). At high cell densities, the V. fischeri genes encoding
the enzymes required for light production are expressed. These
genes are part of the lux ICDABEG operon and are regulated by the
gene products of luxI and luxR (Baldwin, T. O. et al. (1989) J. of
Biolum. and Chemilum. 4:326-341; Eberhard, A., et al. (1991) Arch.
of Microbiol. 155:294-297; Gray, K. M. et al. (1992) J. Bacteriol.
174:4384-4390).
[0005] LuxI is an autoinducer synthase that catalyzes the formation
of the V. fischeri autoinducer (VAI), N-(3oxohexanoyl)homoserine
lactone (Eberhard, A., et al. (1991) Arch. of Microbiol.
155:294-297; Seed, P. C. et al. (1995) J. Bacteriol. 177:654-659).
The autoinducer freely diffuses across the cell membrane and at
high cell densities, reaches a critical concentration (Kaplan, H.
B. et al. (1985) J. Bacteriol. 163:1210-1214). At this critical
concentration, VAI interacts with LuxR, a DNA-binding
transcriptional regulator. The LuxR-VAI complex then binds to an
upstream sequence of the lux operon called the "lux box", and
activates transcription (Devine, J. H. et al. (1989) PNAS 86:
5688-5692; Hanzelka, B. A. et al. (1995) J Bacteriol. 177:815-817;
Stevens, A. M. et al. (1994) PNAS 91:12619-12623). Since one of the
genes of the operon is luxI, an autoregulatory loop is formed.
[0006] Many gram-negative bacteria have been shown to possess one
or more quorum sensing systems (Fuqua, W. C. et al. (1996) Annu.
Rev. Microbiol. 50:727-751; Salmond, G. P. C. et al. (1995) Mol.
Microbiol. 16:615-624). These systems regulate a variety of
physiological processes, including the activation of virulence
genes and the formation of biofilms. The systems typically have
acylated homoserine lactone ring autoinducers, in which the
homoserine lactone ring is conserved. The acyl side chain, however,
can vary in length and degree of substitution. In addition, it has
been recently demonstrated that quorum sensing is involved in
biofilm formation (Davies, D. G. et al. (1998) Science.
280(5361):295-8).
[0007] Biofilms are defined as an association of microorganisms,
single or multiple species, that grow attached to a surface and
produce a slime layer that provides a protective environment
(Costerton, J. W. (1995) J Ind Microbiol. 15(3):137-40, Costerton,
J. W. et al. (1995) Annu Rev Microbiol. 49:711-45). Typically,
biofilms produce large amounts of extracellular polysaccharides,
responsible for the slimy appearance, and are characterized by an
increased resistance to antibiotics (1000- to 1500-fold less
susceptible). Several mechanisms are proposed to explain this
biofilm resistance to antimicrobial agents (Costerton, J. W. et al.
(1999) Science. 284(5418):1318-22). One idea is that the
extracellular matrix in which the bacterial cells are embedded
provides a barrier toward penetration by the biocides. A further
possibility is that a majority of the cells in a biofilm are in a
slow-growing, nutrient-starved state, and therefore not as
susceptible to the effects of anti-microbial agents. A third
mechanism of resistance could be that the cells in a biofilm adopt
a distinct and protected biofilm phenotype, e.g., by elevated
expression of drug-efflux pumps.
[0008] In most natural settings, bacteria grow predominantly in
biofilms. Biofilms of P. aeruginosa have been isolated from medical
implants, such as indwelling urethral, venous or peritoneal
catheters (Stickler, D. J. et al. (1998) Appl Environ Microbiol.
64(9):3486-90). Chronic P. aeruginosa infections in cystic fibrosis
lungs are considered to be biofilms (Costerton, J. W. et al. (1999)
Science. 284(5418):1318-22).
[0009] In industrial settings, the formation of biofilms is often
referred to as `biofouling`. Biological fouling of surfaces is
common and leads to material degradation, product contamination,
mechanical blockage, and impedance of heat transfer in
water-processing systems. Biofilms are also the primary cause of
biological contamination of drinking water distribution systems,
due to growth on filtration devices.
[0010] As noted earlier, many gram-negative bacteria have been
shown to possess one or more quorum sensing systems that regulate a
variety of physiological processes, including the activation of
virulence genes and biofilm formation. One such gram negative
bacterium is Pseudomonas aeruginosa.
[0011] P. aeruginosa is a soil and water bacterium that can infect
animal hosts. Normally, the host defense system is adequate to
prevent infection. However, in immunocompromised individuals (such
as burn patients, patients with cystic fibrosis, or patients
undergoing immunosuppressive therapy), P. aeruginosa is an
opportunistic pathogen, and infection with P. aeruginosa can be
fatal (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74; Van
Delden, C. et al. (1998) Emerg Infect Dis. 4(4):551-60).
[0012] For example, Cystic fibrosis (CF), the most common inherited
lethal disorder in Caucasian populations (.about.1 out of 2,500
life births), is characterized by bacterial colonization and
chronic infections of the lungs. The most prominent bacterium in
these infections is P. aeruginosa--by their mid-twenties, over 80%
of people with CF have P. aeruginosa in their lungs (Govan, J. R.
et al. (1996) Microbiol Rev. 60(3):539-74). Although these
infections can be controlled for many years by antibiotics,
ultimately they "progress to mucoidy," meaning that the P.
aeruginosa forms a biofilm that is resistant to antibiotic
treatment. At this point the prognosis is poor. The median survival
age for people with CF is the late 20s, with P. aeruginosa being
the leading cause of death (Govan, J. R. et al. (1996) Microbiol
Rev. 60(3):539-74). According to the Cystic Fibrosis Foundation,
treatment of CF cost more than $900 million in 1995 (Foundation, CF
http://www.cff.org/homeline199701.htm).
[0013] P. aeruginosa is also one of several opportunistic pathogens
that infect people with AIDS, and is the main cause of bacteremia
(bacterial infection of the blood) and pneumonitis in these
patients (Rolston, K. V. et al. (1990) Cancer Detect Prev.
14(3):377-81; Witt, D. J. et al. (1987) Am J Med. 82(5):900-6). A
recent study of 1635 AIDS patients admitted to a French hospital
between 1991-1995 documented 41 cases of severe P. aeruginosa
infection (Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
Seventeen of these had bacteremia, which was lethal in 8 cases.
Similar, numbers were obtained in a smaller study in a New York
hospital, where the mortality rate for AIDS patients admitted with
P. aeruginosa bacteremia was about 50% (Mendelson, M. H. et al.
1994. Clin Infect Dis. 18(6):886-95).
[0014] In addition, about two million Americans suffer serious
burns each year, and 10,000-12,000 die from their injuries. The
leading cause of death is infection (Lee, J. J. et al. (1990) J
Burn Care Rehabil. 11(6):575-80). P. aeruginosa bacteremia occurs
in 10% of seriously burned patients, with a mortality rate of 80%
(Mayhall, C. G. (1993) p. 614-664, Prevention and control of
nosocomial infections. Williams & Wilkins, Baltimore; McManus,
A. T et al. (1985) Eur J Clin Microbiol. 4(2):219-23).
[0015] Such infections are often acquired in hospitals ("nosocomial
infections") when susceptible patients come into contact with other
patients, hospital staff, or equipment. In 1995 there were
approximately 2 million incidents of nosocomial infections in the
U.S., resulting in 88,000 deaths and an estimated cost of $ 4.5
billion (Weinstein, R. A. (1998) Emerg Infect Dis. 4(3):416-20). Of
the AIDS patients mentioned above who died of P. aeruginosa
bacteremia, more than half acquired these infections in hospitals
(Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
[0016] Nosocomial infections are especially common in patients in
intensive care units as these people often have weakened immune
systems and are frequently on ventilators and/or catheters.
Catheter-associated urinary tract infections are the most common
nosocomial infection (Richards, M. J. et al. (1999) Crit Care Med.
27(5):887-92) (31% of the total), and P. aeruginosa is highly
associated with biofilm growth and catheter obstruction. While the
catheter is in place, these infections are difficult to eliminate
(Stickler, D. J. et al. (1998) Appl Environ Microbiol.
64(9):3486-90). The second most frequent nosocomial infection is
pneumonia, with P. aeruginosa the cause of infection in 21% of the
reported cases (Richards, M. J. et al. (1999) Crit Care Med.
27(5):887-92). The annual costs for diagnosing and treating
nosocomial pneumonia has been estimated at greater than $2 billion
(Craven, D. E. et al. (1991) Am J Med. 91(3B):44S-53S).
[0017] Treatment of these so-called nosocomial infections is
complicated by the fact that bacteria encountered in hospital
settings are often resistant to many antibiotics. In June 1998, the
National Nosocomial Infections Surveillance (NNIS) System reported
increases in resistance of P. aeruginosa isolates from intensive
care units of 89% for quinolone resistance and 32% for imipenem
resistance compared to the years 1993-1997 (NNIS.
http://www.cdc.gov/ncidod/hip/NNIS/AR_Surv1198.htm). In fact, some
strains of P. aeruginosa are resistant to over 100 antibiotics
(Levy, S. (1998) Scientific American. March). There is a critical
need to overcome the emergence of bacterial strains that are
resistant to conventional antibiotics (Travis, J. (1994) Science.
264:360-362).
[0018] P. aeruginosa is also of great industrial concern (Bitton,
G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y.;
Steelhammer, J. C. et al. (1995) Indust. Water Treatm.:49-55). The
organism grows in an aggregated state, the biofilm, which causes
problems in many water processing plants. Of particular public
health concern are food processing and water purification plants.
Problems include corroded pipes, loss of efficiency in heat
exchangers and cooling towers, plugged water injection jets leading
to increased hydraulic pressure, and biological contamination of
drinking water distribution systems (Bitton, G. (1994) Wastewater
Microbiology. Wiley-Liss, New York, N.Y., 9). The elimination of
biofilms in industrial equipment has so far been the province of
biocides. Biocides, in contrast to antibiotics, are antimicrobials
that do not possess high specificity for bacteria, so they are
often toxic to humans as well. Biocide sales in the US run at about
$ 1 billion per year (Peaff, G. (1994) Chem. Eng. News:15-23).
[0019] A particularly ironic connection between industrial water
contamination and public health issues is an outbreak of P.
aeruginosa peritonitis that was traced back to contaminated
poloxamer-iodine solution, a disinfectant used to treat the
peritoneal catheters. P. aeruginosa is commonly found to
contaminate distribution pipes and water filters used in plants
that manufacture iodine solutions. Once the organism has matured
into a biofilm, it becomes protected against the biocidal activity
of the iodophor solution. Hence, a common soil organism that is
harmless to the healthy population, but causes mechanical problems
in industrial settings, ultimately contaminated antibacterial
solutions that were used to treat the very people most susceptible
to infection.
[0020] Regulation of virulence genes by quorum sensing is well
documented in P. aeruginosa. Recently, genes not directly involved
in virulence including the stationary phase sigma factor rpoS and
genes coding for components of the general secretory pathway (xcp)
(Jamin, M. et al. (1991) Biochem J. 280(Pt 2):499-506) have been
reported to be positively regulated by quorum sensing. Furthermore,
the las quorum sensing system is required for maturation of P.
aeruginosa biofilms (Chapon-Herve, V. et al. (1997) Mol. Microbiol.
24, 1169-1170; Davies, D. G., et al. (1998) Science 280, 295-298).
Thus it seems clear that quorum sensing represents a global gene
regulation system in P. aeruginosa. However, the number and types
of genes controlled by quorum sensing have not been identified or
studied extensively.
SUMMARY OF THE INVENTION
[0021] In general, the invention pertains to the modulation of
bacterial cell-to-cell signaling. The inhibition of quorum sensing
signaling renders a bacterial population more susceptible to
treatment, either directly through the host immune-response or in
combination with traditional antibacterial agents and biocides.
More particularly, the invention also pertains to a method for
identifing modulators, e.g., inhibitors of cell-to-cell signaling
in bacteria, and in particular one particular human pathogen,
Pseudomonas aeruginosa.
[0022] Thus, in one aspect, the invention is a method for
identifying a modulator of quorum sensing signaling in bacteria.
The method comprises:
[0023] providing a cell comprising a quorum sensing controlled
gene, wherein the cell is responsive to a quorum sensing signal
molecule such that a detectable signal is generated;
[0024] contacting said cell with a quorum sensing signal molecule
in the presence and absence of a test compound;
[0025] and detecting a change in the detectable signal to thereby
identify the test compound as a modulator of quorum sensing
signaling in bacteria.
[0026] In one embodiment the cell comprises a reporter gene
operatively linked to a regulatory sequence of a quorum sensing
controlled gene, such that the quorum sensing signal molecule
modulates the transcription of the reporter gene, thereby providing
a detectable signal.
[0027] Another aspect of the invention is a method for identifying
a modulator of a quorum sensing signaling in Pseudomonas
aeruginosa. The method comprises:
[0028] providing a wild type strain of Pseudomonas aeruginosa which
produces a quorum sensing signal molecule;
[0029] providing a mutant strain of Pseudomonas aeruginosa which
comprises a reporter gene operatively linked to a regulatory
sequence of a quorum sensing controlled gene, wherein the mutant
strain is responsive to the quorum sensing signal molecule produced
by the wild type strain, such that a detectable signal is
generated;
[0030] contacting the mutant strain with the quorum sensing signal
molecule and a test compound; and
[0031] detecting a change in the detectable signal to thereby
identify the test compound as a modulator of quorum sensing
signaling in Pseudomonas aeruginosa.
[0032] In one embodiment, the endogenous lasI and rhlI quorum
sensing systems are inactivated in the mutant strain of Pseudomonas
aeruginosa. In another embodiment the mutant strain of Pseudomonas
aeruginosa comprises a promoterless reporter gene inserted at a
genetic locus in the chromosome, wherein the genetic locus
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
[0033] A further aspect of the invention is a mutant strain of
Pseudomonas aeruginosa comprising a promoterless reporter gene
inserted at a genetic locus in the chromosome, wherein the genetic
locus comprises a nucleotide sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ D NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.
[0034] In one embodiment, the endogenous lasI and rhlI quorum
sensing systems are inactivated in the mutant strain of Pseudomonas
aeruginosa. In another embodiment the mutant strain of Pseudomonas
aeruginosa is responsive to a quorum sensing signal molecule such
that a detectable signal is generated by the reporter gene. In yet
another embodiment, the reporter gene is contained in a
transposable element.
[0035] Yet another aspect of the invention is a method for
identifying a modulator of quorum sensing signaling in Pseudomonas
aeruginosa. The method comprises:
[0036] providing a wild type strain of Pseudomonas aeruginosa which
produces a quorum sensing signal molecule;
[0037] providing a mutant strain of Pseudomonas aeruginosa which
comprises a promoterless reporter gene inserted at a genetic locus
in the chromosome of said Pseudomonas aeruginosa, wherein the
genetic locus comprises a nucleotide sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36; and
wherein the mutant strain is responsive to the quorum sensing
signal molecule produced by the wild type strain, such that a
detectable signal is generated by the reporter gene;
[0038] contacting the mutant strain with the quorum sensing signal
molecule and a test compound; and
[0039] detecting a change in the detectable signal to thereby
identify the test compound as a modulator of quorum sensing
signaling in Pseudomonas aeruginosa.
[0040] Another aspect of the invention is an isolated nucleic acid
molecule comprising a nucleotide sequence which comprises:
[0041] a regulatory sequence derived from the genome of Pseudomonas
aeruginosa, wherein the regulatory sequence regulates a quorum
sensing controlled genetic locus of the Pseudomonas aeruginosa
chromosome, and wherein the genetic locus comprises a nucleotide
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35
and SEQ ID NO:36; and
[0042] a reporter gene operatively linked to the regulatory
sequence.
[0043] A further aspect of the invention provides an isolated
nucleic acid molecule comprising a quorum sensing controlled
genetic locus derived from the genome of Pseudomonas aeruginosa,
wherein the genetic locus comprises a nucleotide sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID
NO:36, operatively linked to a reporter gene.
[0044] In one embodiment, the invention is an isolated nucleic acid
molecule comprising a polynucleotide having at least 80% identity
to a quorum sensing controlled genetic locus derived from the
genome of Pseudomonas aeruginosa, wherein the genetic locus
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, operatively linked to a
reporter gene.
[0045] In another embodiment, the invention is an isolated nucleic
acid molecule comprising a a polynucleotide that hybridizes under
stringent conditions to a quorum sensing controlled genetic locus
derived from the genome of Pseudomonas aeruginosa, wherein the
genetic locus comprises a nucleotide sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36,
operatively linked to a reporter gene.
[0046] In one embodiment, an isolated nucleic acid molecule of the
invention comprises a reporter gene contained in a transposable
element.
[0047] Accordingly, a further aspect of the invention pertains to a
vector comprising an isolated nucleic acid molecule of the
invention. In another aspect, the invention provides cells
containing an isolated nucleic acid molecule of the invention.
[0048] An additional aspect of the invention is a method for
identifying a modulator of quorum sensing signaling in bacteria.
The method comprises:
[0049] providing a cell containing an isolated nucleic acid
molecule of the invention, wherein the cell is responsive to a
quorum sensing signal molecule such that a detectable signal is
generated;
[0050] contacting said cell with a quorum sensing signal molecule
in the presence and absence of a test compound;
[0051] and detecting a change in the detectable signal to therby
identify the test compound as a modulator of quorum sensing
signaling in bacteria.
[0052] Accordingly, in another aspect, the invention provides a
compound identified by a method of the invention which modulates,
e.g., inhibits, quorum sensing signaling in Pseudomonas aeruginosa.
In one embodiment, the compound inhibits quorum sensing signaling
in Pseudomonas aeruginosa by inhibiting an enzyme involved in the
synthesis of a quorum sensing signal molecule, by interfering with
quorum sensing signal reception, or by scavenging the quorum
sensing signal molecule.
[0053] The invention also pertains to a method for identifing
quorum sensing controlled genes in a cell, and specifically in one
particular human pathogen, Pseudomonas aeruginosa. Thus, in one
aspect, the invention provides a method for identifying a quorum
sensing controlled gene in a cell, the method comprising:
[0054] providing a cell which is responsive to a quorum sensing
signal molecule such that expression of a quorum sensing controlled
gene is modulated, and wherein modulation of the expression of said
quorum sensing controlled gene generates a detectable signal;
[0055] contacting said cell with a quorum sensing signal
molecule;
[0056] and detecting a change in the detectable signal to thereby
identify a quorum sensing signaling controlled gene.
[0057] In one embodiment the cell comprises a reporter gene
operatively linked to a quorum sensing controlled gene or a
regulatory sequence of a quorum sensing controlled gene, such that
modulation of the expression of the quorum sensing controlled gene
modulates the transcription of the reporter gene, thereby providing
a detectable signal. In another embodiment the reporter gene is
contained in a transposable element. In yet another embodiment, the
quorum sensing signal molecule is produced by a second cell, e.g.,
a bacterial cell. In a further embodiment, the quorum sensing
signal molecule is an autoinducer of said quorum sensing controlled
gene, e.g., a homoserine lactone, or an analog thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 depicts the paragdigm for quorum sensing signaling in
the target bacterium, Pseudomonas aeruginosa.
[0059] FIG. 2 depicts patterns of .beta.-galactosidase expression
in representative qsc mutants and in a strain with a lasB::lacZ
chromosomal fusion generated by site-specific mutation. Units of
.beta.-galactosidase are given as a function of culture density for
cells grown without added signal molecules (.largecircle.), with
added 3OC.sub.12-HSL (.circle-solid.), with added C.sub.4-HSL
(.box-solid.), or with both signals added (.quadrature.).
[0060] FIG. 3 depicts the nucleic acid sequence of the quorum
sensing controlled locus on the P. aeruginosa chromosome mapped in
the P. aeruginosa mutant strain qsc102.
[0061] FIG. 4 depicts putative qsc operons. Open reading frames
(ORFs) are indicated by the arrows. ORFs discovered in the qsc
screen are indicated by their qsc number.
[0062] FIG. 5 depicts a growth curve of PAO1/pMW303G. Culture
growth is monitored at 600 nm (closed circles) and
.beta.-galactosidase activity is measured with a chemiluminescent
substrate analog in relative light units (RLU; open circles).
[0063] FIG. 6 is a map of the qsc insertions on the P. aeruginosa
chromosome. Arrowheads indicate the direction of lacZ
transcription. In addition to the qsc mutants, lasR and lasI, rhlR,
and lasB are also mapped. The locations of las-boxes like elements
are shown as black dots between the two DNA strands. The numbers
indicate distance in megabases on the approximately 6 megabase
chromosome.
[0064] FIG. 7 depicts putative las-type boxes in upstream DNA
regions of qsc mutants. ORFs as described in Materials and Methods.
Bases outlined in black represent residues conserved in all
sequences and gray outlines are conserved in 8 of 10 sequences.
[0065] FIG. 8 depicts the principle of a bioassay for modulators of
quorum sensing signaling. Strain PAO1 produces the signal
3-oxo-C12-HSL. Strain QSC102 responds by inducing lacZ.
[0066] FIG. 9 depicts the results of an assay performed using the
test compound acetyl-butyrolactone, which is present in the wells
at increasing concentration (mM, as indicated). There are two rows
and two columns per concentration to show reproducibily of the
assay.
[0067] FIG. 10A depicts the structure of a mobilizable plasmid for
generating an indicator strain. Filled boxes represent chromosomal
DNA derived from the P. aeruginosa locus where lacZ is inserted in
strain QSC102.
[0068] FIG. 10B depicts induction of .beta.-galactosidase as PAQ1
reaches high density. Cell growth is monitored at 600 nm (closed
circles) and expression of .beta.-galactosidase is measured in
Miller units (open circles).
[0069] FIG. 11 depicts the reaction mechanism of the RhlI
autoinducer synthase.
[0070] FIG. 12 depicts a continuous culture bioreactor.
DETAILED DESCRIPTION OF THE INVENTION
[0071] In gram-negative bacteria, such as Pseudomonas aeruginosa,
quorum sensing involves two proteins, the autoinducer synthase--the
I protein--and the transcriptional activator--the R protein. The
synthase produces an acylated homoserine lactone (the
"autoinducer"; see structure 1 below), which can diffuse into the
surrounding environment (Fuqua, C. et al. (1998) Curr Opin
Microbiol. 1(2):183-189; Fuqua, et al. 1994. J Bacteriol.
176(2):269-75). The autoinducer molecule is composed of an acyl
chain in a peptide bond with the amino nitrogen of a homoserine
lactone (HSL). For different quorum sensing systems, the side-chain
may vary in length, degree of saturation, and oxidation state. As
the density of bacteria increases, so does the concentration of
this freely diffusible signal molecule. Once the concentration
reaches a defined threshold, it binds to the R-protein, which then
activates transcription of numerous genes. Of particular interest
are genes involved in pathogenicity and in biofilm formation (see
FIG. 1).
[0072] Pseudomonas aeruginosa has two quorum sensing systems, las
and rhl, named for their role in the expression of elastase, and
the RhlI/RhIR proteins, which were first described for their role
in rhamnolipid biosynthesis. (Hanzelka, B. A. et al. (1996) J.
Bacteriol. 178:5291-5294; Baldwin, T. O. et al. (1989) J. of
Biolum. and Chemilum. 4:326-341; Passador, L., et al. (1993)
Science 260:1127-1130; Pearson, J. P et al. (1994) PNAS 91:197-201;
Pesci, E. C. et al. (1997) Trends in Microbiol. 5(4):132-135;
Pesci, E. C. et al. (1997) J. Bacteriol. 179:3127-3132). The two
systems have distinct autoinducer synthases (lasI and rhlI),
transcriptional regulators (lasR and rhlR), and autoinducers
(N-(3-oxododecanoyl)homoserine lactone (HSL) and N-butyryl HSL)
(Sitnikov, D. M. et al. (1995) Mol. Microbiol. 17:801-812). The rhl
and las systems are involved in regulating the expression of a
number of secreted virulence factors, biofilm development, and the
stationary phase sigma factor (RpoS) (Davies, D. G. et al. (1998)
Science 280:295-298; Latifi, A. et al. (1995) Mol. Microbiol. Rev.
17:333-344; Ochsner, U. A., et al. (1995) PNAS, 92:6424-6428;
Pesci, E. C. et al. (1997) Trends in Microbiol. 5(4):132-135;
Pesci, E. C. et al. (1997) J. Bacteriol. 179:3127-3132). Expression
of the rhl system requires a functional las system, therefore the
two systems in combination with RpoS constitute a regulatory
cascade (Pesci, E. C. et al. (1997) Trends in Microbiol.
5(4):132-135; Pesci, E. C. et al. (1997) J. Bacteriol.
179:3127-3132, Seed et al. 1995).
[0073] The signal in the Las system is 3-oxo-dodecanoyl-HSL
(3-oxo-C12-HSL) 2, while the signal used in the Rhl system is
butanoyl-HSL (C4-HSL) 3. It has been shown that 3-oxo-C12-HSL
increases expression of RhIR, indicating a hierarchy of regulation
systems (Pesci, E. C. et al. (1997) Trends Microbiol. 5(4):132-4).
The Las signal 3-oxo-C12-HSL is synthesized by LasI along with a
small amount of N-(3-oxooctanoyl) HSL and N-(3-oxohexanoyl) HSL,
while RhlI makes primarily the signal C4-HSL and a small amount of
N-hexanoyl (Pearson, J. P. et al. (1997) J. Bacteriol.
179:5756-5757; Winson, M. K. et al. (1995) PNAS 92:9427-9431).
1
[0074] Bacterial signaling triggers the expression of a number of
virulence factors in P. aeruginosa including two elastases, an
alkaline protease and exotoxin A (Pesci, E. C. et al. (1997) Trends
Microbiol. 5(4): 132-4; Pesci, E. C. et al. (1997) J Bacteriol.
179(10):3127-32)--proteins that allow the organism to attack host
tissue. Bacterial signaling also controls the expression of the
antioxidant pyocyanin, a compound that allows the bacteria to
neutralize one important host defense, the generation of superoxide
radicals (Britigan, et al. (1999) Infect Immun. 67(3):1207-12,
Hassan, H. M. et al. (1979) Arch Biochem Biophys. 196(2):385-95,
Hassan, H. M. et al. 1980. J Bacteriol. 141(1):156-63). It has been
shown in a neonatal mouse model that a defined mutant of P.
aeruginosa which lacks the signal receptor protein (LasR) was
significantly less virulent than the wild type PAO1, as measured by
the ability to cause acute pneumonia, bacteremia and death (Tang,
H. B. et al. (1996) Infect Immun. 64(1):37-43).
[0075] The invention is based on the interruption of bacterial
cell-to-cell singaling, i.e., quorum sensing signaling in order to
render a bacterial population more susceptible to treatment, either
through the host immune-response or in combination with traditional
antibacterial agents and biocides. Thus, the invention provides a
bacterial indicator strain that allows for a high throughput
screening assay for identifying compounds that modulate, e.g.,
inhibit bacterial cell-to-cell signalling. The compounds so
identified will provide novel anti-pathogenics and anti-fouling
agents.
DEFINITIONS
[0076] Before further description of the invention, certain terms
employed in the specification, examples and appended claims are,
for convenience, collected here.
[0077] The term "analog" as in "homoserine lactone analog" is
intended to encompass compounds that are chemically and/or
electronically similar but have different atoms, such as isosteres
and isologs. An analog includes a compound with a structure similar
to that of another compound but differing from it in respect to
certain components or structural makeup. The term analog is also
intened to encompass stereoisomers.
[0078] The language "autoinducer compounds" is art-recognized and
is intended to include molecules, e.g., proteins which freely
diffuse across cell membranes and which activate transcription of
various factors which affect bacterial viability. Such compounds
can affect virulence, and biofilm development. Autoinducer
compounds can be acylated homoserine lactones. They can be other
compounds similar to those listed in Table 1. Homoserine
autoinducer compounds are produced in vivo by the interaction of a
homoserine lactone substrate and an acylated acyl carrier protein
in a reaction catalyzed by an autoinducer synthase molecule. In
isolated form, autoinducer compounds can be obtained from naturally
occurring proteins by purifying cellular extracts, or they can be
chemically synthesized or recombinantly produced. The language
"autoinducer synthase molecule" is intended to include molecules,
e.g. proteins, which catalyze or facilitate the synthesis of
autoinducer compounds, e.g. in the quorum sensing system of
bacteria. It is also intended to include active portions of the
autoinducer synthase protein contained in the protein or in
fragments or portions of the protein (e.g., a biologically active
fragment). The language "active portions" is intended to include
the portion of the autoinducer synthase protein which contains the
homoserine lactone binding site.
[0079] Table 1 contains a list of exemplary autoinducer synthase
proteins of the quorum sensing systems of various gram-negative
bacteria.
1TABLE 1 Summary of N-acyl homoserine lactone based regulatory
systems Regulatory Bacterial species Signal molecules.sup.a
proteins.sup.b Target function(s) Vibrio fischeri
N-3-(oxohexanoyl)- LuxI/LuxR luxICDABEG, homoserine lactone luxR
(VAI-1) luminescence N-(octanoyl)-L-homoserine AinS/AinR.sup.c
luxICDABEG, ? lactone (VAI-2) Vibrio harveyi
N-.beta.-(hydroxybutyryl)- LuxM/LuxN luxICDABEG, homoserine lactone
LuxO-LuxR.sup.d luminescence and (HAI-1) polyhydroxybutyrate
synthesis HAI-2 Lux?/LuxPQ- luxCDABEG LuxO-LuxR.sup.d Pseudomonas
N-3-(oxododecanyoyl)-L- LasI/LasR lasB, lasA, aprA, toxA,
aeruginosa homoserine lactone virulence factors (PAI-1)
N-(butyryl)-L-homoserine RhII/RhIR rhlAB, rhamnolipid lactone
synthesis, virulence (PAI-2) factors Pseudomonas (PRAI).sup.e
PhzI/PhzR phz, phenazine aeureofaciens biosynthesis Agroacterium
N-3-(oxooctanoyl)-L- TraI/TraR-TraM tra gens, traR, Ti tumefaciens
homoserine lactone plasmid conjugal (AAI) transfer Erwinia
carotovora VAI-1.sup.f ExpI/ExpR pel, pec, pep, subsp. carotovora
exoenzyme synthesis SCR1193 Erwinia carotovora VAI-1.sup.f
CarI/CarR cap, carbapenem subsp. carotovora antibiotic synthesis
SCC3193 Erwinia carotovora VAI-1.sup.f HsII/? pel, pec, pep, subsp.
carotovora exoenzyme synthesis 71 Erwinia stewartii VAI-1.sup.f
EsaI/EsaR wts genes, exopolysaccharide synthesis, virulence factors
Rhizobium N-(3R-hydroxy-7-cis- ?/RhiR rhiABC, rhizosphere
leguminosarum tetradecanoyl-L-homoserine genes and stationary
lactone, small bacteriocin, phase (RLAI) Enterobacter VAI-1.sup.f
EagI/EagR function unclear agglomerans Yersenia VAI-1.sup.f
YenI/YenR function unclear enterocolitica Serratia liquifaciens
N-butanoyl-L-homoserine SwrI/? swarming motility lacton (SAI-1)
N-hexanoyl-L-homoserine SwrI/? swarming motility lacton (SAI-2)
Aeromonas (AHAI).sup.e AhyI/AhyR function unclear hydrophila
Escherichia coli/?.sup.g ?/SdiA ftsQAZ, cell division
[0080] Autoinducer synthase molecules can be obtained from
naturally occurring sources, e.g., by purifying cellular extracts,
can be chemically synthesized or can be recombinantly produced.
Recombinantly produced autoinducer synthase molecules can have the
amino acid sequence of a a naturally occurring form of the
autoinducer synthase protein. They can also have a similar amino
acid sequence which includes mutations such as substitutions and
deletions (including truncation) of a naturally occurring form of
the protein. Autoinducer synthase molecules can also include
molecules which are structurally similar to the structures of
naturally occurring autoinducer synthase proteins, e.g.,
biologically active variants.
[0081] TraI, LuxI, RhlI are the homoserine lactone autoinducer
synthases of Agrobacterium tumefaceins, Vibrio fischeri, and
Pseudomonas aeruginosa, respectively. The term "RhlI" is intended
to include proteins which catalyze the synthesis of the homoserine
lactone autoinducer of the RhlI quorum sensing system of P.
aeruginosa, butyryl homoserine lactone.
[0082] The term "biofilm" is intended to include biological films
that develop and persist at interfaces in aqueous environments.
Biofilms are composed of microorganisms embedded in an organic
gelatinous structure composed of one or more matrix polymers which
are secreted by the resident microorganisms. The language "biofilm
development" or "biofilm formation" is intended to include the
formation, growth, and modification of the bacterial colonies
contained with the biofilm structures as well as the synthesis and
maintenance of the exopolysaccharide matrix of the biofilm
structures.
[0083] The term "compound" as used herein (e.g., as in "test
compound," or "modulator compound") is intended to include both
exogenously added test compounds and peptides endogenously
expressed from a peptide library. Test compounds may be purchased,
chemically synthesized or recombinantly produced. Test compounds
can be obtained from a library of diverse compounds based on a
desired activity, or alternatively they can be selected from a
random screening procedure. In one embodiment, an indicator cell
(e.g., a cell which responds to quorum sensing signals by
generating a detectable signal) also produces the test compound
which is being screened. For instance, the indicator cell can
produce, e.g., a test polypeptide, a test nucleic acid and/or a
test carbohydrate, which is screened for its ability to modulate
quorum sensing signaling. In such embodiments, a culture of such
reagent cells will collectively provide a library of potential
modulator molecules and those members of the library which either
stimulate or inhibit quorum sensing signaling can be selected and
identified. In another embodiment, a test compound is produced by a
second cell which is co-incubated with the indicator cell.
[0084] The terms "derived from" or "derivative", as used
interchangeably herein, are intended to mean that a sequence is
identical to or modified from another sequence, e.g., a naturally
occurring squence. Derivatives within the scope of the invention
include polynucleotide derivatives. Polynucleotide or nucleic acid
derivatives differ from the sequences described herein (e.g., SEQ
ID Nos.: 1-38) or known in nucleotide sequence. For example, a
polynucleotide derivative may be characterized by one or more
nucleotide substitutions, insertions, or deletions, as compared to
a reference sequence. A nucleotide sequence comprising a quorum
sensing controlled genetic locus that is derived from the genome of
P. aeruginosa, e.g., SEQ ID Nos.: 1-38, includes sequences that
have been modified by various changes such as insertions, deletions
and substitutions, and which retain the property of being regulated
in response to a quorum sensing signaling event. Such sequences may
comprise a quorum sensing controlled regulatory element and/or a
quorum sensing controlled gene. The nucleotide sequence of the P.
aeruginosa genome is available at www.pseudomonas.com.
[0085] Polypeptide or protein derivatives include polypeptide or
protein sequences that differ from the sequences described or known
in amino acid sequence, or in ways that do not involve sequence, or
both, and still preserve the activity of the polypeptide or
protein. Derivatives in amino acid sequence are produced when one
or more amino acids is substituted with a different natural amino
acid, an amino acid derivative or non-native amino acid. In certain
embodiments protein derivatives include naturally occurring
polypeptides or proteins, or biologically active fragments thereof,
whose sequences differ from the wild type sequence by one or more
conservative amino acid substitutions, which typically have minimal
influence on the secondary structure and hydrophobic nature of the
protein or peptide. Derivatives may also have sequences which
differ by one or more non-conservative amino acid substitutions,
deletions or insertions which do not abolish the biological
activity of the polypeptide or protein.
[0086] Conservative substitutions (substituents) typically include
the substitution of one amino acid for another with similar
characteristics (e.g., charge, size, shape, and other biological
properties) such as substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. The non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0087] In other embodiments, derivatives with amino acid
substitutions which are less conservative may also result in
desired derivatives, e.g., by causing changes in charge,
conformation and other biological properties. Such substitutions
would include, for example, substitution of hydrophilic residue for
a hydrophobic residue, substitution of a cysteine or proline for
another residue, substitution of a residue having a small side
chain for a residue having a bulky side chain or substitution of a
residue having a net positive charge for a residue having a net
negative charge. When the result of a given substitution cannot be
predicted with certainty, the derivatives may be readily assayed
according to the methods disclosed herein to determine the presence
or absence of the desired characteristics. The polypeptides and
proteins of this invention may also be modified by various changes
such as insertions, deletions and substitutions, either
conservative or nonconservative where such changes might provide
for certain advantages in their use.
[0088] As used herein, the term "genetic locus" includes a position
on a chromosome, or within a genome, which is associated with a
particular gene or genetic sequences having a particular
characteristic. For example, in one embodiment, a quorum sensing
controlled genetic locus includes nucleic acid sequences which
comprise an open reading frame (ORF) of a quorum sensing controlled
gene. In another embodiment, a quorum sensing controlled genetic
locus includes nucleic acid sequences which comprise
transcriptional regulatory sequences that are responsive to quorum
sensing signaling (e.g., a quorum sensing controlled regulatory
element). Examples of quorum sensing controlled genetic loci of P.
aeruginosa are described herein as SEQ ID NOs.: 1-38.
[0089] The term "modulator", as in "modulator of quorum sensing
signaling" is intended to encompass, in its various grammatical
forms, induction and/or potentiation, as well as inhibition and/or
downregulation of quorum sensing signaling and/or quorum sensing
controlled gene expression. As used herein, the term "modulator of
quorum sensing signaling" includes a compound or agent that is
capable of modulating or regulating at least one quorum sensing
controlled gene or quorum sensing controlled genetic locus, e.g., a
quorum sensing controlled genetic locus in P. aeruginosa, as
described herein. A modulator of quorum sensing signaling may act
to modulate either signal generation (e.g., the synthesis of a
quorum sensing signal molecule), signal reception (e.g., the
binding of a signal molecule to a receptor or target molecule), or
signal transmission (e.g., signal transduction via effector
molecules to generate an appropriate biological response). In one
embodiment, a method of the present invention encompasses the
modulation of the transcription of an indicator gene in response to
an autoinducer molecule. In another embodiment, a method of the
present invention encompasses the modulation of the transcription
of an indicator gene, preferably an quorum sensing controlled
indicator gene, by a test compound.
[0090] The term "operatively linked" or "operably linked" is
intended to mean that molecules are functionally coupled to each
other in that the change of activity or state of one molecule is
affected by the activity or state of the other molecule. In one
embodiment, nucleotide sequences are "operatively linked" when the
regulatory sequence functionally relates to the DNA sequence
encoding the polypeptide or protein of interest. For example, a
nucleotide sequence comprising a transcriptional regulatory
element(s) (e.g., a promoter) is operably linked to a DNA sequence
encoding the protein or polypeptide of interest if the promoter
nucleotide sequence controls the transcription of the DNA sequence
encoding the protein of interest. In addition, two nucleotide
sequences are operatively linked if they are coordinately regulated
and/or transcribed. Typically, two polypeptides that are
operatively linked are covalently attached through peptide
bonds.
[0091] The term "quorum sensing signaling" or "quorum sensing" is
intended to include the generation of a cellular signal in response
to cell density. In one embodiment, quorum sensing signaling
mediates the coordinated expression of specific genes. A "quorum
sensing controlled gene" is any gene, the expression of which is
regulated in a cell density dependent fashion. In a preferred
embodiment, the expression of a quorum sensing controlled gene is
modulated by a quorum sensing signal molecule, e g., an autoinducer
molecule (e.g., a homoserine lactone molecule). The term "quorum
sensing signal molecule" is intended to include a molecule that
transduces a quorum sensing signal and mediates the cellular
response to cell density. In a preferred embodiment the quorum
sensing signal molecule is a freely diffusible autoinducer
molecule, e.g., a homoserine lactone molecule or analog thereof. In
one embodiment, a quorum sensing controlled gene encodes a
virulence factor. In another embodiment, a quorum sensing
controlled gene encodes a protein or polypeptide that, either
directly or indirectly, inhibits and/or antagonizes a bacterial
host defense mechanism. In yet another embodiment, a quorum sensing
controlled gene encodes a protein or polypeptide that regulates
biofilm formation.
[0092] The term "regulatory sequences" is intended to include the
DNA sequences that control the transcription of an adjacent gene.
Gene regulatory sequences include, but are not limited to, promoter
sequences that are found in the 5' region of a gene proximal to the
transcription start site which bind RNA polymerase to initiate
transcription. Gene regulatory sequences also include enhancer
sequences which can function in either orientation and in any
location with respect to a promoter, to modulate the utilization of
a promoter, and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described,
for example, in Goeddel (1990) Methods Enzymol. 185:3-7.
Transcriptional control elements include, but are not limited to,
promoters, enhancers, and repressor and activator binding sites.
The gene regulatory sequences of the present invention contain
binding sites for transcriptional regulatory proteins. In one
embodiment, a regulatory sequence includes a sequence that mediates
quorum sensing controlled gene expression, e.g., a las box. In a
preferred embodiment, gene regulatory sequences comprise sequences
derived from the Pseudomonas aeruginosa genome which modulate
quorum sensing controlled gene expression, e.g., SEQ ID NOs.:38 and
39. In another preferred embodiment, gene regulatory sequences
comprise sequences (e.g., a genetic locus) derived from the
Pseudomonas aeruginosa genome which modulate the expression of
quorum sensing controlled genes, e.g., SEQ ID NOs.: 1-36.
[0093] The term "reporter gene" or "indicator gene" generically
refers to an expressible (e.g., able to be transcribed and
(optionally) translated) DNA sequence which is expressed in
response to the activity of a transcriptional regulatory protein.
Indicator genes include unmodified endogenous genes of the host
cell, modified endogenous genes, or a reporter gene of a
heterologous construct, e.g., as part of a reporter gene construct.
In a preferred embodiment, the level of expression of an indicator
gene produces a detectable signal.
[0094] Reporter gene constructs are prepared by operatively linking
an indicator gene with at least one transcriptional regulatory
element. If only one transcriptional regulatory element is
included, it is advantageously a regulatable promoter. In a
preferred embodiment at least one of the selected transcriptional
regulatory elements is directly or indirectly regulated by quorum
sensing signals, whereby quorum sensing controlled gene expression
can be monitored via transcription and/or translation of the
reporter genes.
[0095] Many reporter genes and transcriptional regulatory elements
are known to those of skill in the art and others may be identified
or synthesized by methods known to those of skill in the art.
Reporter genes include any gene that expresses a detectable gene
product, which may be RNA or protein. Preferred reporter genes are
those that are readily detectable. In one embodiment, an indicator
gene of the present invention is comprised in the nucleic acid
molecule in the form of a fusion gene (e.g., operatively linked)
with a nucleotide sequence that includes regulatory sequences
(e.g., quorum sensing transcriptional regulatory elements, e.g., a
las box) derived from the Pseudomonas aeruginosa genome (e.g., SEQ
ID NOs:38 and 39). In another embodiment, an indicator gene of the
present invention is operatively linked to quorum sensing
transcriptional regulatory sequences that regulate a quorum sensing
controlled genetic locus derived from the Pseudomonas aeruginosa
genome, e.g., a genetic locus comprising a nucleotide sequence set
forth as SEQ ID NOs.: 1-36. In yet another embodiment, an indicator
gene of the present invention is operatively linked to a nucleotide
sequence comprising a quorum sensing controlled genetic locus
derived from the Pseudomonas aeruginosa genome (e.g., SEQ ID NOs.:
1-39). In certain embodiments of the invention, an indicator gene
(e.g., a promoterless indicator gene) is contained in a
transposable element.
[0096] The term "detecting a change in the detectable signal" is
intended to include the detection of alterations in gene
transcription of an indicator or reporter gene induced upon
modulation of quorum sensing signaling. In certain embodiments, the
reporter gene may provide a selection method such that cells in
which the transcriptional regulatory protein activates
transcription have a growth advantage. For example the reporter
could enhance cell viability, relieve a cell nutritional
requirement, and/or provide resistance to a drug. In other
embodiments, the detection of an alteration in a signal produced by
an indicator gene encompass assaying general, global changes to the
cell such as changes in second messenger generation.
[0097] The amount of transcription from the reporter gene may be
measured using any method known to those of skill in the art. For
example, specific mRNA expression may be detected using Northern
blots, or a specific protein product may be identified by a
characteristic stain or an intrinsic activity. In preferred
embodiments, the gene product of the reporter is detected by an
intrinsic activity associated with that product. For instance, the
reporter gene may encode a gene product that, by enzymatic
activity, gives rise to a detection signal based on color,
fluorescence, or luminescence.
[0098] The amount of regulation of the indicator gene, e.g.,
expression of a reporter gene, is then compared to the amount of
expression in a control cell. For example, the amount of
transcription of an indicator gene may be compared between a cell
in the absence of a test modulator molecule and an identical cell
in the presence of a test modulator molecule.
[0099] As used interchangeably herein, the terms "transposon" and
"transposable element" are intended to include a piece of DNA that
can insert into and cut itself out of, genomic DNA of a particular
host species. Transposons include mobile genetic elements (MGEs)
containing insertion sequences and additional genetic sequences
unrelated to insertion functions (for example, sequences encoding a
reporter gene). Insertion sequence elements include sequences that
are between 0.7 and 1.8 kb in size with termini approximately 10 to
40 base pairs in length with perfect or nearly perfect repeats. As
used herein, a transposable element is operatively linked to the
nucleotide sequence into which it is inserted. Transposable
elements are well known in the art, and are described for example,
at www.bact.wisc.edu/MicrotextBook/BactGenetics.
[0100] The present invention discloses a method for identifying
modulators of quorum sensing signaling in bacteria, e.g.,
Pseudomonas aeruginosa. As described herein, the method of the
invention comprises providing a cell which comprises a quorum
sensing controlled gene, wherein the cell is responsive to a quorum
sensing signal molecule such that a detectable signal is generated.
A cell which responds to a quorum sensing signal molecule by
generating a detectable signal is referred to herein as an
"indicator cell" or a "reporter cell". In a preferred embodiment of
the invention, the cell is a P. aeruginosa bacterial cell. In
another preferred embodiment, the cell is from a mutant strain of
P. aeruginosa which comprises a reporter gene operatively linked to
a regulatory sequence of a quorum sensing controlled gene, wherein
said mutant strain is responsive to a quorum sensing signal
molecule, such that a detectable signal is generated. In yet
another preferred embodiment, the cell is a mutant strain of P.
aeruginosa which comprises a promoterless reporter gene inserted in
the chromosome at a quorum sensing controlled genetic locus, e.g.,
a genetic locus comprising a nucleotide sequence set forth as SEQ
ID NOs.: 1-38, wherein said mutant strain is responsive to a quorum
sensing signal molecule such that a detectable signal is generated
by the reporter gene. In a preferred embodiment, the reporter gene
is contained in a transposable element. In a further preferred
embodiment, the cell is from a strain of P. aeruginosa in which
lasI and rhlI are inactivated, such that the cell does not express
the lasI and rhlI autoinducer synthases which are involved in the
generation of quorum sensing signal molecules. A compound is
identified as a modulator of quorum sensing signaling in bacteria
by contacting the cell with a quorum sensing signal molecule in the
presence and absence of a test compound and detecting a change in
the detectable signal.
[0101] Quorum sensing signal molecules that are useful in the
methods of the present invention include autoinducer compounds such
as homoserine lactones, and analogs thereof (see Table 1). In
certain embodiments, the quorum sensing signal molecule is either
3-oxo-C12-homoserine lactone or C4-HSL. In one embodiment, the cell
does not express the quorum sensing signal molecule. For example,
the cell may comprise a mutant strain of Pseudomonas aeruginosa
wherein lasI and rhlI are inactivated. Therefore, the cell is
contacted with an exogenous quorum sensing signal molecule, e.g., a
recombinant or synthetic molecule. In another embodiment, the
quorum sensing signal molecule is produced by a second cell (e.g.,
a prokaryotic or eukaryotic cell), which is co-incubated with the
indicator cell. For example, an indicator cell which does not
express a quorum sensing signal molecule can be co-incubated with a
wild type strain of Pseudomonas aeruginosa which produces a quorum
sensing signal molecule. Alternatively, the indicator strain which
does not express a quorum sensing signal molecule is co-incubated
with a second cell which has been transformed, or otherwise
altered, such that it is able to express a quorum sensing signal
moleucle. In yet another embodiment, the quorum sensing signal
molecule is expressed by the indicator strain.
[0102] Similarly, the test compound can be exogenously added to an
indicator strain, produced by a second cell which is co-incubated
with the indicator strain, or expressed by the indicator strain.
Exemplary compounds which can be screened for activity include, but
are not limited to, peptides, nucleic acids, carbohydrates, small
organic molecules, and natural product extract libraries.
[0103] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:45).
[0104] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example, in: DeWitt et al. (1993)
Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc.
Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med.
Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al.
(1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.
Med. Chem. 37:1233.
[0105] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6378-6382); (Felici (1991) J.
Mol. Biol. 222:301-310); (Ladner supra.).
[0106] In certain embodiments of the instant invention, the
compounds tested are in the form of peptides from a peptide
library. The peptide library may take the form of a cell culture,
in which essentially each cell expresses one, and usually only one,
peptide of the library. While the diversity of the library is
maximized if each cell produces a peptide of a different sequence,
it is usually prudent to construct the library so there is some
redundancy. Depending on size, the combinatorial peptides of the
library can be expressed as is, or can be incorporated into larger
fusion proteins. The fusion protein can provide, for example,
stability against degradation or denaturation. In an exemplary
embodiment of a library for intracellular expression, e.g., for use
in conjunction with intracellular target receptors, the polypeptide
library is expressed as thioredoxin fusion proteins (see, for
example, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT
publication WO94/02502). The combinatorial peptide can be attached
on the terminus of the thioredoxin protein, or, for short peptide
libraries, inserted into the so-called active loop.
[0107] In one embodiment of the instant invention the cell further
comprises a means for generating the detectable signal. For
example, the cell may comprise a reporter gene, the transcription
of which is regulated by a quorum sensing signal molecule. In a
preferred embodiment, the reporter gene is operatively linked to a
regulatory sequence of a quorum sensing controlled gene, e.g. a
nucleotide sequence comprising at least one quorum sensing
controlled regulatory element, e.g., a las box. In another
embodiment, the reporter gene is operatively linked to a quorum
sensing controlled genetic locus, e.g., a quorum sensing controlled
gene, such that transcription of the indicator gene is responsive
to quorum sensing signals. For example, in a preferred embodiment,
a promoterless reporter gene is inserted into a quorum sensing
controlled genetic locus derived from the genome of P. aeruginosa.
Such quorum sensing controlled genetic loci, as described herein,
include the loci in the P. aeruginosa genome which comprise the
nucleotide sequences set forth as SEQ ID NOs.: 1-38. In another
preferred embodiment, the promoterless reporter gene is contained
in a transposable element that is inserted into a quorum sensing
controlled genetic locus in the P. aeruginosa genome.
[0108] Examples of reporter genes include, but are not limited to,
CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979),
Nature 282: 864-869), and other enzyme detection systems, such as
beta-galactosidase (lacZ), firefly luciferase (deWet et al. (1987),
Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and
Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984),
Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al (1989)
Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl.
Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen
and Malim (1992) Methods in Enzymol. 216:362-368), and horseradish
peroxidase. In one preferred embodiment, the indicator gene is
lacZ. In another preferred embodiment, the indicator gene is green
fluorescent protein (U.S. Pat. No. 5,491,084; WO96/23898) or a
variant thereof. A preferred variant is GFPmut2. Other reporter
genes include ADE1, ADE2, ADE3, ADE4, ADE5, ADE7, ADE8, ASP3, ARG1,
ARG3, ARG4, ARG5, ARG6, ARG8, ARO2, ARO7, BAR1, CAT, CHO1, CYS3,
GAL1, GAL7, GAL10, HIS1, HIS3, HIS4, HIS5, HOM3, HOM6, ILV1, ILV2,
ILV5, INO1, INO2, INO4, LEU1, LEU2, LEU4, LYS2, MAL, MEL, MET2,
MET3, MET4, MET8, MET9, MET14, MET16, MET19, OLE1, PHO5, PRO1,
PRO3, THR1, THR4, TRP1, TRP2, TRP3, TRP4, TRP5, URA1, URA2, URA3,
URA4, URA5 and URA10.
[0109] In accordance with the methods of the invention, compounds
which modulate quorum sensing singaling can be selected and
identified. The ability of compounds to modulate quorum sensing
signaling can be detected by up or down-regulation of the detection
signal provided by the indicator gene. Any difference, e.g., a
statistically significant difference, in the amount of
transcription indicates that the test compound has in some manner
altered the activity of quorum sensing signaling.
[0110] A modulator of quorum sensing signaling may act by
inhibiting an enzyme involved in the synthesis of a quorum sensing
signal molecule, by inhibiting reception of the quorum sensing
signal molecule by the cell, or by scavenging the quorum sensing
signal molecule. The term "scavenging" is meant to include the
sequestration, chemical modification, or inactivation of a quorum
sensing signal molecule such that it is no longer able to regulate
quorum sensing gene control. After identifying certain test
compounds as potential modulators of quorum sensing signaling, the
practitioner of the subject assay will continue to test the
efficacy and specificity of the selected compounds both in vitro
and in vivo, e.g., in an assay for bacterial viability and/or
pathogenecity.
[0111] In another aspect, the present invention discloses a method
for identifying a quorum sensing controlled gene in bacteria, e.g.,
Pseudomonas aeruginosa. The method comprises providing a cell which
is responsive to a quorum sensing signal molecule such that
expression of a quorum sensing controlled gene is modulated, and
wherein modulation of the expression of the quorum sensing
controlled gene generates a detectable signal. The cell is
contacted with a quorum sensing signal molecule and a change in the
signal is detected to thereby identify a quorum sensing signaling
controlled gene.
[0112] In one embodiment, the cell further comprises a means for
generating the detectable signal, e.g., a reporter gene. For
example, the cell may comprise a promoterless reporter gene that is
operatively-linked to a quorum sensing controlled genetic locus
such that modulation of the expression of the quorum sensing
controlled locus concurrently modulates transcription of the
reporter gene. The position of the quorum sensing controlled
genetic locus is then mapped based on the position of the reporter
gene.
[0113] In a preferred embodiment of the invention, the cell is a P.
aeruginosa bacterial cell. In another preferred embodiment, the
cell is a mutant strain of P. aeruginosa which comprises a
promoterless reporter gene inserted in the chromosome at a quorum
sensing controlled genetic locus, e.g., a genetic locus comprising
a nucleotide sequence set forth as SEQ ID NOs.:1-39, wherein said
mutant strain is responsive to a quorum sensing signal molecule
such that a detectable signal is generated by the reporter gene. In
a preferred embodiment, the reporter gene is contained in a
transposable element. In a further preferred embodiment, the cell
is from a strain of P. aeruginosa in which lasI and rhlI are
inactivated, such that the cell does not express the lasI and rhlI
autoinducer synthases which are involved in the generation of
quorum sensing signal molecules.
[0114] It is also to be understood that genomic sequences from a
mutant bacterial strain (e.g., P. aeruginosa) in which a
promoterless reporter gene (e.g., a reporter gene contained in a
transposable element) has been inserted at a quorum sensing
controlled locus, can be assayed in a heterologous cell that is
responsive to a quorum sensing signal molecule such that quorum
sensing signal transduction occurs. For example, the genomic DNA of
a strain of P. aeruginosa subjected to transposon mutagenesis, as
described herein, can be engineered into a library, and transferred
to another cell capable of quorum sensing signaling (e.g., a
different species of gram negative bacteria), and assayed to
identify a quorum sensing controlled gene.
[0115] In one embodiment, the cell is contacted with an exogenous
quorum sensing signal molecule, e.g., a recombinant or synthetic
molecule, as described herein. In another embodiment, the quorum
sensing signal molecule is produced by a second cell (e.g., a
prokaryotic or eukaryotic cell), which is co-incubated with the
indicator cell. For example, an indicator cell which does not
express a quorum sensing signal molecule can be co-incubated with a
wild type strain of Pseudomonas aeruginosa which produces a quorum
sensing signal molecule. Alternatively, the indicator strain which
does not express a quorum sensing signal molecule is co-incubated
with a second cell which has been transformed, or otherwise
altered, such that it is able to express a quorum sensing signal
moleucle. In yet another embodiment, the quorum sensing signal
molecule is expressed by the indicator strain.
[0116] Another aspect of the invention provides a mutant strain of
Pseudomonas aeruginosa comprising a promoterless reporter gene
inserted in a chromsome at a genetic locus comprising a nucleotide
sequence set forth as SEQ ID NOs:1-36, e.g., a quorum sensing
controlled genetic locus. In one embodiment the reporter gene is
contained in a transposable element. In another embodiment, the
reporter gene is lacZ or GFP, or a variant thereof, e.g., GFPmut2.
In yet another embodiment, lasI and rhlI are inactivated in the
mutant strain of P. aeruginosa. The above-described cells are
useful in the methods of the instant invention, as the cells are
responsive to a quorum sensing signal molecule such that a
detectable signal is generated by the reporter gene. These cells
are also useful for studying the function of polypeptides encoded
by the quorum sensing controlled loci comprising the nucleotide
sequences set forth as SEQ ID NOs.:1-36.
[0117] Yet another aspect of the invention provides isolated
nucleic acid molecules comprising a nucleotide sequence comprising
a quorum sensing controlled genetic locus derived from the genome
of Pseudomonas aeruginosa operatively linked to a reporter gene. In
one embodiment, a reproter gene is operatively linked to a
regulatory sequence derived from the genome of P. aeruginosa,
wherein the regulatory sequence regulates a quorum sensing
controlled genetic locus comprising a nucleotide sequence set forth
as SEQ ID NO:1-36. In a preferred embodiment such regulatory
sequences comprise at least one binding site for a quorum sensing
controlled transcriptional regulatory factor (e.g., a
transcriptional activator or repressor molecule) such that
transcription of the reporter gene is responsive to a quorum
sensing singal molecule and/or a modulator of quorum sensing
signaling. In another embodiment, a reporter gene is operatively
linked to a quorum sensing controlled genetic locus derived from
the genome of P. aeruginosa, wherein the genetic locus comprises a
nucleotide sequence set forth as SEQ ID NO:1-36. In yet another
embodiment, a reporter gene is operatively linked to a nucleotide
sequence which has at least 80%, and more preferably at least 85%,
90% or 95% identity to quorum sensing controlled genetic locus
derived from the genome of P. aeruginosa, wherein the genetic locus
comprises a nucleotide sequence set forth as SEQ ID NO:1-36. In a
further embodiment, a reporter gene is operatively linked to a
nucleotide sequence which hybridizes under stringent conditions to
quorum sensing controlled genetic locus derived from the genome of
P. aeruginosa, wherein the genetic locus comprises a nucleotide
sequence set forth as SEQ ID NO:1-36.
[0118] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regard to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of
nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. As used interchangeably
herein, the terms "nucleic acid molecule" and "polynucleotide" are
intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA. The term"DNA" refers to deoxyribonucleic acid
whether single- or double-stranded. As used herein, the terms
"gene" and "recombinant gene" refer to nucleic acid molecules which
include an open reading frame encoding a protein, preferably a
quorum sensing controlled protein, and can further include
non-coding regulatory sequences, and introns.
[0119] The present invention includes polynucleotides capable of
hybridizing under stringent conditions, prefereably highly
stringent conditions, to the polynucleotides described herein
(e.g., a quorum sensing controlled genetic locus, e.g., SEQ ID
NOs.:1-36). As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4, and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7,
9, and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
alternatively hybridization in 4.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
1.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of highly stringent hybridization conditions includes
hybridization in 1.times.SSC, at about 65-70.degree. C. (or
alternatively hybridization in 1.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
0.3.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of reduced stringency hybridization conditions includes
hybridization in 4.times.SSC, at about 50-60.degree. C. (or
alternatively hybridization in 6.times.SSC plus 50% formamide at
about 40-45.degree. C.) followed by one or more washes in
2.times.SSC, at about 50-60.degree. C. Ranges intermediate to the
above-recited values, e.g. at 65-70.degree. C. or at 42-50.degree.
C. are also intended to be encompassed by the present invention.
SSPE (1.times.SSPE is 0.15M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25
mM EDTA, pH 7.4) can be substituted for SSC (1.times.SSC is 0.15M
NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes each after
hybridization is complete. The hybridization temperature for
hybrids anticipated to be less than 50 base pairs in length should
be 5-10.degree. C, less than the melting temperature (T.sub.m) of
the hybrid, where T.sub.m is determined according to the following
equations. For hybrids less than 18 base pairs in length,
T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For
hybrids between 18 and 49 base pairs in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M). It will also be recognized by
the skilled practitioner that additional reagents may be added to
hybridization and/or wash buffers to decrease non-specific
hybridization of nucleic acid molecules to membranes, for example,
nitrocellulose or nylon membranes, including but not limited to
blocking agents (e.g., BSA or salmon or herring sperm carrier DNA),
detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP
and the like. When using nylon membranes, in particular, an
additional preferred, non-limiting example of stringent
hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.
(see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995), or alternatively 0.2.times.SSC, 1% SDS.
[0120] The invention further encompasses nucleic acid molecules
that differ from the quorum sensing controlled genetic loci
described herein, e.g., the nucleotide sequences shown in SEQ ID
NO:1-36. Accordingly, the invention also includes variants, e.g.,
allelic variants, of the disclosed polynucleotides or proteins;
that is naturally occuring and non-naturally occuring alternative
forms of the isolated polynucleotide which may also encode proteins
which are identical, homologous or related to that encoded by the
polynucleotides of the invention.
[0121] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologues (different locus), and
orthologues (different organism) or can be non naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product). Allelic
variants result, for example, from DNA sequence polymorphisms
within a population (e.g., a bacterial population) that lead to
changes in the nucleic acid sequences of quorum sensing controlled
genetic loci.
[0122] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, 90% or 95% of the
length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0123] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package (available at http://www.gcg.com), using either a
Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In
yet another preferred embodiment, the percent identity between two
nucleotide sequences is determined using the GAP program in the GCG
software package (available at http://www.gcg.com), using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988) which has been incorporated into the
ALIGN program (version 2.0) (available at
http://vega.igh.cnrs.fr/bin/align-guess.cgi), using a PAM120 weight
residue table, a gap length penalty of 12 and a gap penalty of
4.
[0124] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to nucleic acid molecules of
the invention. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Additionally, the "Clustal" method (Higgins and Sharp, Gene,
73:237-44, 1988) and "Megalign" program (Clewley and Arnold,
Methods Mol. Biol, 70:119-29, 1997) can be used to align sequences
and determine similarity, identity, or homology.
[0125] Accordingly, the present invention also discloses
recombinant vector constructs and recombinant host cells
transformed with said constructs.
[0126] The term "vector" or "recombinant vector" is intended to
include any plasmid, phage DNA, or other DNA sequence which is able
to replicate autonomously in a host cell. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. A vector may be
characterized by one or a small number of restriction endonuclease
sites at which such DNA sequences may be cut in a determinable
fashion without the loss of an essential biological function of the
vector, and into which a DNA fragment may be spliced in order to
bring about its replication and cloning. A vector may further
contain a marker suitable for use in the identification of cells
transformed with the vector. Recombinant vectors may be generated
to enhance the expression of a gene which has been cloned into it,
after transformation into a host. The cloned gene is usually placed
under the control of (i.e., operably linked to) certain control
sequences or regulatory sequences, which may be either constitutive
or inducible.
[0127] One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "expression vectors". Expression
systems for both prokaryotic and eukaryotic cells are described in,
for example, chapters 16 and 17 of Sambrook, J. et al. Molecular
Cloning: A Laboratory Manual. 2.sup.nd, ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0128] In the present specification, "plasmid" and "vector" can be
used interchangeably as the plasmid is the most commonly used form
of vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
Protocols for producing recombinant retroviruses and for infecting
cells in vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene
Publishing Associates, (1989), Sections 9.10-9.14 and other
standard laboratory manuals. Examples of suitable retroviruses
include pLJ, pZIP, pWE and pEM which are well known to those
skilled in the art. Examples of suitable packaging virus lines
include .psi.crip, .psi.Cre, .psi.2 and .psi.Am. The genome of
adenovirus can be manipulated such that it encodes and expresses a
transcriptional regulatory protein but is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle. See
for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et
al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell
68:143-155. Suitable adenoviral vectors derived from the adenovirus
strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2,
Ad3, Ad7 etc.) are well known to those skilled in the art.
Alternatively, an adeno-associated virus vector such as that
described in Tratschin et al. ((1985) Mol. Cell. Biol. 5:3251-3260)
can be used.
[0129] In general, it may be desirable that an expression vector be
capable of replication in the host cell. Heterologous DNA may be
integrated into the host genome, and thereafter is replicated as a
part of the chromosomal DNA, or it may be DNA which replicates
autonomously, as in the case of a plasmid. In the latter case, the
vector will include an origin of replication which is functional in
the host. In the case of an integrating vector, the vector may
include sequences which facilitate integration, e.g., sequences
homologous to host sequences, or encoding integrases.
[0130] Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts are known in
the art, and are described in, for example, Powels et al. (Cloning
Vectors: A Laboratory Manual, Elsevier, New York, 1985). Mammalian
expression vectors may comprise non-transcribed elements such as an
origin of replication, a suitable promoter and enhancer linked to
the gene to be expressed, and other 5' or 3' flanking
nontranscribed sequences, and 5' or 3' nontranslated sequences,
such as necessary ribosome binding sites, a poly-adenylation site,
splice donor and acceptor sites, and transcriptional termination
sequences.
[0131] The vectors of the subject invention may be transformed into
an appropriate cellular host for use in the methods of the
invention.
[0132] As used interchangeably herein, a "cell" or a "host cell"
includes any cultivatable cell that can be modified by the
introduction of heterologous DNA. As used herein, "heterologous
DNA", a "heterologous gene" or "heterologous polynucleotide
sequence" is defined in relation to the cell or organism harboring
such a nucleic acid or gene. A heterologous DNA sequence includes a
sequence that is not naturally found in the host cell or organism,
e.g., a sequence which is native to a cell type or species of
organism other than the host cell or organism. Heterologous DNA
also includes mutated endogenous genetic seqeunces, for example, as
such sequences are not naturally found in the host cell or
organism. Preferably, a host cell is one in which a quorum sensing
signal molecule, e.g, an autoinducer molecule, initiates a quorum
sensing signaling response which includes the regulation of target
quorum sensing controlled genetic sequences. The choice of an
appropriate host cell will also be influenced by the choice of
detection signal. For example, reporter constructs, as described
herein, can provide a selectable or screenable trait upon
activation or inhibition of gene transcription in response to a
quorum sensing signaling event; in order to achieve optimal
selection or screening, the host cell phenotype will be
considered.
[0133] A host cell of the present invention includes prokaryotic
cells and eukaryotic cells. Prokaryotes include gram negative or
gram positive organisms, for example, E. Coli or Bacilli. Suitable
prokaryotic host cells for transformation include, for example, E.
coli, Bacillus subtilis, Salmonella typhimurium, and various other
species within the genera Pseudomonas, Streptomyces, and
Staphylococcus. In a preferred embodiment, a host cell of the
invention is a mutant strain of P. aeruginosa in which lasI and
rhlI are inactivated.
[0134] Eukaryotic cells include, but are not limited to, yeast
cells, plant cells, fungal cells, insect cells (e.g., baculovirus),
mammalian cells, and cells of parasitic organisms, e.g.,
trypanosomes. Mammalian host cell culture systems include
established cell lines such as COS cells, L cells, 3T3 cells,
Chinese hamster ovary (CHO) cells, embryonic stem cells, and HeLa
cells. Other suitable host cells are known to those skilled in the
art.
[0135] DNA can be introduced into prokaryotic or eukaryotic cells
via conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2.sup.nd, ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989), and other laboratory manuals.
[0136] Host cells comprising an isolated nucleic acid molecule of
the invention (e.g., a quorum sensing controlled genetic locus
operatively linked to a reporter gene) can be used in the methods
of the instant invention to identify a modulator of quorum sensing
signaling in bacteria.
[0137] Exemplification
[0138] The invention is further illustrated by the following
examples which should not be construed as limiting.
EXAMPLE 1
Identification of Quorum Sensing Genes of P. Aeruginosa
[0139] Materials and Methods
[0140] Bacterial Strains, Plasmids, and Media. The bacterial
strains and plasmids used in this example are listed in Table
2.
[0141] E. coli and P. aeruginosa were routinely grown in
Luria-Bertani (LB) broth or LB agar (Sambrook, et al. (1989)
Molecular Cloning: a Laboratory Manual. (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.)), supplemented with
antimicrobial agents when necessary. The antimicrobial agents were
used at the following concentrations: HgCl.sub.2, 15 .mu.g/ml in
agar and 7.5 .mu.g/ml in broth; nalidixic acid 20 .mu.g/ml;
carbenicillin, 300 .mu.g/ml; tetracycline, 50 .mu.g/ml for P.
aeruginosa and 20 .mu.g/ml for E. coli; and gentamicin, 100
.mu.g/ml for P. aeruginosa and 15 .mu.g/ml for E. coli. Synthetic
acyl-HSL concentrations were 2 .mu.M for 30C.sub.12-HSL and 5 .mu.M
for C.sub.4-HSL, and
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (X-Gal) was
used at 50 .mu.g/ml.
[0142] DNA Manipulations and Plasmid Constructions. DNA treatment
with modifying enzymes and restriction endonucleases, ligation of
DNA fragments with T4 ligase, and transformation of E. coli were
performed according to standard methods (Ausubel, F. et al. (1997)
Short Protocols in Molecular Biology. (John Wiley & Sons, Inc.,
New York, N.Y.)). Plasmid isolation was performed using QIAprep
spin miniprep kits (Qiagen Inc.) and DNA fragments were excised and
purified from agarose gels using GeneClean spin kits (Bio101
Corp.). DNA was sequenced at the University of Iowa DNA core
facility by using standard automated sequencing technology.
[0143] To construct pMW10, the pBR322 tetA(C) gene-containing
Clal-NotI DNA fragment in pJPP4 was replaced with a
tetA(B)-containing BstB1-NotI fragment from Tn10. It was necessary
to use tetA(B) rather than tetA(C) to inactivate lasI because the
tetA(C) gene from pBR322 was a hot spot for Tn5::B22 mutagenesis
(Berg, D. E. et al. (1983) Genetics 105, 813-828).
[0144] To construct pMW300 a 1.6 kb SmaI fragment from pGM.OMEGA.1
that contained the aacC1 gene (encoding gentamicin
acetyltransferase-3-1) was cloned into EagI digested pTL61T, which
had been polished with T4 polymerase. The resulting plasmid
pTL61T-GM.OMEGA.1 was digested with SmaI and MscI to release a
6.5-kb lacZ-aacC1 fragment. A
2TABLE 2 Bacterial strains and plasmids Strain or plasmid Relevant
characteristics Source (reference) Strains P. aeruginosa PAO1
Parental strain (1) P. aeruginosa PDO100 .DELTA.rhlI::Tn501
derivative of PAO1, Hg.sup.r (2) P. aeruginosa PAO-MW1 .DELTA.lasI,
.DELTA.rhlI derivative of PDO100, Hg.sup.r, Tc.sup.r This study P.
aeruginosa PAO-MW10 lasB::lacZ chromosomal insertion in PAO-MW1
This study E. coli DH5.alpha. F.sup.- .phi.80.DELTA.lacZ,
.DELTA.M15, .DELTA.(lacZYA-argF)U169, (3) endA1, recA1, hsdR17,
deoR, gyrA96, thi-1 relA1, supE44 E. coli HB101 F.sup.- mcrB, mrr
hsdS20, recA13, leuB6, ara-14 (3) proA2, lacY1, galK2, xyl-5,
mtl-1, rpsL20 (Sm.sup.r), supE44 E. coli SY327 .lambda.pir
(.lambda.pir), .DELTA.(lac pro), argE(Am), rif, nlA, recA56 (4) E.
coli S17-1 thi, pro, hsdR, recA, RP4-2 (Tet::Mu) (Km::Tn7) (5)
Plasmids pJPP4 oriR6K, mobRP4, .DELTA.lasI, Tc.sup.r, Ap.sup.r (6)
pTL61T lacZ transcriptional fusion vector, Ap.sup.r (7) pGM.OMEGA.1
Contains aacl flanked by transcriptional (8) and translational
stops, Gm.sup.r pTL61T-GM.OMEGA.1 pTL61T with aacl gene from
pGM.OMEGA.1 This study upstream of lacZ, Ap.sup.r, Gm.sup.r pMW100
pJPP4 with 2.7-kb tetA(B) from Tn10 in place of This study the
pBR322 tetA(C), Tc.sup.r, Ap.sup.r pRK2013 ori (ColE1), tra.sup.+,
(RK2)Km.sup.r (9) pSUP102 pACYC184 carrying mobRP4, Cm.sup.r,
Tc.sup.r (10) pSUP102-lasB pSUP102 carrying lasB on a 3.1-kb P.
aeruginosa This study chromosomal DNA fragment, Cm.sup.r, Tc.sup.r
pMW300 pSUP102-lasB containing lacZ-aacl from This study
pTL61T-GM.OMEGA.1 (lasB-lacZ transcriptional fusion knockout
plasmid), Cm.sup.r, Gm.sup.r pTn5-B22 pSUP102 with Tn5-B22 (`lacZ),
Gm.sup.r (28) Abbreviations tor antibiotics are as follows:
kanamycin, Km; gentamicin, Gm; ampicillin, Ap; tetracycline, Tc;
streptomycin, Sm.
[0145] 3.1-kb P. aeruginosa PAO1 chromosomal DNA fragment
containing the lasB gene was amplified by PCR using the Expand.TM.
Long Template PCR System (Boehringer Mannheim). This fragment was
cloned into BamHI-digested pSUP102. The resulting plasmid,
pSUP102-lasB was digested with NotI, polished with T4 polymerase
and ligated with the 6.5-kb lacZ-aacC1 fragment from
pTL61T-GM.OMEGA.1 to generate pMW300. The promoterless lacZ gene in
pMW300 is 549 nucleotides form the start of the lasB ORF, it is
flanked by 1.5 kb upstream and 1.6 kb downstream P. aeruginosa DNA,
and it contains the p15A ori, which does not support replication in
P. aeruginosa.
[0146] Construction of P. aeruginosa Mutants. A lasI, rhlI mutant
strain of P. aeruginosa PAO-MW1 was generated by insertional
mutagenesis of lasI in the rhlI deletion mutant, PDO100. For
insertional mutagenesis, the lasI::tetA(B) plasmid, pMW100 was
mobilized from E. coli SY327 .lambda.pir into PDO100 by triparental
mating with the help of E. coli HB101 containing pRK2013. Because
pMW100 has a .lambda.pir-dependent origin of replication, it cannot
replicate in P. aeruginosa. A tetracycline-resistant,
carbenicillin-sensitive exconjugant was selected, which was shown
by a Southern blot analysis to contain lasI:tetA but not lasI or
pMW100. To confirm the inactivation of the chromosomal lasI in this
strain, PAO-MW1, the amount of 3OC.sub.12-HSL in the fluid from a
stationary phase culture (optical density at 600 nm, 5) was
assessed by a standard bioassay (Pearson, J. P. et al. (1994) PNAS,
91, 197-201). No detectable 3-OC.sub.12-HSL (<5 nM) was
found.
[0147] A mutant strain, P. aeruginosa PAO-MW10, which contains a
lacZ reporter in the chromosomal lasB gene was constructed by
introduction of pMW300 into PAO-MW1 by triparental mating as
described above. Exconjugants resistant to gentamicin and sensitive
to chloramphenicol were selected as potential recombinants.
Southern blotting of chromosomal DNA with lasB and lacZ probes
indicated that the pMW300 lasB-lacZ insertion had replaced the wt
lasB gene.
[0148] Southern Blotting. Chromosomal DNA was prepared using the
QIAMP tissue kit (Qiagen Inc.). Approximately 2 .mu.g of
chromosomal DNA was digested with restriction endonucleases,
separated on a 0.7% agarose gel, and transferred to a nylon
membrane according to standard methods (Ausubel, F. et al. (1997)
Short Protocols in Molecular Biology. (John Wiley & Sons, Inc.,
New York, N.Y.). DNA probes were generated using
digoxigenin-11-dUTP by random primed DNA labeling or PCR. The
Southern blots were visualized using the Genius.TM. system as
outlined by the manufacturer (Boehringer Mannheim).
[0149] Tn5 Mutagenesis. Tn5::B22, which carries a promoterless lacZ
gene, was used to mutagenize P. aeruginosa PAO-MW1 (Simon, R. et
al. (1989) Gene 80, 161-169). Equal volumes of a late logarithmic
phase culture of E. coli S17-1 carrying pTn5::B22 grown at
30.degree. C. with shaking and a late logarithmic phase culture of
P. aeruginosa PAO-MW1 grown at 42.degree. C. without shaking were
mixed. The mixture was centrifuged at 6000.times.g for 10 minutes
at room temperature, suspended in LB (5% of the original volume),
and spread onto LB plates (100 .mu.l per plate). After 16 to 24
hours at 30.degree. C., the cells on each plate were suspended in
500 .mu.l LB and 100 .mu.l volumes were spread onto LB agar plates
containing HgCl.sub.2, gentamicin, tetracycline and nalidixic acid.
The nalidixic acid prevents growth of E. coli but not P.
aeruginosa. After 48 to 72 hours at 30.degree. C., 20 colonies were
selected from each mating and grown on LB selection agar plates
containing X-gal. Ten of the 20 were picked for further study. The
colonies picked showed a range in the intensity of the blue color
on the X-gal plates. In this way, the selection of siblings in a
mating were minimized. A Southern blot using a probe to lacZ was
performed on 20 randomly chosen transconjugants indicated that the
Tn5 insertion in each was in a unique location.
[0150] The Screen for qsc Fusions. A microtiter dish assay was used
to identify mutants showing acyl-HSL-dependent .beta.-galactosidase
expression (quorum sensing-controlled or qsc mutants). Each
transconjugant was grown in four separate wells containing LB broth
without added autoinducer, with added 3OC.sub.12-HSL, C.sub.4-HSL,
or both 30C.sub.12-HSL and C.sub.4-HSL for 12-16 hours at
37.degree. C. Inocula were 10 .mu.l of an overnight culture and
final culture volumes were 70 .mu.l. The .beta.-galactosidase
activity of cells in each microtiter dish well was measured in
microtiter dishes with a luminescence assay (Tropix) Luminescence
was measured with a Lucy I microtiter dish luminometer
(Anthos).
[0151] Patterns of Acyl-HSL Induction of .beta.-galactosidase
Activity in qsc Mutants. The pattern of .beta.-galactosidase
expression was examined in response to acyl-HSLs in each of 47 qsc
mutants identified in the initial screen. Each mutant was grown in
1 ml of MOPS (50 mM, pH 7.0) buffered LB broth containing one, the
other, both, or neither acyl-HSL signal in an 18 mm culture tube at
37.degree. C. with shaking. A mid-logarithmic phase culture was
used as an inoculum and initial optical densities (ODs) at 600 nm
were 0.1. Growth was monitored as OD at 600 nm and
.beta.-galactosidase activity was measured in 0.1 ml samples taken
at 0, 2, 5, and 9 hours after inoculation.
[0152] DNA Sequencing and Sequence Analysis. To identify DNA
sequences flanking Tn5::B22 insertions, arbitrary PCR was performed
with primers and conditions as described (Caetano-Annoles, G.
(1993) PCR Methods Appl. 3, 85-92; O'Toole, G. A. et al. (1998)
Mol. Microbiol. 28, 449-461). Tn5 flanking sequences that could not
be identified using arbitrary PCR were cloned. For cloning, 3 .mu.g
of chromosomal DNA was digested with EcoRI and ligated with
EcoRI-digested, phosphatase treated pBR322. E. coli DH5.alpha. was
transformed by electroporation with the ligation mixtures and
plasmids from gentamicin resistant colonies were used for
sequencing Tn5-flanking DNA.
[0153] DNA sequences flanking Tn5-B22 insertions were located on
the P. aeruginosa PAO1 chromosome by searching the chromosomal
database at the P. aeruginosa Genome Project web site
(www.pseudomonas.com). The ORFs containing the insertions are those
described at the web site. Functional coupling from the Argonne
National Labs (http://wit.mcs.anl.gov/WIT2), sequence analysis, and
expression patterns of the qsc mutants were used to identify
potential operons (Overbeek, R. et al. (1999) PNAS 96,
2896-2901).
[0154] Results
[0155] Identification of Pseudomonas aeruginosa qsc Genes. Seven
thousand Tn5::B22 mutants of P. aeruginosa PAO-MW1 were screened.
Tn5::B22 contains a promoterless lacZ. P. aeruginosa PAO-MW1 is a
lasI, rhlI mutant that does not make acyl-HSL signals. Thus,
transcription of the Tn5::B22 lacZ in a qsc gene was expected to
respond to an acyl-HSL signal. The screen involved growth of each
mutant in a complex medium in a microtiter dish well with no added
acyl-HSL, 30C.sub.12-HSL, C.sub.4-HSL, or both 3OC.sub.12-HSL and
C.sub.4-HSL. After 12-16hours, .beta.-galactosidase activity in
each culture was measured. Two hundred-seventy mutants showed
greater than 2 fold stimulation of .beta.-galactosidase activity in
response to either or both acyl-HSL. Of these, 70 showed a greater
than5-fold stimulation of .beta.-galactosidase activity in response
to either or both acyl-HSL, and were studied further. Each mutant
was grown with shaking in culture tubes and 47 showed a
reproducible greater than5-fold stimulation of .beta.-galactosidase
activity in response to either or both of the acyl-HSL signals.
These were considered to have Tn5::B22 insertions in qsc genes. It
was shown by a Southern blot analysis with a lacZ probe that each
mutant contained a single Tn5: :B22 insertion.
[0156] This collection of 47 mutants is not believed to represent
the entire set of quorum sensing regulated genes in P. aeruginosa.
The threshold of greater than 5-fold induction may be too
stringent, enough mutants may not have been screened to be assure
that insertions in all of the genes in the chromosome have been
tested, and there may be conditions other than those which were
employed that would have revealed other genes which were not
detected in the present screen. Nevertheless, a set of 47
insertions in genes have been identified that show significant
activation in response to acyl-HSL (qsc genes).
[0157] Responses of qsc Mutants to Acyl-HSL Signals. For cultures
of each of the 47 qsc mutants, .beta.-galactosidase activity was
measured at different times after addition of acyl-HSL signals. The
basal levels of .beta.-galactosidase varied depending on the
mutant. The responses to the acyl-HSL signals could be grouped into
4 general classes based on which of the two signals was required
for activation of lacZ, and whether the response to the signal(s)
occurred immediately or was delayed until stationary phase. A
response was considered immediate if there was a 5-fold or greater
response within 2 hours of acyl-HSL addition (the optical
densities(ODs) of the cultures ranged from 0.5-0.7 at 2 hours). A
response was considered delayed or late if there was <5-fold
induction at 2 hours but greater than 5-fold induction of
.beta.-galactosidase at 5 hours or later (ODs of 2 or greater). In
some strains activation of lacZ required 30C.sub.12-HSL, others
required both 30C.sub.12-HSL and C.sub.4-HSL for full activation of
lacZ. A number of strains responded to either signal alone but
showed a much greater response with both 30C.sub.12-HSL and
C.sub.4-HSL. None of the mutants responded well to C.sub.4-HSL
alone (Table 3). This was expected because expression of RhIR,
which is required for a response to C.sub.4-HSL is dependent on
30C.sub.12-HSL (Pesci, E. C. et al. (1997) J. Bacteriol. 179,
3127-3132). Therefore at least some of the insertions exhibiting a
response to both acyl-HSLs may be responding to the rhl system,
which requires activation by the las system.
[0158] Class I mutants responded to 3OC.sub.12-HSL immediately,
Class II responded to 30C.sub.12-HSL late, Class III respond best
to both signals early, and, Class IV to both signals late. There
were 9 Class I, 11 Class II, 18 Class III, and 9 Class IV mutants.
FIG. 2 shows responses of representative members of each class to
acyl-HSLs. Generally, most early genes (Class I and III genes)
showed a much greater induction than most late genes (Class II and
IV). Many of the Class III mutants showed some response to either
signal alone but showed a greater response in the presence of both
signals (Table 3 and FIG. 2).
[0159] Identity and Analysis of qsc Genes. The Tn5-B22-marked qsc
genes were identified by coupling arbitrary PCR or transposon
cloning with DNA sequencing. The sequences were located in the P.
aeruginosa PAO1 chromosome by searching the Pseudomonas aeruginosa
Genome Project web site (www.pseudomonas.com). To confirm the
locations of the Tn5-B22 insertions in each qsc mutant, a Southern
blot analysis was performed with Tn5-B22 as a probe. The sizes of
Tn5-B22 restriction fragments were in agreement with those
predicted based on the P. aeruginosa genomic DNA sequence (data not
shown). The 47 qsc mutations mapped in or adjacent to 39 different
open reading frames (ORFs). For example FIG. 3 depicts the nucleic
acid sequence of the quorum sensing controlled locus on the P.
aeruginosa chromosome mapped in the P. aeruginosa mutant strain
qsc102.
3TABLE 3 Quorum sensing-controlled genes in Pseudomonas aeruginosa
Signal response.sup.b Genomic Classification Identity.sup.a
3OC.sub.12-HSL C.sub.4-HSL Both Position.sup.e Class I qsc100
Peptide synthetase 65 3 69 5801998 qsc101 No match 145 1 184 7730
qsc102 No match 350 1 400 5547 qsc103 No match 85 1 95 3961920
qsc104 Polyamine binding protein 7 2 8 5402505 qsc105 FAD-binding
protein 40 1 42 5410045 qsc106A&B No match 9 1 10 2870317
qsc107 No match 44 2 50 5799641 Class II qsc108 ORF 5 13 1 7
5617382 qsc109 Bacitracin synthetase 3 13 1 8 5651872 qsc110A&B
Pyoverdine synthetase D 10 1 7 5661697 qsc111 Pyoverdine synthetase
D 11 1 7 5666282 qsc112A&B Aculeacin A acylase 15 1 12 5701004
qsc113 Trransmembrane protein 5 1 5 3771157 qsc114.sup.c No match 9
1 7 5209051 qsc115.sup.d No match 4 1 5 1941557 qsc116 No match 5 1
5 1138940 Class III qsc117.sup.d ACP-like protein 22 22 186 41430
qsc118 RhlI 38 14 70 4447967 qsc119 RhlB 9 7 100 4446918 qsc120
Chloramphenicol resistance 3 7 24 4592102 qsc121 3-Oxoacyl ACP
synthase 13 27 105 4594988 qsc122A&B Cytochrome p450 2 10 90
4595538 qsc123 9-Cis retinol dehydrogenase 14 28 96 4597340
qsc124A&B Pyoverdine synthetase D 35 50 148 4598281 qsc125
Zeaxanthin synthesis 20 65 140 4600099 qsc126 Pristanimycin I
synthase 3 & 4 3 5 24 4603518 qsc127.sup.c No match 5 2 15
4608787 qsc128 Hydrogen cyanide synthase HcnB 19 12 42 5924799
qsc129A&B Cellulose binding protein p40 15 1 100 1141723 qsc130
glc operon transcriptional activator 5 1 14 2313744 qsc131 PhzC 50
168 742 1110 Class IV qsc132A&B Unknown (B. pertusis) 1 1 40
3616599 qsc133A&B AcrB 1 1 9 3628342 qsc134 Saframycin Mx1
synthetase A 6 1 28 3781254 qsc135 Cytochrome C precursor 3 1 6
4942182 qsc136.sup.c No match 7 3 45 851491 qsc137 Asparagine
synthetase 1 1 10 2007007 qsc138 No match 3 5 32 2459178 .sup.aThe
bold letters indicate matches were to known P. aeruginosa genes.
.sup.bThe signal response is given as .beta.-galactosidase activity
in cells grown in the presence of the indicated signal(s) divided
by the .beta.-galactosidase activity of cells grown in the absence
of added signals. Maximum responses are indicated. .sup.cThe lacZ
insertions in these strains are in the opposite orientation of the
ORFs described in the P. aeruginosa Genome Project web site. The
insertions are which in locations with no reported identity are
been indicated. .sup.dInsertions do not lie in but are near the
putative ORFs indicated. In qsc117 the insertion is 129 bp
downstream of the ACP ORF and interrupts a potential
rho-independent transcription terminator. The qsc115 insertion is
60 bp upstream of the ORF listed in Materials and Methods.
.sup.eGenomic position as identified using sequence information
described in the P. aeruginosa Genome Project web site (Jul. 15,
1999 release).
[0160] The genomic sequences comprising the ORFs in Table 3 are
described in the Pseudomonas aeruginosa Genome Sequencing Project
web site, as detailed in Table 4.
[0161] Only 2 genes were identified that already were known to be
controlled by quorum sensing, rhlI and rhlB. Several other genes
potentially involved in processes known to be regulated by quorum
sensing were also identified including phzC (phenazine synthesis),
a putative cyanide synthesis gene (related to the Pseudomonas
fluorescens hcnB), and ORF 5 (pyoverdine synthesis) (Latifi, A. et
al. (1995) Mol. Microbiol. 17, 333-344; Cunliffe, H. E. et al.
(1995) J. Bacteriol. 177, 2744-2750). Interestingly, lasB was not
identified by the assay, yet the LasI-LasR quorum sensing system
was originally described as regulating lasB (Gambello, M. J. et al.
(1991) J. Bacteriol. 173, 3000-3009). A lasB-lacZ chromosomal
fusion in P. aeruginosa PAO-MW1 was constructed, so that regulation
of lasB by quorum sensing could be compared to the genes identified
by the assay. The lasB7lacZ fusion only responded slightly to
3OC.sub.12-HSL (3-fold stimulation). The full response (12-13-fold
over background) required both C.sub.4-HSL and 30C.sub.12-HSL, and
the response was late (FIG. 2). Thus, lasB shows the
characteristics of a Class IV gene.
[0162] Some of the qsc mutants had obvious phenotypes. Unlike the
parent, on LB agar, colonies of the Class II mutants qsc108, 109,
110A and B, and 111 were not fluorescent. Because pyoverdine is a
fluorescent pigment, and because the qsc110 and 111 mutations were
in genes coding for pyoverdine synthetase-like proteins, it was
suspected that these mutations define a region involved in
pyoverdine synthesis. The insertion in qsc131 is in phzC which is
required for pyocyanin synthesis. Although the parent strain
produced a blue pigment in LB broth, qsc131 did not. The two qsc132
mutants also did not produce detectable levels of pyocyanin but did
produce a water-soluble red pigment.
[0163] Functional coupling and sequence analysis were used to
identify 7 putative qsc operons, one of which is the previously
described rhlAB operon (FIG. 4). Functional coupling will not
organize genes encoding polypeptides without known relatives into
operons, and organization of genes in an operon was disallowed in
cases where there was greater than 250 bp of intervening sequence
between two adjacent ORFs. The
4TABLE 4 ORFs of quorum sensing-controlled genes in Pseudomonas
aeruginosa Insertion Insertion Jul. 15, 1999 Dec. 15, 1999 Open
Reading Frame QSC release release Dec. 15, 1999 release Orientation
SEQ ID NO 131 1110 4715256 4714774-4715991 Forward 1 102 5547
2067716 2066736-2068517 Reverse 2 101 7730 2065297 2064803-2065495
Reverse 3 117 41430 2031833 2031245-2031655 Forward 4 136 851491
1221771 1221374-1221961 Reverse 5 116 1138940 934322 934191-935210
Reverse 6 129 1141723 931539 930603-931772 Reverse 7 115 1941557
131753 131583-131792 Reverse 8 137 2007007 66507 66264-68135
Forward 9 130 2313744 6023975 6023787-6024542 Forward 10 138
2459178 5878418 5877776-5878597 Forward 11 106 2870317 5467402
5466520-5467887 Forward 12 132 3616599 4721118 4720249-4721457
Forward 13 133 3628342 4709375 4707483-4710572 Forward 14 113
3771157 4566558 4565369-4567903 Reverse 15 134 3781254 4556461
4555202-4558177 Forward 16 103 3961920 4375793 4375589-4376680
Forward 17 119 4446918 3890793 3890724-3892004 Reverse 18 118
4447967 3889744 3889088-3889738 Reverse 19 120 4592102 3745609
3744850-3746016 Forward 20 121 4594988 3742723 3742643-3743635
Forward 21 122 4595538 3742173 3740961-3742217 Forward 22 123
4597340 3740171 3740054-3740968 Forward 23 124 4598281 3739430
3738724-3740052 Forward 24 125 4600099 3737612 3737561-3738727
Forward 25 126 4603518 3734193 3730455-3737564 Forward 26 127
4608787 3728924 Reverse 135 4942182 3395532 3395274-3396677 Reverse
27 114 5209051 3128663 3127731-3129116 Forward 28 104 5402505
2935208 2934490-2935593 Forward 29 105 5410045 2927668
2926722-2927972 Reverse 30 108 5617382 2720329 2718890-2720643
Reverse 31 109 5651872 2678258 2671678-2679012 Reverse 32 110
5661697 2676014 2671678-2679012 Reverse 32 111 5666282 2671429
2669119-2671674 Reverse 33 112 5701004 2636707 2636467-2638800
Reverse 34 107 5799641 2538070 2532619-2539008 Reverse 35 100
5801998 2535711 2532619-2539008 Reverse 35 128 5924799 2412909
2412807-2414201 Forward 36
[0164] qsc101 and 102 genes are an example of a putative operon
that was not identified by functional coupling (FIG. 4). These two
ORFs did not show significant similarities with other polypeptides.
Nevertheless, they are transcribed in the same direction, closely
juxtaposed, qsc101 and 102 are both Class I genes, and there is a
las box-like element upstream of these ORFs. Expression of the
qsc102 insertion is controlled by an upstream ORF (SEQ ID NO:37)
which comprises the sequences between postions 2068711 to 267911 of
the P. aeruginosa genome (Dec. 15, 1999 release) which in turn is
preceded by a las box regulatory element (SEQ ID NO:38) which
comprises the sequences between postions 2068965 to 2068946 of the
P. aeruginosa genome (Dec. 15, 1999 release). The las box is a
palindromic sequence found upstream of and involved in
LasR-dependent activation of lasB (Rust, L. et al., (1996) J.
Bacteriol. 178, 1134-1140).
[0165] The qsc133A and B insertions are in a putative 3-gene operon
with similarity to acrAB-tolC from E. coli and the mex-opr family
of efflux pump operons in P. aeruginosa, one of which (mexAB-oprN)
has been shown to aid 3OC.sub.12-HSL efflux (Kohler, T., et al.
(1997) Mol. Microbiol. 23, 345-354; Poole, K, et al. (1993) J.
Bacteriol. 175, 7363-7372; Poole, K. et al. (1996) Mol. Microbiol.
21, 713-724; Evans, K., et al. (1998) J. Bacteriol. 180, 5443-5447;
Pearson, J. P. et al. (1999) J. Bacteriol. 181, 1203-1210). The
qsc133 mutations are within a gene encoding a MexF homolog. The
qsc133 mutants show typical Class IV regulation. Expression of lacZ
is late and dependent on the presence of both acyl-HSL signals
(Table 3 and FIG. 2). No las box-like sequences upstream of this
suspected efflux pump operon were identified.
[0166] A third possible operon identified by functional coupling is
about 8 kb and contains 10 genes. Eight strains with insertions in
6 of the 10 genes were obtained, all of which are Class III mutants
(Table 3). A las box-like sequence was identified upstream of the
first gene of this operon. The function of these 10 genes is
unknown but the similarities shown in Table 2 suggest that they may
encode functions for synthesis and resistance to an antibiotic-like
compound.
[0167] The qsc128 mutation is within a gene coding for a
polypeptide that shows similarity to the P. fluorescens hcnB
product and appears to be in a 3-gene operon (Table 3, FIG. 4). By
analogy to the P. fluorescens hcn operon, this operon is likely
required for the production of the secondary metabolite, hydrogen
cyanide. Previous investigations have shown that hydrogen cyanide
production is reduced in P. aeruginosa rhl quorum sensing mutants.
Consistent with this, qsc128 is a Class III mutant (Table 2). Full
induction required both acyl-HSL signals, however, some induction
of lacZ resulted from the addition of either signal alone (Table
3). A las box-like sequence was identified in the region upstream
of the translational start codon of the first gene in this operon.
This las-type box may facilitate an interaction with either LasR or
RhlR.
[0168] The phz operon, required for phenazine biosynthesis, has
been described in other pseudomonads and the insertion in strain
qsc131 is located in a gene encoding a phzC homolog. Analysis of
the sequence around this phzC homolog revealed an entire phenazine
biosynthesis operon, phzA-G (Georgakopoulos, D. G. et al. (1994)
Appl. Environ. Microbiol. 60, 2931-2938; Mavrodi, D. V. et al.
(1998) J. Bacteriol. 180, 2541-2548). As discussed above, qsc131
does not produce the blue phenazine pigment pyocyanin. PhzC is part
of an operon of several genes including PhzABCDEFG, and
transcription of this operon is controlled by the promoter region
(SEQ ID NO:39) in front of the first gene in the operon, PhzA. The
phz operon in P. aeruginosa also contains a las-box like sequence
upstream of the first gene of the operon. The PhzA promoter region
(SEQ ID NO:39) has been cloned into a plasmid, transcriptionally
fused to lacZ. The resulting plasmid (pMW303G) was transformed into
PAO1 and used as a reporter strain. The resultant bacterial strain
generates a quorum sensing signal and responds to it by increased
.beta.-galactosidase activity. As shown in FIG. 5, this strain
displayed a high level of induction between early and late growth,
thus providing a dynamic range for detecting modulation (e.g.,
inhibition) of quorum sensing signaling. Accordingly this strain
may be useful for a single strain assay for identifying for
inhibitors of quorum sensing singaling, as described herein.
[0169] The final putative operon consists of 2 or 3 genes,
qsc109-111, which appear to be involved in pyoverdine synthesis.
These ORFs were not identified in the P. aeruginosa genome project
web site but were identified and shown to be functionally coupled
with the Argonne National Laboratory web site.
[0170] For three of the qsc insertions, the lacZ gene was in an
orientation opposite to the ORF described in the Genome Project web
site (qsc114, 127, and 136).
[0171] Locations of qsc Genes on the P. aeruginosa Chromosome. The
qsc genes were mapped to sites on the P. aeruginosa chromosome
(FIG. 6). In addition lasB, lasR and lasI, and rhlR were placed on
this map. The distribution of currently identified qsc genes is
patchy. For example, 16 of the 39 qsc genes representing 3 of the
classes mapped to a 600-kb region of the 6 megabase chromosome. A
140-kb island of 12 Class III genes, 8 transcribed in one direction
and 4. transcribed in the other direction (including the rhl genes)
formed another cluster on the chromosome.
[0172] Identification of las Box-Like Sequences that Could be
Involved in qsc Gene Control. As discussed above, the las box is a
palindromic sequence found upstream of and involved in
LasR-dependent activation of lasB (Rust, L. et al. (1996) J.
Bacteriol. 178, 1134-1140). The las box shows similarity to the lux
box, which is the promoter element required for quorum control of
the V. fischeri luminescence genes (Devine, J. et al. (1989) PNAS
86, 5688-5692). Elements similar to a las box were identified by
searching upstream of qsc ORFs. A search was done for sequences
with at least 50% identity to the las box found 42 bp upstream of
the lasB transcriptional start site (Rust, L. et al. (1996) J.
Bacteriol. 178, 1134-1140). las box-like sequences were identified
which are suspected to be involved in the regulation of 14 of the
39 qsc genes listed in Table 1 (FIG. 7). Because there is little
information on the transcription starts of most of the genes
identified in the screening assay, some relevant las boxes may have
been missed and some of the identified sequences may not be in
relevant positions.
[0173] Discussion
[0174] By screening a library of lacZ promoter probes introduced
into P. aeruginosa PAO1 by transposon mutagenesis, 39 quorum
sensing controlled (qsc) genes were identified. Most of these genes
were not identified as quorum sensing-controlled previously.
Mutations were not found in every gene in putative qsc operons
(FIG. 4). Mutants that showed only a small degree of
acyl-HSL-dependent lacZ induction in the initial screen were not
studied. Thus, it is presumed that all of the quorum sensing
controlled (qsc) genes have been identified. A conservative
estimate is that about 1% of the genes in P. aeruginosa are
controlled by quorum sensing (39 out of about 5,000-6,000 genes in
the P. aeruginosa chromosome were confirmed to be qsc without
saturating the mutagenesis). A more liberal estimation of 3-4% can
be drawn from the finding of 270 mutants showing at least a 2-fold
induction in response to one or both of the acyl-HSL signals in the
initial screen of 7,000 mutants.
[0175] Several mutants, for example qsc101 and 102 showed an
immediate and relatively large response to 3OC.sub.12-HSL (Class I
mutants, Table 3). The qsc101 and 102 genes code for proteins with
no matches in the databases. Several mutants showed a relatively
large and immediate response when both signals were supplied in the
growth medium. Examples are qsc119 (rhlB), 121-125, and 129A and B.
The qsc mutant showing the largest response was qsc131. The level
of .beta.-galactosidase activity when this mutant was grown in the
presence of both signals was greater than 700 times that in the
absence of the signals (Table 3). The qsc131 mutation is in phzC,
which is a phenazine biosynthesis gene, and the qsc131 mutant did
not produce the blue phenazine pigment pyocyanin at detectable
levels. Many of the mutants that responded best to both signals
early (Class III mutants) showed a small response when exposed to
one or the other signal. The reasons for the small response to
either signal are unclear at present but the data suggest that
these genes may be subject to signal cross talk, or they may show a
response to either LasR or RhlR. One reason they may respond to
both signals better than they respond to C.sub.4-HSL alone is that
3OC.sub.12-HSL and LasR are required to activate RhlR, the
transcription factor required for a response to C.sub.4-HSL
(Latifi, A. et al. (1996) Mol. Microbiol. 21, 1137-1146; Pesci, E.
C. et al. (1997) J. Bacteriol. 179, 3127-3132). There were two
mutant classes that showed a delayed response to the signals; Class
II mutants which required only 3OC.sub.12-HSL, and Class IV
mutants, which required both signals for full induction. These
mutants showed between 5 and 45-fold activation of gene expression
(Table 3). There are a number of possible explanations for a
delayed response to signal addition. It is possible that some of
these genes are stationary phase genes. It is also possible that
some are iron repressed. For example, it is known that the
synthesis of pyoverdine is regulated by iron and the Class II,
delayed response, qsc108-111 mutations are in genes involved in
pyoverdine synthesis (Cunliffe, H. E. et al. (1995) J. Bacteriol.
177, 2744-2750; Rombel, I. et al. (1995) Mol. Gen. Genet. 246,
519-528). It is also possible that some of these genes are not
regulated by quorum sensing, directly. The acyl-HSL signals might
control other factors that influence expression of any of the genes
that have been identified and this possibility seems most likely
with the late genes in Classes II and IV. Indirect regulation may
not be common for late genes. This is known because the lasB-lacZ
chromosomal insertion which was generated by site-specific mutation
was in Class IV, and it is known from other investigations that
lasB responds to LasR and 3OC.sub.12-HSL, directly (Passador, L. et
al. (1993) Science 260, 1127-1130; Rust, L. et al. (1996) J.
Bacteriol. 178, 1134-1140). The two classes of late qsc genes may
be comprised of several subclasses.
[0176] Las boxes are genetic elements which may be involved in the
regulation of qsc genes. Although sequences with characteristics
similar to las boxes were identified, (FIG. 7), the locations of
these sequences have not provided insights about the differences in
the patterns of gene expression among the four classes of genes. It
is possible, that when the promoter regions of the qsc genes are
studied that particular motifs in the regulatory DNA of different
classes of genes will be revealed.
[0177] Many of the qsc genes appear to be organized in two patches
or islands on the P. aeruginosa chromosome (FIG. 7). Because LasR
mutants are defective in virulence it is tempting to speculate that
these gene clusters may represent pathogenicity islands. The
rhlI-rhlR quorum sensing modulation occurs on one of the qsc
islands, but none of the qsc genes are tightly linked to the
lasR-lasI modulon. Genes representing each of the 4 classes occur
over the length of the chromosome and on both DNA strands. This is
consistent with the view that quorum sensing is a global regulatory
system in P. aeruginosa. Of interest there is a third LuxR family
member in P. aeruginosa. This gene is adjacent to and divergently
oriented from qsc103.
[0178] Quorum sensing is critical for virulence of P. aeruginosa
and for the development of mature biofilms. The methodology
disclosed herein for identification of qsc genes provides a
manageable group of genes to test for function in virulence and
biofilm development. Furthermore, the availability of the P.
aeruginosa genome sequence will very likely lead to the development
of a gene expression microarray for this organism. The methods
described herein provide a set of 39 genes that respond to specific
treatments in a predictable fashion (Table 3).
EXAMPLE 2
Screening Assay for Quorum Sensing Inhibiting Compounds
[0179] In this example, the screening assay used two strains of P.
aeruginosa: a wild type P. aeruginosa (PAO1) and QSC102, from
Example 1 (see FIG. 8). This assay will detect inhibition of all
aspects of quorum sensing signaling, e.g., signal generation and
signal reception.
[0180] Procedural Overview
[0181] Microtiter plates are prepared by adding 200 .mu.L Luria
Broth ("LB") agar, containing 0.008%
5-bromo-4-chloro-3-indolyl-.beta.-D-galact- ose (X-gal) to each
well. Overnight cultures of PAO1 and QSC102 are subcultured in LB
to a starting absorbance at 600 nm ("A600") of 0.05 and grown at
37.degree. C. to an A600 of 1.0. PAO1 is diluted
2.5.times.10.sup.5-fold in LB and 5 .mu.L of this is applied to the
surface of the LB agar in each well. Plates are then dried in a
laminar flow hood for 60 minutes. A tenfold dilution of QSC102 in
LB is used to inoculate each well using a replicator. Plates are
then sealed and incubated at 37.degree. C. for 40 hours. Growth and
color development are evaluated visually and the data is recorded
with a camera.
[0182] The test compound was present in a microtiter well and
overlaid with LB agar and
5-bromo-4-chloro-3-indolyl-.beta.-D-galactose (X-gal). Both strains
were spotted on the agar in each well. PAO1 emitted the acyl-HSL
signal (3-oxo-C12-HSL), to which QSC102 responded by turning blue.
QSC102 expressed galactosidase only in response to the LasI signal
(3-oxo-C12-HSL); the lacZ fusion in QSC102 did not respond to the
RhlI signal (C4-HSL). Hence, the assay was selective for inhibitors
of the Las system. Inhibition of signaling was evaluated
qualitatively by the absence or weakening of the blue color
development.
[0183] The assay was used to test 6 product analogs, two of which
showed an inhibitory effect: butyrolactone and
acetyl-butyrolactone. Although bacterial growth was not inhibited,
the color development was reduced. Color reduction correlated
directly with test compound concentration, although relatively high
concentrations (.about.20 mM) were required to suppress color
development completely (FIG. 9). 2
EXAMPLE 3
Development of a P. aeruginosa Strain for a High Throughput
Screening Assay
[0184] A. Construction of Reporter Strain-Chromosomal Insertion of
Reporter
[0185] A strain for use in high-throughput screening was
constructed by inserting the lacZ transcriptional fusion, linked
gentamicin resistance marker, and about 2 kb of flanking DNA from
strain QSC102 into a mobilizable plasmid (such as pSUP102) as
depicted in FIG. 10A. Plasmid pSUP102 confers tetracycline
resistance and does not replicate in P. aeruginosa (Simon, R. et
al. (1986) Meth. Enzym. 118:640-659). The pSUP102-derivative was
then transferred into PAO1 by bi- or triparental mating, selecting
for gentamicin resistance (Suh, S. J. et al. (1999) J Bacteriol.
181(13):3890-7). Gentamicin resistant isolates were screened for
tetracycline sensitivity (i.e., a double cross-over event has
resulted in a chromosomal insertion). Southern blotting was used to
confirm the nature of the recombination event and to rule out
candidates with more than one insertion. The resultant bacterial
strain-generates the signal (3-oxo-C12-HSL) and responds to it by
increased .beta.-galactosidase activity. A similar strategy is used
to create a reporter strain that expresses gfp instead of lacZ. The
initial GFP variant is the stable and bright variant GFPmut2
(Cormack, B. P. et al. (1996) Gene. 173(1):33-38).
[0186] Procedural Overview of Assay
[0187] A culture of PAQ1 reporter strain (carrying the reporter
gene lacZ transcriptionally fused to the regulatory sequence of
qsc102 in the wildtype background, PAO1) was grown in LB, 100
.mu.g/ml gentamicin overnight, such that the A600 was around 0.1.
The culture was washed in LB twice and used to subculture at a
1:1000 dilution in LB. The subculture was grown in the presence or
absence of test compound. Growth was monitored at A600 and
expression of .beta.-galactosidase activity is measured according
to the Miller assay (Miller, J. A. (1976) in Experiments in
Molecular Genetics pp 352-355, Cold Spring Harbor Lab. Press,
Plainview, N.Y.).
[0188] The reporter strain was tested by growing it in microtiter
plates in the presence and absence of known inhibitors of bacterial
signaling. Examples of known inhibitors are: acetyl-butyrolactone,
butyrolactone, and methylthioadenosine, a product of the synthase
reaction that was shown to be inhibitory to the RhlI synthase
(Parsek, M. R. et al. (1999) Proc. Natl. Acad. Sci. USA.
96:4360-4365). Initial characterization of the assay entailed
following the optical density (cell growth) in individual sample
wells and measuring induction levels at different time points. FIG.
10B shows the induction of .beta.-galactosidase as PAQ1 reaches
high density, wherein cell growth is measured at 600 nm (closed
circles) and expression of .beta.-galactosidase is measured in
Miller units (open circles). For GFP fusions, the fluorescence of
the culture is determined after excitation at 488 nm.
[0189] B. Construction of Reporter Strain-Reporter on a Plasmid
[0190] The PAO1/pMW303G strain is constructed as described in
Example 1 above.
[0191] Procedural Overview of the Assay
[0192] An overnight culture of PAO1/pMW303G was diluted to an A600
of 0.1 in LB, 300 .mu.g/ml carbenicillin. Of this, 50 .mu.L were
added to microtiter plate wells and grown at 37 .degree. C.,
shaking at 250 rpm, in the presence or absence of test compounds.
Culture growth was monitored directly in the microtiter plate at
620 nm. Expression of the reporter gene, .beta.-galactosidase was
measured with the Galacton substrate by Tropix as follows. 12A 20
.mu.L aliquot of the culture was added to 70 .mu.L of 1:100 diluted
Galacton substrate (Tropix, PE Biosystems, Bedford, Mass.) and
incubated in the dark at room temperature for 60 minutes. The
reaction was stopped and light emission was triggered by the
addition of 100 .mu.L Accelerator II (Tropix, PE Biosystems,
Bedford, Mass.), and luminescence was read with plate reader
(SpectrofluorPlus, Tecan). Timepoints were; taken at 5, 8 and 12
minutes.
[0193] In either embodiment of the assay (chromosomal insertion of
reporter, or reporter on a plasmid), a satisfactory -assay shows
normal cell growth but reduced .beta.-galactosidase activity or gfp
expression in the presence of a known signaling inhibitor. Possible
problems associated with the use of fluorescence in whole-cell
systems are interference by turbidity as cell density increases and
the production of pyocyanin and pyoverdine, fluorescent molecules
that are excreted by wild type P. aeruginosa. However, interference
due to endogenous fluorescent pigments may be reduced by using
mutants that lack these pigments (Byng, G. S. et al. (1979) J
Bacteriol. 138(3):846-52).
EXAMPLE 4
Screening Assay to Determine Inhibition of the Signal Synthase
[0194] An assay was developed to measure inhibition of RhlI
activity, based on a previously published enzyme assay for RhlI
(Parsek, M. R. et al. (1999) Proc. Natl. Acad. Sci. USA.
96:4360-4365). It was shown that the substrates for RhlI are
S-adenosylmethionine (SAM) and butanoyl-acyl carrier protein
(C4-ACP). It is proposed that RhlI can be used as a model enzyme to
study inhibition of acyl-HSL synthases. This is based on the
observation that TraI from Agrobacterium tumefaciens (Mor, M. I. et
al. (1996) Science. 272(5268):1655-8) and LuxI from Vibrio fischeri
(Schaefer, A. L. et al. (1996) Proc Natl Acad Sci USA.
93(18):9505-9), two homologs of RhlI and LasI, that also utilize
SAM and the respective acylated-acyl carrier protein as their
substrates.
[0195] RhlI activity assay. Studies of autoinducer synthases have
been hampered by the low solubility of the enzyme. It is only in
the past year that the first rigorous characterization of an
autoinducer synthase was published (Parsek, M. R. et al. (1999)
Proc. Natl. Acad. Sci. USA. 96:4360-4365). This study was performed
on RhlI, which had been slightly overproduced in a LasI minus
strain of P. aeruginosa, thereby avoiding previously encountered
problems of solubility. The reaction mechanism deduced for RhlI is
summarized in FIG. 11. The substrates for the synthase are
butanoyl-acyl carrier protein (C4-ACP) and S-adenosylmethionine
(SAM). The amino-group of SAM attacks the thioester of C4-ACP to
form a peptide bond between butanoic acid and SAM. The first
product, acyl carrier protein (ACP) is released. Next, the
SAM-moiety undergoes internal ring closure to form a homoserine
lactone (HSL). Methylthioadenosine (MTA) and butanoyl-HSL (C4-HSL)
are released.
[0196] The enzyme assay reaction mixture contains 60 .mu.M
.sup.14C-labeled SAM and 40 .mu.M C4-ACP in a final volume of 100
.mu.L. (buffer: 2 mM dithiothreitol, 200 mM NaCl, 20 mM Tris-HCL,
pH 7.8). The reaction is started with the addition of 70 ng RhlI,
incubated at 37.degree. C. and quenched after 10 min by addition of
4 .mu.L of 1 M HCl. Product formation is quantitated by extracting
the reaction mixtures with 100 .mu.L ethyl acetate and
scintillation counting the radiolabeled C4-HSL, which partitions
into the organic phase. (SAM remains in the aqueous phase.)
[0197] Other variations on the assay include detection of the
non-acylated ACP (i.e., ACP with a free thiol group). Non-acylated
ACP can be detected through the use of a thiol reagent such as
dithionitrobenzoic acid (DTNB), which releases a highly colored
thiolate (.epsilon..sub.412=13 600 cm.sup.-1 M.sup.-1) upon
reaction with thiol groups (Ellman, G. L. (1959) Arch. Biochem.
Biophys. 82:70-77). Another variation of this assay uses an even
more sensitive reagent, 4,4'-dithiobipyridyl which has a
.epsilon..sub.324=20 000 cm.sup.-1 M.sup.-1 (Jamin, M. et al.
(1991) Biochem J. 280(Pt 2):499-506). Use of DTNB eliminates the
need for radioactivity and allows for a continuous assay.
[0198] Another variation on the assay includes using a substitute
for the substrate C4-ACP. It has already been found that RhlI turns
over butanoyl-CoA in lieu of C4-ACP (Parsek, M. R. et al. (1999)
Proc. Natl. Acad. Sci. USA. 96:4360-4365). The K.sub.M for the CoA
substrate is 230 .mu.M, compared to 6 .mu.M for C4-ACP, but
v.sub.max is only one order of magnitude slower. N-Acetylcysteamine
represents a truncated moiety of CoA and acylated
N-acetylcysteamines often function as substrate analogs for
CoA-dependent enzymes (Bayer et al. (1995) Arch Microbiol.
163(4):310-2; Singh, N. et al. (1985) Biochem Biophys Res Commun.
131(2):786-92; Whitty, A. (1995) Biochemistry. 34(37):11678-89). It
will be determined whether butanoyl-N-acetylcysteamine is turned
over by RhlI. If so, an assay will be developed for the release of
free thiol groups with a thiol reagent such as DTNB.
Butanoyl-N-acetylcysteamine is readily synthesized from the
commercially available precursors butyrylchloride and
N-acetylcysteamine. 3
[0199] LasI activity assay. In analogy with khlI, TraI, and LuxI,
proposed substrates for LasI are SAM and 3-oxo-C12-ACP. In this
assay, compounds are tested for inhibiting the activity of LasI.
This assay is based on observations that bacterial strains
incubated with .sup.14C-labeled methionine produce radiolabeled
acylated-HSLs, which can be isolated from the culture supernatant
and identified by their retention times (in comparison to known
standards) when eluted over a high pressure liquid chromatography
(HPLC) reversed phase column. A synthase-inhibitor assay has been
set up using this methodology.
[0200] A Pseudomonas strain that expresses lasI but not rhlI, such
as PDO100, is grown in the presence and absence of the test
compound (Brint, J. M. et al. (1995) J Bacteriol. 177(24):7155-63).
Cells are pulsed for 10-30 minutes with .sup.14C-labeled methionine
(available from American Radiochemicals) and pelleted by
centrifugation. The supernatant liquid is extracted with ethyl
acetate and the products separated by HPLC. If the test compound
inhibits LasI synthase, the amount of 3-oxo-C12-HSL produced will
be significantly reduced when compared to the control.
[0201] An in vitro assay for LasI activity similar to the
radiometric assay used to study RhlI will be developed. The
substrates for this assay are .sup.14C-labeled SAM (available
Amersham Pharmacia) and 3-oxo-C12-ACP (similar methodology in Mor,
M. I. et al. (1996) Science. 272(5268):1655-8). LasI activity is
monitored by the appearance of radiolabeled 3-oxo-C12-HSL, after
extraction into ethyl acetate and scintillation counting.
Initially, crude extracts of LasI overexpressed in E. coli serve as
the source of enzyme. Once a satisfactory assay is in place, a
purification protocol will be developed to obtain LasI in a soluble
and active form. The purification may involve expression at low
levels (low plasmid copy number, weak promoter, low growth
temperature) in a P. aeruginosa rhlI mutant. Purification will
follow standard techniques such as ammonium sulfate precipitation,
anion exchange chromatography, cation exchange chromatography and
size-exclusion chromatography.
EXAMPLE 5
IN Vivo Assays to Determine Inhibition of Signal Binding
[0202] In vivo assays were also used to determine whether a test
compound inhibits signal reception by LasR.
[0203] One assay used the P. aeruginosa strain QSC102 (Table 3),
which responds to the presence of exogenous 3-oxo-C12-HSL by
inducing .beta.-galactosidase activity up to 400-fold (Example 1).
Cells were grown in the presence of a minimal concentration of
3-oxo-C12-HSL and in the presence and absence of the test compound.
If the test compound interferes with signal reception,
.beta.-galactosidase activity is reduced. Interference can be a
result of any of several mechanisms. The simplest is, if the test
compound prevents the 3-oxo-C12-HSL from binding to LasR.
Alternatively, the test compound may prevent LasR from binding to
DNA or interacting productively with RNA polymerase.
[0204] A further in vivo assay is used to determine whether a test
compound inhibits binding of 3-oxo-C12-HSL to LasR. This assay is
based on an observation originally made with LuxR of Vibrio
fischeri. Namely, the autoinducer binds to Escherichia coli cells
in which LuxR is produced, provided that LuxR is co-expressed with
Hsp60 (Adar et al. (1993) J Biolumin Chemilumin. 8(5):261-6). This
finding was used to develop a competition-assay for binding of
inhibitors to LuxR (Schaefer, A. L. et al. (1996) J Bacteriol.
178(10):2897-901) and LasR (Passador, L. et al. (1996) J Bacteriol.
178(20):5995-6000). Briefly, cultures of E. coli harboring
expression plasmids for Hsp60 and LasR (or LuxR) are induced for
several hours, at which time an aliquot of cells is added to
tritiated signal molecule, alone or in combination with a potential
inhibitor. After 10-15 minutes, cells are pelleted by
centrifugation, washed, and the amount of radioactivity bound to
the cells is determined by scintillation counting.
[0205] Plasmids for expression of LasR (pKDT37) (Passador, L. et
al. (1996) J Bacteriol. 178(20):5995-6000) and Hsp60 (pGroESL) have
been made. A simple method for preparing .sup.14C-labeled
3-oxo-C12-HSL has been developed. E. coli cells expressing lasI
excrete .sup.14C-labeled 3-oxo-C12-HSL into the medium when
incubated in the presence of .sup.14C-labeled methionine. The
.sup.14C-labeled 3-oxo-C12-HSL can be recovered by extraction into
ethyl acetate and purified by HPLC. The correct product is
identified by its radioactivity and by the correct HPLC retention
time compared to an unlabeled standard.
EXAMPLE 6
Assay for Inhibition of Biofilms
[0206] This assay tests whether compounds useful for inhibiting
quorum sensing also inhibit or modulate the formation or growth of
biofilms. The LasI/LasR signaling system was found to regulate not
only the expression of virulence factors, but also the development
of mature biofilms (Davies, D. G. et al. (1998) Science.
280(5361):295-8). This was demonstrated by using a simple
flow-through system, as shown in FIG. 12, that allows fresh medium
to be pumped through a small chamber in a Plexiglas body.
[0207] Cultures of P. aeruginosa expressing green fluorescent
protein (GFP) were grown in a chamber that was sealed with a
coverslip and flushed with fresh medium. Surface attachment and
biofilm maturation were determined by examining the coverslip by
epifluorescence and confocal microscopy. Both wild type PAO1 and a
rhlI mutant strain were able to attach to the surface and form the
mushroom-shaped structure characteristic of a biofilm. However, a
lasI mutant that cannot synthesize the signal molecule
3-oxo-C12-HSL was only able to attach to the surface. It did not
encase itself in an extracellular matrix or form any kind of
three-dimensional structure. It also remained susceptible to 0.2%
sodium dodecyl sulfate, which was used to mimic the susceptibility
to a biocide. When the 3oxo-C12-HSL signal was added back to the
lasI mutant cells, the wild type phenotype was restored. The cells
formed biofilms and remained resistant to sodium dodecyl
sulfate.
[0208] Accordingly, the bioreactor depicted in FIG. 12 is
inoculated with wild type P. aeruginosa PAO1 that expresses GFP.
Test compounds (signaling inhibitors) are added to the flow-through
medium to determine whether they prevent formation of the
three-dimensional structures typical of a bacterial biofilm.
Biofilm formation is monitored using a confocal microscope.
REFERENCES
[0209] 1. Holloway, B. W., Krishnapillai, V. & Morgan, A. F.
(1979) Microbiol. Rev. 43, 73-102.
[0210] 2. Brint, J. M. & Ohman, D. E. (1995) J. Bacteriol. 177,
7155-7163.
[0211] 3. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989)
Molecular cloning: a laboratory manual (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0212] 4. Miller, V. L. & Mekalonos, J. J. (1988) J. Bacteriol.
170, 2575-2583.
[0213] 5. Simon, R., Priefer, U. & Puhler, A. (1983)
Bio-Technology 1, 37-45.
[0214] 6. Pearson, J. P., Pesci, E. C. & Iglewski, B. H. (1997)
J. Bacteriol. 179, 5756-5767.
[0215] 7. Linn, T. & St Pierre, R. (1990) J. Bacteriol. 172,
1077-1084.
[0216] 8. Schweizer, H. P. (1993) Biotechniques 15, 831-833.
[0217] 9. Figurski, D. H. & Helinski, D. R. (1979) Proc. Natl.
Acad. Sci. USA 76, 1648-1652.
[0218] 10. Simon, R., O'Connell, M., Labes, M. & Puhler, A.
(1986) in Methods in Enzymology, Vol. 118, pp. 640-659.
[0219] Incorporation by Reference
[0220] The contents of all references, patents and published patent
applications cited throughout this application, as well as the
figures and the sequence listing, are incorporated herein by
reference.
[0221] Equivalents
[0222] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments and methods described
herein. Such equivalents are intended to be encompassed by the
scope of the following claims.
Sequence CWU 1
1
39 1 1218 DNA Pseudomonas aeruginosa 1 atggacgatc tattgcaacg
cgtacggcgc tgcgaagcgc tgcagcaacc cgaatggggc 60 gatccgtcgc
gcctgcgcga cgtgcaggcg tacctgcgcg gcagtccggc gctgatccgc 120
gccggcgaca tcctggccct gcgcgcgacc ctggcgcggg tcgcccgcgg cgaggcgctg
180 gtggtacagt gcggcgactg cgccgaggac atggacgacc accatgccga
gaacgtggcg 240 cgcaaggccg ccgtgctgga actgctggcc ggcgccctgc
gcctggccgg ccggcggccg 300 gtgatccgcg tcgggcgcat cgccgggcag
tacgccaagc cgcgttccaa gccgcacgag 360 caggtcggcg agcagaccct
gccggtctat cgcggcgaca tggtcaacgg ccgcgaggcc 420 catgccgaac
agcgccgggc cgatccgcag cggatcctca agggctatgc ggcggcgcgc 480
aacatcatgc gccacctggg ctgggacgcc gcgtccgggc aggaggcgaa tgcctcgccg
540 gtctggacca gccacgagat gctgctgctc gactacgagc tgtcgatgct
gcgcgaggac 600 gagcagcgcc gggtctatct cggttcgacc cactggccgt
ggatcggcga gcgcacccgc 660 caggtcgacg gcgcccatgt ggcgctgctg
gccgaggtgc tcaacccggt ggcctgcaag 720 gtcggtccgg agatcggccg
cgaccagttg ctggcgctct gcgagcgcct cgatccgcgc 780 cgcgagccgg
gacgcctgac gctgatcgcg cggatgggcg cgcagaaggt cggcgagcgc 840
ctgccgccgc tggtggaggc ggtgcgcgcg gccgggcacc cggtgatctg gctgagcgac
900 ccgatgcacg gcaacaccat cgtcgcgccc tgcggcaaca agacccgcct
ggtgcgcagc 960 atcgccgagg aggtggcggc gttccgcctg ggatcctcta
gccgaggcgg cgtgctcaac 1020 ggactgcacc tggaaaccac cccggacgac
gtcaccgagt gcgtcgccga ttccagcggc 1080 ctgcaccagg tcagccggca
ctacaccagc ctctgcgatc cgcggctgaa cccctggcag 1140 gcgctcagcg
cggtgatggc ctggtccggc gcagaagcga tccagagcgc aaccttcccc 1200
ctggagaccg tggcatga 1218 2 1782 DNA Pseudomonas aeruginosa 2
atggatgatg gggcacagcc tgctgcacac ctcggatgcc ctgccaccac tgctggccgg
60 cttcgccgcc tacttcgtca acaccttcgt cacctactgg tggcatcgcg
cgcgccacgc 120 caacgacacg ctctggcggc tgttccacca gttgcaccac
gcgccgcaac gcatcgaggt 180 attcacctcc ttctacaagc atccgaccga
gatggtcttc aactcgctgc tgggcagctt 240 cgtcgcctac gtggtgatgg
gcatcagcat cgaggccggc gcctactaca tcatgttcgc 300 cgcgctcggc
gagatgttct accactcgaa cctgcgcacc ccgcacgtcc tcggctacct 360
gttccagcgc ccggagatgc accgcatcca ccaccagcgc gaccgtcacg agtgcaacta
420 cagcgacttc ccgatctggg acatgttgtt cggcacctac gagaaccccc
gccgcatcga 480 cgagccgcag ggcttcgccg gcgacaagga gcagcagttc
gtcgacatgc tgctgttccg 540 cgacgtgcac agcctccccg gaaaaaccca
gcccgctccc gtcctggtca agcccgacgt 600 caggtgaacg ccatgattcc
agacatcgat tcccgtctca gccggaacat attgaaatcc 660 atctcgtatg
gcctccccct cgccgaagtg gtccccgacc atacctatgc gcaactggaa 720
acgcgcctcg gcgaactgaa acgcaggtat ctggagctgc gcatctccca cggcgcgcgc
780 gagctgccgt tcagcaacta cctgttctac ctgatcctcc agtcgcgcca
ccaggaattc 840 gacttcaagc tgcgccaggg caactcggtg gtcaccaaca
tccaccgatt caagagcaag 900 ggacgcatcc cgtccctgac caccctgctc
ctggccgatg cggtcaacgc caagagcgag 960 ctggagctca agcatccgga
catcccgcag ctcgaccgcc acgctcgcga catcgagcgc 1020 tggctggccg
ccggcaacgt catgccgccc agcgagcggg ccctgcgcgg cctggttgag 1080
gcgctggagc gcgccgctgg cgaaggccgt ccgttgcacc tggtgagcgc ggtatgcccg
1140 gactactcgc actccagcga tgccgagggc aagccgcgct acaccttcga
gcgagtcggc 1200 gaccagcccg gcctggccgg cgccaagctg gtcagcgccg
gccaggcggt ggcggagctg 1260 gccagggcgc gccaggtgga aatccgccac
gcgatcctcg gcggcgagtt cgagtaccta 1320 tcgttcaacc gcaaccccgc
caccggcgag acccgcgagg gtttcctcgg caaggtcgag 1380 cgccagctcg
agcggatcgc cggggccctg ccctgcccgg ccgcgacctg ctcgttcttc 1440
gagatgtgcg gcggcgagga cggctggcac caggcccacg gcgagatcgt ccagcgcctg
1500 gaacagggcg actacggcca gaccgggctg gactacccgg ccctggaatc
gatcttcctg 1560 tcgcgcctgc cgctctacga gaaatggttc gccagccagt
cgcgcgagca gatctgggcc 1620 agcttcgtct cccaggccgc cgagtacgca
ttgatgggaa aactcttcgg cgagcgcttc 1680 gacaacttcg tcgtgctggc
cgtcgatcac taccggatgg agccgttcta ctcgttcttc 1740 gcgaccgtcc
cgacgctcta catccgaacc gactacctgt aa 1782 3 693 DNA Pseudomonas
aeruginosa 3 atgccgccaa ccagccccac accaaccaac ccgcatccca ggctaccgcc
tgatcacaca 60 ggaaacccca tgaatactca gattgcccag atcacccaga
gcctggcagc caacggctgc 120 gcctatatca cccccagcga cgcgctctac
gacgagcagg actgggaact gatgaaccag 180 gtcctggcca actcgaccct
gccgtgggag aagatcctga tcggcgacgc cgacgaggag 240 aacgacctct
acgtggcccg tttcatgacc gaccgcgacc gtcccacggt ggtcaaccat 300
gcgctgtcgg agctgatcat cccgcgcgtc tgcaacgaca acgtgatgag cctgttccgc
360 aagctgatgg gcgacgacgc cttctacgtc cggcgcatgc aggtgaaccg
gatgaaggcc 420 ggctcgttca tcggccggca cctggatacc gacagcaacc
cggactacca gtactccatc 480 gtcctgcagc tcggcaccta cttctccggc
ggccagttcg tggtctacga ccgcgacggc 540 aacctgcgca acgacatcaa
gccggagccg cgctcggtga tcatcagcga ctgtagctat 600 ccccacgagg
tccagcaggt gaccgccggc gagcgcgtct cgctggtgtt cttcgtcagt 660
cgccatgcgg accggaaccg gcgggtctat tga 693 4 411 DNA Pseudomonas
aeruginosa 4 gtgacggact tcgaatcctc gcgtcgcgct ccgtccaccg gattgtccgg
cgcgctgcgg 60 cggccgcgct ccagcgcagc gccactgcca gatctggcag
ttgtcgctgg cggggacgga 120 ggctttagta gccgcacttt tttccagggc
cgggcagtgg gaccgcaatg catggacgac 180 atcgagacca gagtgaggaa
actggtagcc gcccggttcg gcgtggagga atgcgacatc 240 cggctggaca
gcgacttccg taacgacttc ggtgccgagt cgctcgaggt agtcgaactg 300
gtcatggccc tggaagcgga gttcggcgtc gagatcgccg atgacgatgc ggaacggatc
360 gagaccgtgc gccaggccat cgactatctc gaggaagccg tgccgacctg a 411 5
588 DNA Pseudomonas aeruginosa 5 atggccgtcc gggtcgagga agtagaacga
atcgccctcg ctgcggttct gcttccattc 60 gcgcacgcca tgcgcgcgca
gctgcgcggc gaagcgggcg aaatccgcgg cggcgatgcc 120 gaaggcgtag
tgcgtgtagt ccgcggccgg cccgccgtac tgcggctccc gggacaggca 180
cagccacagc gaacccagtt cgagataggc gccctggtcc cagcgcgctt ccaggcgaaa
240 gccgagaaga tcgcggtaga aggcgatgct ggccggcagg tcggcgaccg
ccagggtcag 300 gtgattgaga ccggtaagca tgggggctcc ttgcaagatg
tggcgggagg tcgattcagg 360 cacgtcccag ccagtcgccg cggatcattt
ccatcagttg gcgcaagccg ggttgcggct 420 ggcgtcggct cggatagtag
aggcagaacg gcgcgcccat cgaggtccag tccggcaata 480 ccagttgcag
ccggccgcta cgcagctcct cggcgattcc cacctccagg cagtaggcca 540
ggccgacacc gtccagggcc gcggcaaccg ccgtattgct ttcgttga 588 6 1020 DNA
Pseudomonas aeruginosa 6 atgaacggaa ccgccgccga taccctcgcc
gtatcgcccc cgcccctgcg caacctctgc 60 gacggccacg gccggctcga
tccccgggcg gtcggctggt ccgcccggcc gcgggtgctc 120 tgccacatcc
ccggccactt cggccggcgc aagcgctgga accactggtg catcgtcagc 180
cccggctgga tgctctcgct gaccatcgcc gacctcgact acctgaccta cggcgccgcc
240 tatttcctcg acctggacag cggccaggcg gtagcgcaca cgcagatccg
cttcttcggc 300 ctcggctgcc agttgcccga cgagccgcag gccagccatg
ccttcgagca tccccgcctg 360 caattgcgct tcgacgaaca gcccgggcgc
ctgcgcgtca ccggccaggc cccggacctc 420 ggtggcctgc cgctggagct
ggcgctggaa gtgcgacgac cgtcgcacct ggagtcggtg 480 aacctggtgg
tgccgatggg cgaacacacc ttccatgcct gcagccgcca gctcggcctg 540
ccgatcagcg gctgcctgca gctcggccgc cgacgctacg actgccaggc gggccagagc
600 ttcgccgcgc tggacttcgg ccgcggtgtc tggccgctgc atacctactg
gacccgcgcc 660 gccttcgccg cccccggcgg catcgccggc aacttcggca
ccggctggac cgaagccagc 720 gacctgcgcg agaacgccct gtggttcggc
ggcaagctca gccgcgtgct cgacgacgtg 780 cacatccgcg agcctcgcga
cccgctggcc gaatggcgcc tggacagcgc ctgcggtcgc 840 gtcgagctgc
tcttccgtcc cgaacagctg caccaggcgc ggcccagcgt cggcctgttc 900
tatgccaata cccgccagtg gttcggccgt ttcaacggca ccctgcgcca cgacgacggc
960 gactgcgtgc cggtggacgg cgccctcggc tggatcggtt cgacccgcgc
gcgctggtga 1020 7 1170 DNA Pseudomonas aeruginosa 7 atgaaacact
actcagccac cctggcactc ctgccactca ccctcgccct gttcctgccc 60
caggcagccc atgcccacgg ctcgatggaa acgccgccca gtcgggtcta cggctgcttc
120 ctcgaaggtc cggagaatcc caagtcggcc gcctgcaagg ccgccgtcgc
cgccggcggc 180 acccaggcac tgtacgactg gaatggcgtc aaccagggca
acgccaacgg caaccaccag 240 gcggtggtcc ccgacggcca gctctgcggc
gccggcaagg cactgttcaa gggcctgaac 300 ctggctcgca gcgactggcc
cagcactgcc atcgcgccgg acgccagcgg caacttccag 360 ttcgtctaca
aggccagcgc gccgcacgcg acccgctact tcgacttcta catcaccaag 420
gacggctata accccgagaa gccgctggcc tggagcgacc tggaacccgc gccgttctgc
480 tcgatcacca gcgtcaagct ggagaacggc acctaccgga tgaactgccc
gctgccccag 540 ggcaagaccg gcaagcatgt gatctataac gtctggcagc
gctcggacag cccggaagcc 600 ttctacgcct gcatcgacgt gagcttcagc
ggcgccgtcg ccaacccctg gcaagcgctg 660 ggcaacctgc gcgcgcagca
ggacctgcca gccggtgcta ccgtcaccct gcgtctgttc 720 gatgcccagg
gccgcgacgc ccagcgtcac agcctgaccc tggcccaggg cgccaacggt 780
gccaagcaat ggccgctggc gctggcgcag aaggtcaacc aggactccac cctggtcaac
840 atcggcgtgc tggatgccta cggggcggtc agcccggtgg ccagctcgca
ggacaaccag 900 gtctacgtgc gccaggccgg ctaccgcttc caggtcgaca
tcgaactgcc ggtcgagggc 960 ggcggcgagc aaccgggcgg cgacggcaag
gtcgacttcg actatccgca aggcctgcag 1020 caatacgacg ccgggaccgt
agtgcgcggt gccgatggca agcgctacca gtgcaagccc 1080 tacccgaact
ccggctggtg caagggctgg gacctctact acgccccggg caagggcatg 1140
gcctggcagg acgcctggac cctgctgtaa 1170 8 210 DNA Pseudomonas
aeruginosa 8 atgttgaaag tggcgatcgt cctgctactg ctggctaccc tggtgagcct
gttcagcggc 60 ctgttcttcc tggtcaagga ccagggccat ggttcccgcg
tggtcaattc gctgaccgtc 120 cgcgtggtgc tcgccgcggc gaccctggtg
ctggtcgcct ggggcttcta cagcggcgag 180 ctgaacagcc acgcgccctg
gcatttctga 210 9 1872 DNA Pseudomonas aeruginosa 9 atgagtttcc
cgataaacat caattatagg agtttcccta tgtgcggtct cgcgggttgg 60
gtggattaca cgcgcaagct cgacgacgaa tttccggcga tcttcgccat gaccgatacg
120 ctcgccttgc gcgggccgga tgccgagggc atctggaagc accgcaacgc
cctgctgggt 180 caccggcggc tggcggtcat cgacctcagc ggcggcgtgc
agccgatgtc ctatcgcttt 240 cccaccggcc aggaggtcac cctcgtctac
accggcgagg tgtacaacca cgatgccctg 300 cgcgagcggt tgcgccgggc
cggacatgag ttccgcaccc gcagcgatac cgaggtggtc 360 ctgcacgcct
atctgcaatg gggcgagcgt tgttgcgagt acctgaccgg gatgttcgcc 420
ttcgccgtct tcgatggccg cgacggccac ctgctgctgg tgcgcgaccg cctgggcatc
480 aagccgctgt attacgcgcg gcaccgcgag ggactgctgt tcggctcgga
gatcaagtcc 540 atcctggcgc atccggaatt cgccgccagg ctcgacgcgg
tcggcctggt cgacctcctg 600 acgctgtccc ggggcacttc gcagacgccg
ttccgcgagg tccaggaact gctgcccggc 660 cacctgctgt cctggcgtcc
caattcccag gcgaagttgc gccgctactg ggaggtgcgc 720 cgccaggagc
atgccgacga cctgcagagc accgtgcagc gcacccgcga actggtcacc 780
cgcgccctgg gggcgcaatt gcacgccgac gttccggtgt gttcgctgct atcgggtggg
840 ctcgattcga ccgccctgac cggcatcgcc cagcgcatcg cgaaggcgga
gcacggcggc 900 gacatcaatt cgttctcggt ggacttcgtc ggccaggccg
agcagttccg cagcgacgac 960 ctgcgtcccg accaggacca gccgttcgcc
ctgctggccg cgcagtacat cggcagccgt 1020 catcgcaccg tgctcatcga
caatgccgaa ctggtctgcg aacgagcgcg cgaagaggta 1080 ttccgggcca
aggacgtacc tttcaccttc ggcgacatgg atacctcgct gcacctgatg 1140
ttcggcgaga tccgccggca ttccacggtg gccatctccg gtgaaggcgc cgacgagctg
1200 ttcggtggct acggctggtt ccgcgatccg caggcggtgg ctgcggcgcg
cttcccctgg 1260 gcctccaggg tgcgcctgcc ggccggcttc atcgacgccg
gtttcaaccg ccgctgcgat 1320 ctcctccagt accagcaggc cagctacgac
gatgggctgc gccaggtcga acacctggcc 1380 ggcgacagcc cggaggagcg
gcggatgcgc gagttcagcc acctgcatct gaagcgctgg 1440 atggtgctgc
tgctcgaacg caaggatcgc ctgagcatgt gcaacggcct ggaggtgcgg 1500
gtgccctaca ccgaccatga gctggtggag tacgtctaca acgtgccctg gtcgatcaag
1560 agccgggacg gcgaggagaa gtggctgctc aagcgggcct gcgccgacta
tgtcccggaa 1620 gccgtgctca agcgccgcaa gagcccttat ccgacttctg
ccaacctcgg ctacgagcgt 1680 ttcctgcgcg ggagcgtgcg gcgcctgctg
gaggacgcgg tgaacccggt gttcggcatc 1740 gtttcgcgag agttcctggc
cgccgaactg gagcatccgg aggggtactt caacacccag 1800 gtgagccgcc
acaacctgga gaccgcactg gcgctggaag gctggctcag gttgtacggg 1860
ctctccgcct ga 1872 10 756 DNA Pseudomonas aeruginosa 10 atgcagaaac
agcgggtggc cgaccaggtc gcagagcgta tcgagcggtt gatcgtcgac 60
ggcgtgctca aggtcggcca ggcactgccg tccgagcggc gcctggtggc caagctcggc
120 tgctcgcgct cggccctgcg cgagggcctg cgggcgctgc gcgggcgcgg
catcatcgac 180 accgagcatg gccgtgggtc gttcgtcgcc gacctcgacc
gcaacgccga cgtcagcccg 240 ctgatgcacc tgttcggctc ccagccgcgc
accctctacg acctgctcga agtccgcgcc 300 ctgctggagg gcgaggcggc
ccgcctggca gcgctacgcg gcaccgaggc agacttcgtc 360 ctgctcgccc
ggcgctacga agagatgctc gccagccacg aggaaaccca gccgatcgat 420
ccccgcgagc acgcccgccg cgaccacgcg ttccaccggg cgatcagcga ggcatcgcac
480 aatccggtgc tggtgcatac cctgcaatcg ctcaacgaac tgctgctgag
cacggtgttc 540 gcctcagtga acaacctcta ccaccgaccg ccgcagaaac
gccagatcga ccgccagcac 600 gcgcgcctct acgcggccct ccgcgagcgc
cagccggacc aggcgcaacg ggcggcgcgc 660 gaacatatcc acagcatccg
cgacaacctg cgggagatcg agcaggaaga acagcgcctg 720 gtccgcgcca
ccctgcgcct gaacggctgg ggctga 756 11 822 DNA Pseudomonas aeruginosa
11 atgaaccatg tcatcacccc ccacagcaag ctgctcggcg tcatcgagcc
ggtcctcaac 60 gacatgcccg ccggaaccct gcgccacgca ctgttccggg
ccttctggga cgagacggcg 120 tcgttgctgg acatcgagga cgccttcgcc
cgggtcaccg cccggcgcca ggcggtcgag 180 ccgctgcgca agttcttcgc
cagttggtcg aagaccaaca actcggcggc cagcgtttcc 240 ggactggcca
atcgccttac cctgctggcc cgttcggaac agggttcggc agcggcagac 300
cagctctatc gagcctgcgg cagcctgcaa cggatcaccg acgaagacct cggcgccctc
360 ggcaacaccg tgcatgccga tcttttctac accatggcca ccaccctttg
cggcgacgac 420 cgctggctgc tgcgcgagaa ctgcctgcct tcggcgcagg
cgttcaagga ctggaccgac 480 cgccagcgcc tgtgcgagcg cgacctgatg
cagggactgc tgaccacgct ggtacacgag 540 gtctataccc acggcgaggt
ggagtacatc cacccgctgt acaaggaatg gttcagccgc 600 gacatgggcg
tacccgccga acgcgcccgc gccaccgtgg cctgggtaac ggtgcacacc 660
ggcggcaccg agagcaatca cttcgcccac gccacggcgg cggtgaacgc cttcgtcgag
720 gcgatggaaa tcgaggtgaa cgaagaagcc gcgcgcaacc tgttcgggct
ctacctgcgc 780 aacaaggcgc aggtcatgcg tgactgcgcg gcgctgttct ga 822
12 1368 DNA Pseudomonas aeruginosa 12 atgtcctccc gccaatcgtc
ccgcaacgct tccaccccgt atctgaccaa ggccttccag 60 gcaacggcca
tcgtcgtggt gagctacttc ctctactgga cctaccagct ctaccagtac 120
ggcgagattc ccatcagcaa gaaggacgtg atgctgcgcc aagccatcct cgcgcgcttt
180 ccggcggact acgaggtgga gatcaagggc gccgacctgc tcggcttcgg
cgagaaattc 240 ctggtcgcct acggcaatcg gcgcttcgtc ggcaaggcct
tcgccatgga cgaccaggtc 300 atcgagcgcc tggagcggaa ccagggacgg
accaacctgc cgctggtgaa ggtgttctac 360 atcgccgaac ccggcctcct
ctcctcgctg ctcaacctct ccccgttcct ggatatccag 420 aagaacatgg
tcgagctgag cctccgggaa taccggaaga tccagttggt ccccttcgat 480
ccggacgcga agcggaaacc gcgcgagcag ttcgaaaccg attatgcctt cccccagctg
540 ttcagcctca gccaactgga agtcgccgac tacgacggcg acggccgcga
cgaactgcgc 600 ctgggctacc tgtcctacgc cggcggttcg ggagggacgc
gctggtcggt gatctacgac 660 ctgaaggacg gcgcgctgac cgcccattcc
ggctatccgg aaatgctcga catcgacgtc 720 gcccggttca tccaggcggt
caacctgtac gccggcctcg acggcacctt gccgcgcgac 780 cagcgtcagc
tggaagacgt ggtcggccga ggcagcgagc gcttcgccct gaccgccgcg 840
gagcgccagg cactggtcgc cgacccgccg cagcgggacg actacgccag ggtcctgatg
900 agcctttcgc cgcgctcgcc ctacgccccc gatcgcttca tcgacctcgg
cgacggcagc 960 cgactgaccc tggccccgcg ccataccgac gattactcga
ccttcctcga catcggcggg 1020 aagaaaatct tcgtcgaagc cttctacgtc
gacgacgacg cctgccactg gtgcgagcat 1080 cgctggcgag tgatggcttt
ccattacgac gacggtcgct ggatctcgga ccgcaccatc 1140 aacggcgaca
gcttcaacgg gcaatggctg cgcaacgcgg agccgctggg cctcaacgac 1200
gttttcggta cctaccgcga ccagggcccg acgggcctgg cctggtcctt catcgacccg
1260 cgctggaccg cctccagcca gcatgacatg gacgatccgc tgggcgtggg
aatgcgcacc 1320 ctgtcgccgg tggagcaatg ggtgaaggaa cgctatcggg
aaaactga 1368 13 1209 DNA Pseudomonas aeruginosa 13 atgagcgaac
ccatcgatat cctcatcgcc ggcgccggca tcggcggcct cagttgcgcc 60
ctggccctgc accaggccgg catcggcaag gtcacgctgc tggaaagcag cagcgagata
120 cgcccccttg gcgtcggcat caatatccag ccggcggcgg tcgaggccct
tgccgaactg 180 ggcctcggcc cggcgctggc ggccaccgcc atccccaccc
acgagctgcg ctacatcgac 240 cagagcggcg ccacggtatg gtccgagccg
cgcggggtgg aagccggcaa cgcctatccg 300 cagtactcga tccatcgcgg
cgaactgcag atgatcctgc tcgccgcggt gcgcgagcgc 360 ctcggccaac
aggcggtacg caccggtctc ggcgtggagc gtatcgagga gcgcgacggc 420
cgcgtgctga tcggcgcccg cgacggacac ggcaagcccc aggcgctcgg tgccgatgtg
480 ctggtcggcg ccgacggtat ccattcggcg gtccgcgcgc acctgcatcc
cgaccagagg 540 ccgctgtccc acggtgggat caccatgtgg cgcggcgtca
ccgagttcga ccgcttcctc 600 gacggcaaga ccatgatcgt cgccaacgac
gagcactggt cgcgcctggt cgcctatccg 660 atctcggcgc gtcacgcggc
cgaaggcaag tcgctggtga actgggtgtg catggtgccg 720 agcgccgccg
tcggccagct cgacaacgag gccgactgga accgcgacgg gcgcctggag 780
gacgtgctgc cgttcttcgc cgactgggac ctgggctggt tcgacatccg cgacctgctg
840 acccgcaacc agttgatcct gcagtacccg atggtagacc gcgatccgct
gccgcactgg 900 ggccggggac gcatcaccct gctcggcgac gccgcccacc
tgatgtatcc gatgggcgcc 960 aacggcgctt cgcaagcaat cctcgacggc
atcgagctgg ccgccgcgct ggcgcgcaac 1020 gccgacgtgg ccgcagccct
gcgcgaatac gaagaagcgc ggcggccgac cgccaacaag 1080 atcatcctgg
ccaaccgaga acgggaaaaa gaggaatggg ccgcggcttc gcgaccgaag 1140
accgagaaga gcgcggcgct ggaagcgatc accggcagct accgcaacca ggtggaacgg
1200 ccacgctag 1209 14 3090 DNA Pseudomonas aeruginosa 14
atgaccttta ccgacctgtt cgtccgccgg ccggtgctgg cgctggtggt cagcacgctg
60 atcctgctgc tcggcctgtt ctccctgggc aagctgccga tccgccagta
cccgctgctg 120 gaaagctcga ccatcaccgt caccaccgag taccccggcg
cctccgccga tctcatgcaa 180 ggcttcgtca cccagccgat cgcccaggcg
gtgtcgtcgg tggagggcat cgactacctt 240 tcctcgacct cggtgcaggg
gcgtagcgtg gtgaccatcc gcatgctgct caaccgcgat 300 tcgacccagg
cgatgaccga gaccatggcc aaggtcaact cggtgcgcta caagctgccc 360
gagcgtgcct acgactcggt gatcgaacgc tcttccggcg agaccaccgc ggtagcctac
420 gtcggctttt ccagcaagac cctgccgatc ccggcgttga ccgactacct
gtcgcgggtg 480 gtcgagccga tgttctcttc catcgacggc gtggccaagg
tccagacctt tggcggccag 540 cgcctggcca tgcgcctctg gctcgacgcc
gaccgcctcg ccgggcgcgg cctgaccgcc 600 tccgacgtgg ccgaggcgat
ccgccgcaac aactaccagg cggcgccggg gatggtgaag 660 gggcagtacg
tgctgtccaa cgtgcgggtc aacaccgacc tgaccaacgt cgacgacttc 720
cgcgagatgg tcatccgcaa cgatggcaac ggcctggtgc gcctgcgcga cgtcggtacc
780 gtcgaactgg gcgccgcggc caccgagacc agcgcactga tggacggcga
cccggcggtg 840 cacctggggt tgttcccgac gcccaccggc aacccgctgg
tgatcgtcga cggcatccgc 900 aagctgctgc cggagatcca gaagaccctg
ccgccggatg tccgcgtcga cctcgcctac 960 gagacttcgc gcttcatcca
ggcctccatc gacgaggtgg tgcggaccct ggtggaagcg 1020 ctgctgatcg
tggtgctggt gatctacctc tgcctcggct cgctgcgcag cgtgctgatc 1080
ccggtggcga ccattcccct gtcgatgctc ggcgccgccg cgctgatgct ggccttcggc
1140 ttcagcgtca
acctgctgac cctgctggcg atggtgctgg ccatcgggct ggtggtggac 1200
gacgccatcg tggtggtgga gaacgtccac cgccacatcg aggaaggcaa gtcgccggtg
1260 gcggcggcgc tgatcggcgc ccgcgaagtg gccggcccgg tgatcgccat
gaccatcacc 1320 ctggccgccg tgtacacccc catcggcctg atgggcggcc
tcaccggcgc gctgttccgc 1380 gagttcgccc tgaccctggc gggcgcggtg
atcgtgtccg gggtggtggc gctgaccctg 1440 tcgccggtga tgagttcgct
gctgctccag gcgcaccaga acgaggggcg catgggccgc 1500 gccgccgagt
ggttcttcgg cggcctgacg cggcgctacg ggcaggtcct ggagttctcc 1560
ctgggccacc gctggctgac cggcggcctg gcattgctgg tgtgcatcag cctgccgctg
1620 ctgtattcga tgcccaagcg cgaactggcg ccgaccgagg accaggccgc
ggtgctcacc 1680 gcgatcaagg cgccgcagca cgccaacctc gactatgtcg
aactgttcgc gcgcaagctc 1740 gaccaggtct acaccagcat cccggaaacc
gtgagcacct ggatcatcaa cggcaccgac 1800 ggaccggcgg cgagcttcgg
cgggatcaac ctggcggcct gggaaaaacg cgagcgcgac 1860 gcctcggcga
tccagtccga gctgcaaggc aaggtcggcg atgtcgaggg cagcagcatc 1920
ttcgccttcc agttggccgc cctgcccggc tccaccggcg gcctgccggt gcagatggtg
1980 ctgcgcagcc cgcaggacta tccagtgctc taccggacca tggaagagat
caagcagaag 2040 gcccgacaga gcgggctgtt cgtggtggtc gacagcgacc
tcgactacaa caacccggtg 2100 gtccaggtcc gcatcgaccg cgccaaggcc
aacagcctgg gcatccgcat gcaggacatc 2160 ggcgagtcgc tggcggtgct
ggtgggcgag aactacgtca accgcttcgg catggagggc 2220 cgctcctacg
acgtgatccc acagagcctg cgcgaccagc gtttcactcc gcaagcgctg 2280
gcacgacagt tcgtgcgcac ccaggacggc aacctggtgc cgctgtcgac ggtggtccgg
2340 gtggcgcttc aggtcgaacc gaacaagctg atccagttcg accagcagaa
cgccgcgacc 2400 ctccaggcga tccccgcgcc cggcgtctcc atgggccagg
cggtggcctt cctcgacgac 2460 gtggcgcgcg gcctgccggc cggcttcagc
cacgactggc aatccgactc gcggcaatac 2520 acccaggaag gcaacaccct
ggtgttcgcc ttcctcgccg ccctggtggt gatctacctg 2580 gtgctcgccg
cgcagtacga gagcctggcc gacccgctga tcatcctgat caccgtgccg 2640
ctgtcgatct gcggcgcgct gctgccgctg gcgctgggct acgcgacgat gaacatctat
2700 acgcagatcg gcctggtcac cctgatcggc ctgatcagca agcacggcat
cctcatggtc 2760 gagttcgcca acgaactgca actccacgag cgcctcgacc
gccgcgcggc gatcctgcgc 2820 gccgcgcaga tccgcctgcg gccggtgctg
atgaccaccg cggcaatggt cttcggcctg 2880 gtgccgctgc tcttcgccag
cggcgccggc gccgccagcc gcttcggcct gggcgtggtg 2940 atcgtctccg
ggatgctggt cggcaccctc ttcaccctgt tcgtgctgcc caccgtctat 3000
accctgctgg cgcgcaacca cgcggaagtc gacaagagcc cgcgcagccg gcaactggcc
3060 gaggccgatc tgctggtgaa caaggcatga 3090 15 2535 DNA Pseudomonas
aeruginosa 15 gtggccgttg cgtcaccggc cggcgggttg gacgcaccgt
cgcggcggat cgtcttcgac 60 gcgcagatgc tggccctggg gccgggcgga
cgctcgatcg atacgtcgcg tttcgagcgc 120 ggcgacgtca tcgagccagg
ccgctatcgc ctcgacctgc tgctcaacag ccgatggcgt 180 ggcgtcgagg
aagtcgagct gcgccgccag ccggggcggg aaagcgcggt cttctgctac 240
gaccggggcc tgctggagcg ggcgggcatc gacctggaga agagcgcgcg tggccaggac
300 cgttcctcgg ctcgcgatcc tctgcccgaa ggtttgcact gcgaccctct
cgagcgctat 360 gtgccggggg cccgggtcaa gctcgatatc gccgagcagt
cgatctatgt ctcggtgccc 420 agctattacc tgagcctgga ttcttcgaag
acctatgtcg atccggcgag ctgggacagc 480 ggcatttccg ccgccttgct
caactacaac agcaatctcc acgtcaggga aaaccacggc 540 aggagcgcca
ccagcggcta tgccgggatg aacgccggct tcaatttcgg gcgggcgcgc 600
ctgcgccaca acggcacggc cacctggtcg cgccgcatgg gcagccatta ccagcgtagc
660 gcaacctatg tgcagaccga cctgccggcc tggcgtgcgc agttattgct
gggagaaaac 720 tccaccagca gcgagttctt cgatgcggtg tccttccgtg
gagtgcagct atccagcgat 780 gaccggatgc tgccggattc gctgcgctac
tacgctccgg tggtccgtgg gaccgccagt 840 accaatgcgc gggtatcggt
ctaccagcgc ggctacctca tctacgaaac cacggtggca 900 cccggggcgt
tcgctctcga cgaactgcag accgccagct atggcgggga cctggaagtg 960
cgggtgaccg aagccagcgg ggaagtccgc agtttcatcg tgccgttcgc caccaccgta
1020 caactgctgc gccccgggac cacgcgctac agcctgacgg ccgggcggct
caacgatccc 1080 agcctggagc gtcggccgaa catgctgcag ggcgtctacc
agcgcggcct gggcaacgac 1140 gtcaccgcat acgcgggcgg ggccttcacc
ggcagctaca tgtccgggtt gatgggcgcg 1200 gcgctgaaca cgccggtggg
cggattctcc ggtgacgtga cgctggcgcg taccgaggtt 1260 cccggcgacg
accgccttag cggctccagc taccgtctcg cctacagcaa gaacctgccg 1320
aacaccggca ccaacttttc gctgctcgcc tatcgttact ccaccggtgg ctatctcggc
1380 ctgcgcgacg cggccttcat gcaggaccgg gtagagcgag gcgagccgct
ggagtcgttc 1440 tcgcgcttgc gcaatcgtct cgacgccaac atcagccagc
aactgggcaa cggcggcaac 1500 ctttacctga acggctcctc gcagcgctac
tggagcggcg gcgggcgggc ggtcaacttc 1560 tccgtcggct acagcaacca
gtggcgcgac gtcagttact ccatttccgc gcaacgcctg 1620 cgcagccagt
acgaaggctt ttccagcggt gacaggcgcg gcgagaccag cacgctgttc 1680
agcctgaacc tgtccattcc gctcggcggc gctggacgcg ggtcgccgac cctgagcagc
1740 tacctgaccc gcgacagcaa cagcggaacc cagctcacca gcggggtttc
cggcatgctg 1800 ggcaagcgtg gcgaggcctc ctactcgctg tcggcctccc
atgaccgcga cagccggcag 1860 acctcgaaga gcgccagcct cgactatcga
ctgccgcagg tcgaactcgg ctccagcctc 1920 tcgcagggac cgggctatcg
gcagttgtcg gtcaaggccg cggggggcct ggtcgcgcac 1980 agcggcggga
tcaccgcggc acaaaccctg ggcgagacga tcggcctggt ccacgcgcca 2040
aatgccaggg gcgcggctgc cggctactcg ggaagccgga tcgaccgcca cggctatgcg
2100 gtgattccca acctgctgcc ctaccagttg aacagcgtcg acctcgaccc
caacggcatg 2160 gccgacgaga tcgaactgag gtccagttcg cgcaacgtgg
cgcccaccgc cggagcggtg 2220 gtgcgcctcg actatccgac gcgggtggca
aggcccttgc tggtggatag ccggatgccc 2280 agcggcgagc ccctgccgtt
cgccgcggaa gtgctcgatg cccacagcgg gcagtcggtg 2340 ggcgccgtcg
gccagggcag ccgcctggtg ctgcgggtcg agcaggatcg cggctcggtt 2400
cgggtgcgct ggggcaacga gccgcagcag cagtgcctgg tcgactatgc gttgggcccg
2460 cgcgagacga cgcctcccgt cctgcaactg gcatgtcgcc cggcgtcggc
cgccgaccgg 2520 gagcgcacgc tgtga 2535 16 2976 DNA Pseudomonas
aeruginosa 16 atgtcgaaag attctgttct tggcttattc aggcaacacg
cagataccca tcccgaacgc 60 cccgccctcg tcgatcgcga gcgctcgttc
agctaccgcg aactcgaccg gctcagcgac 120 cggctggccg cccacctggc
caggcgcggc gtcgcccggg gcgagctgct gcccctgctg 180 gccgaacgct
cggccgaact ggtcatcgcc atcctggcgg ccgccaagtg cgcagcggcc 240
tacgtaccgg tggaccgtcg gcaacccgac aggcgcaagc gggaagtcct ccgccagtgc
300 caagccccct tggccctcgc cacccatgcc gaggacctgc cggggcaacc
ggtggaggtc 360 atcgcacagg cgctcgcgac gagtgcggcg ggtgccgcgc
cgagaccggc gctcgacggc 420 agcgaagcgc tgtatgtgat cttcacctcg
ggcaccaccg gcgaacccaa gggcgtggtg 480 atcgagagtc gctccctggc
caacctcgtg ggctggcaca accggcgctt caatatggat 540 caacggagcc
gcaccaccct gatggccggc gtcggcttcg acgtttccca atgggaaatc 600
tggtccaccc tgtgcgcagg cgcctgcctc cacctggtgc ccgacgaggt gcgcccagac
660 ccggcggcgc tgctggcatt cttcgccgag cagcggatca gccacgcctt
cgcgcctacc 720 gtgatggtgc ccgcgctggc ggagcagccc gccccgccgt
cgctggcgct gcgctacctg 780 ttctgcgccg gggaaaaact gccgccggtc
gcaaccggcg ggctgcccta taccgtggtg 840 gattactacg gcccgaccga
ggccacggtc ttcgccacct gccgcatcgt cgacgccgaa 900 gcacatcggc
gacccgcctc gatcggcacg cccatcgacg gctgcgaggc attcatcctc 960
gacgccgacg accggccttg ccatggcgac cgacccggtg aactgaacct ggcgggcgtc
1020 tgcctggcgc gcgaatacct gcgcgacccg gacatgaccg ccaggcgctt
ccactactcg 1080 caggcactgc ggcgtcggct ctaccgcacc ggcgacaagg
cccgctggtt ggccgatggc 1140 agcctgcagt tcctcggtcg gctggacgac
caggtgaaga tccgcggcca ccgcgtcgaa 1200 ctcggcgacg tcgaggccgc
gctgttgcgc cagccggcta tccacggcgc ggtggtgctg 1260 gcgcatgccg
acccacgctc cggtagccag caattgagcg ccttcgtggt cccccgccag 1320
caggacggcg atgccagggc cgtgctcgcc gccatcaaga ccgcactgcg ccaggaactg
1380 cccgactaca tgctgcccag ccgctacctg tcgctggaca gcctgccgac
cacggtcaac 1440 ggcaagatcg accgccaggc cctgcgtcga cacctggacg
aacaatgcca ggaacgactc 1500 gacgagcaac gcttcggcac ccccggcgaa
ctgcaagtgg ccctgtcctg gcaggaagtg 1560 ctggggcata ccgacttcgg
cctggacgac agcttcttcg aggtcggcgg ccattccctg 1620 ctggccgccg
ccctggtgcg cgaattgagc cgacgcttcg gcaaccgtgc ctacatccac 1680
gacatctacc gcaccccgag cgtgcgccaa ctggcggcca gcctggcgcg gcgcgccggc
1740 gaagcgccgc cggcgctgga cagcgaaccg gcccaggagc tgcaacggga
cgtgcgcctg 1800 cccgccgacg tggatttcag ccgccccacg gacaccgccc
aattgctggc gccacggcac 1860 atcctgctca ccggcgccag cgggctgatg
ggcgcccacc tgctcgccga gctgctggcc 1920 agccgcgagg ccgacctgca
ttgtccggtc cgtgcgcaaa acgacgccca tgccctcgaa 1980 cgcctgcgcc
aggccgcccg gcagcaccgc atcgaactcg ccgagacgga ctggcgacgg 2040
gtcagggcct acgccgccga cctcgcagaa ccaggtttcg gactaccggc ggaaacctat
2100 cgcgagctgg ccggcagcgt cgaccaggtc ttccattccg ccagcgcggt
gaacttcatc 2160 cagccataca gctacatgaa gcgcgacaac gtcgaggggc
tcggccaggt cctgcgcttc 2220 tgcgccagcg gccgctgcaa gccgctgatg
ctgctgtcga gcatctcggt gtacagctgg 2280 ggccacctgc ataccggcaa
gcgcctgatg cgcgaggacg acgacatcga ccagaacctg 2340 ccggcggtgg
tcaccgacat gggctacgtg cgcagcaaat gggtgatgga aaagatcgcc 2400
gacctcgccg ccgaacgcgg cctgccgctg atgaccttcc gcctcggcta cgccacctgc
2460 cacagccgta ccggcgccta cgccgactac cagtggtgga gccggctggc
gcggacctgc 2520 ctggagtacc gggccgtgcc gctcctgcgc gagctgcgcg
agggcctgac cacggtggac 2580 tacatggtag aggcgatcag cgtcatcgcc
cgccagcctt cggcgctggg caagaaattc 2640 aacctggtgc cgagcattcc
gcgctgcctg accctggacg agttcttcgg ccgtctcggg 2700 cgacgcgccg
ggcgtcccct tcggcagatg ccgttcgacg actgggtaag tctctgggaa 2760
gacaatcgcg acgccccgct ctatcccctg ctgagcatgt tccgcgacaa catgtacgcc
2820 ggccgcagca ccgtcgagtt gtaccaggac acctatctct gggactgcac
caacgtcgag 2880 gaacacctgc gcgggagcgc cgtgcgcgag ccggagttcg
acgaccgcct gctcgacctg 2940 tacctcgccg gcctgggcgg cagcgccatg cggtaa
2976 17 1092 DNA Pseudomonas aeruginosa 17 gtgggacggc ttcgacgagc
cgcgctgcac cctgctggag gcgaaggcca actacgcctt 60 cctgttcgtc
ccgctgctcg gcgtgcccag gccctgggca cgggccaagg tgaagtcgga 120
cctgctgcag aaggccgagg tccacagcga caaggcccga ccgaccccgc cggtgttcgt
180 cgaatggcac ttcctgcagc ggatcgtcta cgagtactgc gccgcggagt
acctgcgcat 240 gggactggcc aacctgaagg cattctggaa tccgatgccg
ggaacggacg agcacgacga 300 ctaccaggaa acccgcgcga aggaacagga
agagatgaaa aggttttgcg aagagaaccc 360 ggggtattgc gcatgacgga
cgccaaggct ttcaggcgct acatattcga gctgtacttc 420 gatccggcac
ggctcctcga actggacgac gaccagcacc tgcaacggat agaacgcttc 480
ctcgatgccc tcgcgcccct ccatccggtg ctggagaact ggtatctgtg cggcgactcc
540 ctgcgcgatg ccctcagcca caacgtcacc gagcaccgcc aggatctcgc
caaggccctg 600 tcgcgtgacc gacgcacccg ggcggtggaa ctggtgctat
ggaacggcga ggaggatccg 660 ctcaagggcg ggttgtcgct ggactacgag
gccagcggca gggccgtctc gtccaggctc 720 cagttggaag atgccggcag
cctgctgcag gtgttcgacg caccggcgtc ctccttcgtc 780 gcgatcttcc
tcgcggtgct ggaaatctgg cccgaaacga cctggggcat gctcgctccg 840
catgcgtact tcgtacacca gcggaccttc ccggaccgcc gcagcatcgg ctggatcggc
900 ttctgcccgc atccgctaag ggccacggac ttcccggcgg ctacggagct
ggtcgacatt 960 cccggccgtg gcaccctgct gctgaacggc cgcgaaccga
tggacgaaac ccgtcgcgaa 1020 catttcgagc gcgtcggcga agcggacatc
aagctgatgg aactgggcta cctgccgccg 1080 ctgcgcggct ga 1092 18 1281
DNA Pseudomonas aeruginosa 18 atgcacgcca tcctcatcgc catcggctcg
gccggcgacg tatttccctt catcggcctg 60 gcccggaccc tgaaactgcg
cgggcaccgc gtgagcctct gcaccatccc ggtgtttcgc 120 gacgcggtgg
agcagcacgg catcgcgttc gtcccgctga gcgacgaact gacctaccgc 180
cggaccatgg gcgatccgcg cctgtgggac cccaagacgt ccttcggcgt gctctggcaa
240 gccatcgccg ggatgatcga gccggtctac gagtacgtct cggcgcagcg
ccatgacgac 300 atcgtggtgg tcggctcgct atgggcgctg ggcgcacgca
tcgctcacga gaagtacggg 360 attccctacc tgtccgcgca ggtctcgcca
tcgaccctgt tgtcggcgca cctgccgccg 420 gtacacccca agttcaacgt
gcccgagcag atgccgctgg cgatgcgcaa gctgctctgg 480 cgctgcatcg
agcgcttcaa gctggatcgc acctgcgcgc cggagatcaa cgcggtgcgc 540
cgcaaggtcg gcctggaaac gccggtgaag cgcatcttca cccaatggat gcattcgccg
600 cagggcgtgg tctgcctgtt cccggcctgg ttcgcgccgc cccagcagga
ttggccgcaa 660 cccctgcaca tgaccggctt cccgctgttc gacggcagta
tcccggggac cccgctcgac 720 gacgaactgc aacgctttct cgatcagggc
agccggccgc tggtgttcac ccagggctcg 780 accgaacacc tgcagggcga
cttctacgcc atggccctgc gcgcgctgga acgcctcggc 840 gcgcgtggga
tcttcctcac cggcgccggc caggaaccgc tgcgcggctt gccgaaccac 900
gtgctgcagc gcgcctacgc gccactggga gccttgctgc catcgtgcgc cgggctggtc
960 catccgggcg gtatcggcgc catgagccta gccttggcgg cgggggtgcc
gcaggtgctg 1020 ctgccctgtg cccacgacca gttcgacaat gccgaacggc
tggtccggct cggctgcggg 1080 atgcgcctgg gcgtgccgtt gcgcgagcag
gagttgcgcg gggcgctgtg gcgcttgctc 1140 gaggacccgg ccatggcggc
ggcctgtcgg cgtttcatgg aattgtcaca accgcacagt 1200 atcgcttgcg
gtaaagcggc ccaggtggtc gaacgttgtc atagggaggg ggatgctcga 1260
tggctgaagg ctgcgtcctg a 1281 19 651 DNA Pseudomonas aeruginosa 19
atgccgcctt ttttttctcg gccggcacga cacggggact tggtcatgat cgaattgctc
60 tctgaatcgc tggaagggct ttccgccgcc atgatcgccg agctgggacg
ctaccggcat 120 caggtcttca tcgagaagct gggctgggac gtggtctcca
cctccagggt ccgcgaccag 180 gaattcgacc agttcgacca tccgcaaacc
cgctacatcg tcgccatgag ccgccagggc 240 atctgcggtt gcgcccgcct
gctgccgacg accgacgcct acctgctcaa ggacgtcttc 300 gcctacctgt
gcagcgaaac cccgccgagc gatccgtcgg tctgggagct ttcgcgctac 360
gccgccagcg cggcggacga tccgcagctg gcgatgaaga tattctggtc cagcctgcaa
420 tgcgcctggt acctgggcgc cagttcggtg gtggcggtga ccaccacggc
catggagcgc 480 tatttcgttc gcaacggcgt gatcctccag cgcctcggcc
cgccgcagaa ggtcaagggc 540 gagacgctgg tcgcgatcag cttcccggcc
taccaggagc gcggcctgga gatgctgctg 600 cgctaccacc cggaatggct
gcagggcgta ccgctgtcga tggcggtgtg a 651 20 1167 DNA Pseudomonas
aeruginosa 20 atgcctttga ttgtctatgt gctcggtgcc gcgatcttcg
ccctgaccac cagcgaatac 60 atggtcgccg ggctgatgcc ggcgctggcc
gccgaattcg gcgtgtcctt cgccgcgatc 120 ggctacctgg tcaccttcta
cgccggtgcg atggccgtcg gcggcccgct gttgaccacc 180 gccctgctcc
gggtgccgcg caagaacgcc ctgctcggcc tgatcgcgct gttcgtggtc 240
ggccaggtca tcggcgccct ggcgccgggc tatgcggtga tggtcgcggc gcgactggtc
300 accgcggtcg ccgccgcggc cttcttcggc gtggcgctga ccgcctgcgc
cgaactggtc 360 gaaggcaacc agttcggccg cgcgtcgtcg ctggtgctcg
gtggcctgat ggtcggcacc 420 gtgctcggcc tgcccgtcgc cacctggctg
ggcgaatggt acggctggcg cgcgagcttc 480 ttcgcggtgg cgctggtggc
ggtgctggtc ggcctgctgg tgttgcagct gatgccggcg 540 atcccggggt
cggcgggcag cggctcgctg cgcgaggaac tgaaggtgtt caggaacgcc 600
catctatggt gggtctacgc caccagcctg ctgctgatcg gcgccacctt cgccggcttc
660 acctatttcg tgccgatcct caccgaggtc agcggcttct ccgcctcgac
cgtaccgctg 720 ctgctggtgg tctacggcct ggcgacgctg gtgggcaaca
acatcgtcgg ccgcctggcc 780 gaccgccata ccatcgcggt cctggccttc
ggcctgctgg cggccatcgc cgcgatggtg 840 gccttcgccc tgttcggaca
ggttccggcg gtggcggtgg cggcgctggt ggtgatcggc 900 ctgaccgggg
tgtcgatgaa cccggcgctg gtgacccgcg gcgcacgggt cggccataac 960
aacatgctgg tcaactcggt gcacactgcc tgcatcatgc tcggcgtaat ggccggttcc
1020 tggatcggcg gcctgggcat cgccggcgga ttcggcctgc agggcgcgct
ctgggtcggc 1080 gcggccctcg gagtactggc gctgctgacc ctgctgccgg
agctgcgctt cgcccgcgcc 1140 ccggtaggcg gggcgctggg ccgctga 1167 21
993 DNA Pseudomonas aeruginosa 21 atgccgcgcg ccgccgtggt ctgcggcctg
ggcagctacc tgcccgaggc cgtgctcagc 60 aacgacatgc tcgccgccga
gctggacact tccgacgcct ggatcagcag ccgcaccggc 120 gtgcgccagc
ggcatatcgc cggcgacctc ggcagcggcg acctggccct gcgggcggcc 180
tccgccgcgc tcgcctcggc ggggctggag cgagtcgatg cggtggtgct ggcaaccagc
240 accggcgact tctgctgccc ggccaccgcg cccagggtcg cggcgcgcct
ggggttggtc 300 ggcgcgctcg cgttcgacct gtccgccgcc tgcaccggct
tcgtctacgg cctggccagc 360 gtcggctcgc tgatcagcgc cgggctggcg
gacagcgcgc tgctggtcgg ggtggacact 420 ttcagccata ccctcgaccc
cgccgatcgc tcgacccgcg cactgttcgg cgacggcgcc 480 ggagcggtgg
tgctgcgtgc cggcgatgcc gaggaagaag gcgcgctgct ggccttcgac 540
ctcggcagcg acggccacca gttcgacctg ctgatgaccc ccgccgtcag tcgcgccgaa
600 cgcagttccg gacaggcctc caactacttc cggatggacg gcaaggcagt
gttcggccag 660 gcggtgacgc agatgagcga ctcggtgcgg cgggtgctcg
accgggtcgg ctggcaagct 720 tcggacctcc atcacctggt cccgcaccag
gccaacacac gcattctcgc ggcggtcgcc 780 gaccagctcg accttcccgt
cgagcgagtg gtgagcaaca tcgccgaggt gggcaatacc 840 gtcgccgcct
cgattcccct ggccctggcc cacggcctgc gccaaggcat cctgcgcgac 900
ggcggcaaca tggtcctcac cggtttcggt gccggactga cctggggttc ggtcgccctg
960 cgctggccga agatcgttcc gacaatggac tga 993 22 1257 DNA
Pseudomonas aeruginosa 22 gtgcctgatc gcaaactgag actgggcgag
gaactgatct cgccactgca cgcgctctac 60 gacggcctgc aggtggacgg
cgcgccgcgt cccgcgcatc gcgccgccga gcatccggtg 120 tgggtggtga
cgcgctaccg cgacgcgcgc aaggtcctca accatccggg cgtccgccgc 180
gacgcccggc aggccgccga actctacgcg aagcgtaccg gcagcccgcg cgcggggatc
240 ggcgagggac tcagccacca catgctcaac ctcgacccgc cggaccatac
ccgcctgcgc 300 tcgctggttg gccgcgcgtt caccccgcgc caggtggagc
gcctgcaacc gcatatagaa 360 cggatcaccg aggcattgct ggacgccatg
gccggccgcg aacaggccga cctgatggcc 420 gacttcgcga tcccgctgac
catcgcggtg atcttcgagc tgctgggcat tcccgaggcc 480 gagcgcgaac
acgcccgcca gtcctgggag cgccaggcgg aactgctgtc gccggaggag 540
gcccaggccc tggccgatgc gcaggtcgac tacctgcgcg tgctgctcga ggccaagcgc
600 cggcagccgg ccgacgacgt ctacagcggg ctggtgcagg ccgccgacga
gagcggccag 660 ttgagcgaag cggaactcgt ctccatggcc cacctgctga
tgatgagcgg cttcgagacc 720 accatgaaca tgatcggcaa cgcgctggtc
accctgctgg tcaacccgga gcaactggcg 780 ttgctgcggg cgcagccgga
actcctgccc aacgccatgg aagaactggt ccgccacgac 840 agtccggtgc
gcgcctcgat gttgcgcttc accgtggaag acgtggaact ggacggggtc 900
accattcccg ccggcgaata catcctggtc tccaacctga ccgccaacca cgatgccgag
960 cgcttcgacg atcccgaccg cctcgacctc acccgcaaca ccgatggcca
tctcggctac 1020 ggcttcggcg tgcactactg cgtcggcgcc tcgctggccc
ggctggaggg gcggatcgcc 1080 atccagcgcc tgctcgcgcg cttccccgac
ctccagttgg cggtgcccca cgcggagctg 1140 cagtggctgc cgatcacctt
cctccgcgcc ctgatcagcg tgccggtgcg caccggatgc 1200 agcgccccgg
cgaacaccgc ctcccacgcc aacccgatcg agaggatcgc ccaatga 1257 23 915 DNA
Pseudomonas aeruginosa 23 atgttattca ccagcaaacc tctctcgccc
cagggccgcc acgtactgat caccggcgcc 60 tccagcggcc tcggccggga
aaccgcgctg cacctggccg aacagggttt ccaggtgatc 120 gccggggtgc
gccgccagga ggatggcgag cgcctggcga acgcctgccc gtccggccgg 180
atcagcacgc tgctgatcga tgtcaccgac gaggaatcca ttggccgggc cgccgcgcag
240 gtggcggaga aagtcggcga taccgggctc tggggcctgg tgaacaacgc
cgggatctgc 300 atttccgcgc cgctggaatg cgtctccagc gacctgctgc
ggcgccagct ggaagtcaac 360 ctgatcggcc agctcgcggt gacccgggcg
atcctgccgc tgctgcgccg tggcggcgcg 420 gcgcgcctgg tgaacgtcac
ctcgggcctc ggctcggtcg ccattcccta cctgggcgcc 480 tactccgccg
cgcagttcgc caaggaggga gtgagcgacg ccctgcgccg cgagctggca 540
cccatgggca tccaggtctc ggtggtcagc cccggggcga tctggacgcc
gatctggggc
600 aagatcgcca gcgagggcga gcgcgccctg gccgacgccc ccgacgccgt
cgccgacctc 660 tatcgcgata cctacctgcg cttcctccag gccaacgagg
acggcgcgcg caacagcgcg 720 accaagcccg ccgatgtcgc cgccgcggtg
catgccgcgc tcaccgcggc caagccgcgg 780 acccgctacc gggtcggcgc
cgacgtgcgc cgcggtaccc tgctggcgcg gctgctgccc 840 gatagcgtga
tcgacgggat gttccgcccc atcgtcaccg ccgccccggc ggcgaaggag 900
gagcaacgtg cctga 915 24 1329 DNA Pseudomonas aeruginosa 24
atgatggccg agatacgacg cccgctgtcc gcggtggaac gctggtactg gctcagcgac
60 cagttctccg cgctgaacgt gatttcccgg gtgcgggtcc atggccggtt
gtccatcgac 120 gacctgcgcc gcggcctcga cgcgctgcag gcgcggcatc
cgctgctgcg cgcgcggatc 180 gagcacgatg ccgggctcga tccgcgctgg
gtgccctgcg agcggcccat cccgctgcgc 240 gaggtgcgcg gcggcggcga
ggagcaatgg ctgcgggaaa tcaacgagcg cgaattgccg 300 gaacgcatcg
atccggacag cgggccactg atccgtaccg tggcgatcgc caccgacgcc 360
ggcgcccacg acctgctggt cgtggtaccg cacatcatcg ccgacggcac taccgtgctg
420 accctcgccg aacaatggct gaccctggcc gccgaccccg ccgcgcaacc
ctggaccgcc 480 agcgccctgc cgccggcgga ggatctgcgt ccgcgccgct
tcaccggcga cgaaggcgcg 540 gcgcgcctgg ccgagcagac cgcccaggac
gaagcgctgg tcggccgcca ccgcccgggc 600 cggatcgagc cgagcaaccc
ggtgccgctg gaagcgcggc gtacccgcct gctgcaccgg 660 gagctggacg
gcgcgcagct ggaacagctg caacgacgcg cccgcgaaca cggcaccacg 720
gtacacggcg cgctgaccgc ggcgctggcc atcgccgccg gccacgacca ccagcgccgc
780 cctagccaca tcgccatcgg ctcgccgatc gacttccgcg acgaactgga
gccgccggtg 840 cgccccgacg aagtaggcac ctacgtcgcc acggtaccgg
tggtgctgga catcgcccgg 900 ccgttctggg aggtcgcccg cgcgctcacc
gacgacctcg gcgaacgccg tcgccagggc 960 catcatttca acctggtcac
cctggtcgcc agcgctgcgc cgcgctgcat ggccgacgcg 1020 cggccattca
tggccttcat ggaagccgaa gggccgatca acctgtgctc ctccaacatc 1080
ggtcgctatc cgttccccga gcggatcggc gccttgcgcc tctccgacgc gcagttcctc
1140 accggcatct cggtgaacgg ctacttcgtg gccgccatca actccagcca
tggccggctg 1200 ttctggaact tcacctatat cgacgaagcg gtccccggcg
aacgcgccga acgcctggcc 1260 gaagattgcc tgggcaccct gctgtcggcg
atccacgccc cccaacgatc cgccctcgag 1320 gagcaatga 1329 25 1167 DNA
Pseudomonas aeruginosa 25 atgagcagac atcccctgaa gatcgtcatc
gccggcgccg gcatcggcgg gctcgccgcg 60 gccgcctgcc tgaaagccgc
cggcttcgag gtcgaactct acgagcgggc cagggagctg 120 cgcgcggtcg
gctcggcgct gtcgctgatg cccaacgcgc tgaccgccct ggagagggtc 180
ggcgtgcgcc ctgaccttac ccgcgcccag gccttcgact cgctgcggtt cctcacccgg
240 cgcgggcgac cgatccgcgc catcgacttc ggcggcctgg cccgtcagct
cggccagccg 300 agcctggcga tccaccgcgc gagcctgcag caggcgctgc
tggaacaggc ccgcgactgc 360 cgcatcgaac tgggcgtgag cgccaccggc
tacctgcgcc acgccgacgg cgaaggcgtc 420 accgtgctct gcagcgacgg
ccgcgaagtg cacgccgacg tgctgatcgg cgccgacggc 480 ttcaactcgg
cgatccgcgc caccatgacc ggcccggagc gtcccaccga ctggcactac 540
gtgatctggc gtgccacgcc ggcgttccgc catccgaagg tgacgccggg ctacgtcgcc
600 cattactggg gccgtgggca gcgcttcggt ctcgccgaca tcggcgaagg
caacgtctat 660 tggtggggca cccgcaacat gccggccgaa caggcgaagg
actggcgcgg cggcaaggcg 720 ggcatccagc gcctctacgc cggctgggcc
gacgaagtgc aggcggtcat cgaggcgacc 780 ccggaggccg acatcagcag
cctgccggcc caggaccgac cgttcctgga gcgctggggc 840 gacggcccgg
tgaccctgct cggcgatgcc gcgcatccga tgctgaccag cctcggccag 900
ggcgccgcca tcgccatcga agacgccgcg gtgctggccc actgcctggc caccatcgac
960 gacccgcaag ccgccctgcg cgcctacgag aaccgccgtc gcgaccgcgc
cagggcgatg 1020 gtcgagacct cgcgggcgct gagccgcatc gagcagttgg
agcatccgct gcgcaccgtc 1080 gcccgcgatc tctacttccg cttcgctccg
gagcgaacct tcgcccggca gaacgaactg 1140 gcactgacct tcccaggagt cgaatga
1167 26 7110 DNA Pseudomonas aeruginosa 26 gtgcggtgcc cgtgttcgcc
gaatcgatgg attgttgaag aggacgcagg gatggttcgt 60 ttcgctcgct
tgccgctatc gccctaccaa cgggacatct gggtcgccgc cgcgcagttt 120
ccggaactcg accagtacac catcttcagc tacgaccgct tcaccggcga ggtcgatacc
180 caggccctgg aacgagcgct gctgcaggcg gcgcgagaca ccgaggcgtt
ccgcctgcgc 240 ctcggcgaga cggacggtac gccgtaccag tggctggaca
cggatgccga gttcgaggcg 300 cgccacgtcg acctgcgcgc cgaccgcgac
cccgaggccg ccgtgcgatc ctggctgcgc 360 gacgccttcc gtcacgccta
cccgctggac ggccgcagcc tggtggacct ggccctgctg 420 catagcgacc
aggcgctcta cgtctacgtg cgcacccacc atatcgtcag cgacgcctgg 480
ggcctgcagc tattcctcag ccgggtgcgc gccggctacc tgggtgagct aggcgagccg
540 caggcgcaga tgccgacggc ttccctcctg gcgcagctcg agaccgacga
ctactccggt 600 tcggaacagt accgcggcga ccgcgcctat ttcgccgagg
ccctggaggg cctggagccg 660 gccctcttca cccgcaggcg cccggccggg
ctgcgccgca ccgcgcgcca caggctgacg 720 ctggaacgca cactgctcga
tgcgatccgc gatcgtggcg aatcgccctt cctgttcctc 780 tccgccgccg
tggcgctgta cctggcgcgg atccaccaga acgacgacgt ggtcctcggc 840
gtaccggtgt tgaaccgcgc cgaccgcgcg gccaagcaag tggtcgggca cttcgccaat
900 accctgccgc tacgcatccg caccgcgccg gaacagaccg tcgacgaatt
cctggcgcag 960 ttgcgcgagg cgacccggac gctgctgcgc caccagaaga
tgcccctcgg cgacctgttg 1020 cgcggcgcct cgccactgtt cgacaccacc
ctttcctaca tgcgctggcc cgccgcccag 1080 gcgatcccga acgccagcgt
cgagaccgtg gcgcaaaccc acgcccatga cccggacgcg 1140 ctggccatct
gggtctccga gttcgacggg cacagcgacg cgcaggtgga tttcgaatac 1200
gcctgcgatg tgttcgacgc cgacttcccc atggacgccg cggcgcggca tatcgaaacc
1260 ttcctgcgcg ccctggtgga gggcggcgag cgccgcctcg gcgaactcga
tccgctgtcg 1320 gccgccgagc gcgaggaact gatccacacc cgcaacgcca
ccgaccaggc attccccgag 1380 caggctaccc tgcccacact gttcgccgag
caggtggcgc gcaccccgca acgcaccgcg 1440 ctgctggaag ccgacggcgg
cacgctcagc tatgccgagc tggacgccaa ggtccaggcc 1500 gtggccgacg
ccctgcgcgc agcgggtgtg aggaccgacg agcgggtagc gctactggtc 1560
gcccgcggtc cccacctgct gccggcgatc cttggcgtgc agcgcgccgg cggcgcctat
1620 gtgccgatca atcccgacca tcccctggag cgcgtccgcc tgctgttgga
agactgcggt 1680 gcccgcgtgg tgctggtgga cgagcgcgca gcgacactcg
gcgagagcct cggcgagacg 1740 cgcgtgctgc acctcgaacg cctgccgcag
agcaccggcg acctgccggc ggccaacgtg 1800 gcgcccggcg acctggccta
tgtcatctat acctccggtt cgaccggcat gcccaagggc 1860 gtcatggtcg
agcaccgctc ggtggtcaac cgcctgaact ggatgcagcg tcgttatccg 1920
atcggcgaac gcgacgtgct tctgcaaaag actccggtga cgttcgacgt gtccgtctgg
1980 gaactgttct ggtggagttt caccggcgcc cgcctgtcgc tgttgccgcc
cggcgccgag 2040 aaggacccgc gggaaatgct gcggagcatc cagcgcgacg
cggtcacggt catccacttc 2100 gtgccgtcga tgctgacgcc gttcctcgac
ctgctcgacg gcgacccgac cgcccgcgcg 2160 gcggcaagct cgctgcgcct
ggtgttctgc agcggcgaag ccctcgcgcc gttgcaggtc 2220 gcgcgcttcc
gccggctgtt cggcgacgcc gtgcgactgg tcaacctgta cggaccgacc 2280
gaggccaccg tcgacgtgtc cgaccatgaa tgcgccagcg acaaccccac gcgggtcccg
2340 atcggccggc cgatcgacaa cctgcgcctg tacgtcctcg accgcgcgct
caggccgcag 2400 cccctcggtg ccgtcggcga gctatatata ggaggcgtcg
gcgtcgcccg cggctacctg 2460 aaccggccgg agctgaacgc cgagcgcttc
ctcgtcgacc ccttcgtcgc cggcggccgt 2520 ctctaccgta ccggcgacct
ggcccgctgg ctggccgacg gcaacctcga atacctcggc 2580 cgcgccgacg
accaggtgaa gatccgcggc aaccgggtcg aacccgacga agtacgcgac 2640
cgcctcgccg cgcttcccgg cgtacgcgac gccgcggtcg tggcacgcga ttcggcggta
2700 cgcggcacgc acctggtcgg ctactacgtg gctgcggcgg aactcgaccc
cggtcaattg 2760 cgcgccggac tttcggcgac gctgccggac ttcatgctgc
cagccttctt cgtgcgcatc 2820 gacagcctcc cgctcagcgc caacggcaag
ctcgaccgcc ggcaactgcc ggcaccgccg 2880 gaacaggtgg cggcggttgc
gccgcgcacg gcgaccgagg ccgaactggc ggcggtgtgg 2940 gccgatgtcc
tcggcgtggc ggaggtcggc gtgcacgacg acttctacgc cctcggcggc 3000
gactcgatcc tgatgctgcg catccgcgcc gccgcacagc ggcgcggcct gggcttcgaa
3060 ctcgccgacc tgatgcgcaa cccgacggtg gcgggcctcg ccgagcgcct
ggtgcgtccg 3120 ctcgcggagc gaagctacca gcccttcgaa ctggtttccg
aagtcgacaa gccgcgcctg 3180 gaagggctgg aggacgcctt cccgaccagc
cggctgagtc tcggcctgct cttccatagc 3240 cgccagcgcc ccgactcgtc
ggtctaccac gacgtgttcc actaccgctt cgacctggcc 3300 tgggacgaag
ccgcgttccg ccacgcgctg gaccgggtgg tcgccgccta tcccgcgctg 3360
cgttcgtcgt tcgacctcag cggtgcatcc gaaccgctgc aactggtgca tacccaggcg
3420 cgcagcgaac cgctgatcct ggacctgcgc ggcaacccgg aggccgggac
ggtgctcgac 3480 gagcacatcc gccaacgccg cttccatcgc tattcgctgc
aacagcccgg gctattcctg 3540 ttcgccgcgt tcgtccgcga ggacggcctg
gacctggtat tcagcttcca ccatgcgatc 3600 ctcgacggct ggagcgtggc
caacctgatc gtcgcgctgg tcgccgccta ccgtggcgag 3660 ccgctgccgg
gccccgcgcc ggcgctggcc tgccatgtcc gcgaggagct ggccgcgctg 3720
gcttcgccgg ccgccgtggg gtactggacc gggctgctgg agggcgcgag gatgacccgc
3780 ctcgacggct tcggcgccca cgagccgcaa gccgcgcaag gtccggccag
ccatcgcgaa 3840 gcgctgccgg acgggctgct cgaacgactc aaggccactg
cggcgcaacg cggactgccg 3900 ttgaagtcgc tgctgctcgc cgcccattgc
ctgaccctgc atctgttctc ccgcagcgac 3960 agcgtggtca ccggcgcgat
cagcaacggc cgccccgaac tgcccgacgc cgaccgcatg 4020 gtcggcctgt
tcctgaatac cgtgccggtc cgctcggaga ttgccgggtg tagctggatc 4080
gaggtagccg atgcgctgtt ccgccaggag cgcgacggac acgcccaccg ccgctatccg
4140 ctcagcgcca tccagcagat cgtcggcgac gaactgagca gcgccttcaa
ctacgtcaac 4200 ctgcatgtcc tcgaaccgct gtggcaattg cgcgacttcc
gcgtctggga agaaaccaac 4260 ttcgccctgc tggtcaacgt gatcgccacg
cccagcgacg gcatgtacct gcgcatcgac 4320 agcgacggcc gcggcatcag
ccgcagccag gccgcgctga tcggcgcgac cttcgtcgag 4380 ctcctgtggc
gcctcgccga tcatcccgac gaagccgccg acttcgcctt cctcgcccct 4440
cgccgcgacg ccgcttccca gcccgagccg ctggtcgacg tcgtcagcct gttcgaacgc
4500 caggtcgagg cgctgccggg cagcgccgcg ctggccttcg aggagcaacg
ctggacctat 4560 cgcgacctcg accatgtggc gcgctgcgtg gccacccgcc
tggtccgcgc cggcgcgcgc 4620 cgcggcgatg cgatcggagt ggcgctgaac
cgttcgccgg agatgatcgc gacgatctgg 4680 ggcatcctgc gcgccggcct
ggtctgcgtg ccgctggacg tcagctatcc cgcgcagcgc 4740 ctggcgctga
tcctggagac cgcacagccg ttccgggtgg tcgcgcatcc cgagcacgcc 4800
catgtcgccg cggcggaacg ggtgctgccg gtagaggaac tggtcgccga catcgagccc
4860 gagaccttcg ccgcgccgca gctcgacgag ctggccatgc tgctgttcac
ctctggttcc 4920 accgggcggc cgaagggcgt cgagcttagc caccggatgt
gggccaacta cacccagtgg 4980 caattgcgcg tcgccagcgg cgtaccgggg
ctgcgcacac tgcagttcgc gccgctgagc 5040 ttcgacatgg ccttccagga
gatcttctcc acgctgtgcg gcggcggcga gctgcaactc 5100 atctccaacc
gcgagcggat ggacccctcc gcgttgctgc atgtcctcga acgccgccag 5160
gtccagcgcg tgctgttgcc cttcgtcgcc ctgcaacgcc tcgccgaggc ctccaacgcg
5220 ctgggcgtgc gccccggcgc cctgcgcgtg gtggtgtcct ccggcgagca
gttgcgcatc 5280 accgaagacg tccgcgcgtt ctgcgcggcg atgcccgggc
tgctgctgga gaaccagtac 5340 ggtcccaccg agacgcacca ggtcacctac
cactcgctga gcggcgatcc ggcgcactac 5400 ccggacctgc cgccgatcgg
ccggccgctg gacggggtcg aggtgcaggt gctcgacgcc 5460 gcgctgcgcc
cggtaccggt cggcgttacc ggcgagctgt acttcggcgg cgactgcctc 5520
gcgcgcggct accaccgcgc ccccaaactc accgccgagc gcttcgtcga acatccctgg
5580 cgccccggcg ccaggctcta ccgcaccggc gacctcgggc gcatcctcgg
caacggcgag 5640 atcgtctggc tcggccgcgc cgatacccag gtcaaggtcc
gcggcttccg catcgagccg 5700 gccgaggtcg agctggcgat catgcgccag
gccgagcgcc agccgggcct gcgcggcgcg 5760 gcggtggtgg ctcgcgagcg
ccagggcaac gatgcattcc tcgctgcctt cctgctcggc 5820 gagcccgagg
cggtggatct cgccgaactg aagcaggcac tgcgcagcga actgccggaa 5880
cacatggtgc cggcacactt cgcctgggtc gacggcttcg ccctcacccc cagcggcaag
5940 cgcgacgacg ccgccctgcg cgcactgccg ctggagcacg ggacgaacat
cgagtacctg 6000 gccccgcgcg acgactacga gcgcaccctg gccggactcc
tcggcgagtt gctggatcgt 6060 ccccgggtag gcatccgcga cagcttcttc
gacctcggcg gcacctcgct cagcgcgatg 6120 cgcttcatgc tgctgatcga
gaagcgctat ggcgtcgacc tgccgatggc cgcgctgatc 6180 gagacgccga
ccgtggaggg cctggccgaa cgcctgcggg aacgctcggc ggtgcgcgcc 6240
ttcgacccgc tggtaccgat ccgtgccggc ggcagccgcc cgccgctgtt cctcgtccac
6300 ccgctcggcg gccacgtgct ctgctacctg ccgctggtcc gcgcactgcc
gccggaccag 6360 ccggtatatg ccctgcaggc ggccggcacc ggccagggca
gtacgccgct ggcggtcctc 6420 gaggacatcg ccgccagtta cctcgcggcc
atccgccggg tgcagccgga aggcccctat 6480 tacctcggcg gctggtcgtt
cggcggcttc gtcgcctacg agatggcccg gcaactgcgc 6540 gcgctcgacc
cgcaggcggt cgcccaactg atcgtgctcg actccatcac cgtcgaccgc 6600
aaccacgccg gcagcgccag cgacgaagcc ctgctgctgt tcttctactg ggaactggtc
6660 tggttcgagc gcagcgacaa ggaggtcgag ccgctgcctg aaggcgcgag
cctggagcag 6720 aaactcgacc acatcgtcga acgcgccatc gaggccggcg
tacttcccgc cggcaccccg 6780 cgcgccaccg tgcagcggct ctacgagctg
ttccgggcga gctggcaggc actcatcggc 6840 tatcgcccgg aagtcagcga
ccaggacatg accctgctgc gcgcggacgg cccgctgccg 6900 ctggcgctga
agccgatgca cgacgccgcc ggcacccact acggcgaccc gaagaacggc 6960
tggcagcact ggaccagtgg ccgcctcgat gtgatcgacg tccccggcga ccacctggtg
7020 ctgatgaaag aaccctatgt cgagacggtc gcggcagaga tcgccgcgtt
gctcgaaccc 7080 tccacctcca gcgaacggac ccgcccatga 7110 27 1404 DNA
Pseudomonas aeruginosa 27 atgaaaacgc ccgcctggac gcgccatgcc
ctctgggtca tgccgctcgc cctggggctg 60 caatccgccg tggtcgcggg
ggatgagcag ccaagcaaga cttccagcta ttcgccggtg 120 gtgatcaatg
aggacttcgc caccatcatg aagcgcatga cggcgaacaa accgtcgatc 180
gaacaggccc acaagacgct tctcgagcag cgttacgatc tcagcgacag gccggccaag
240 ggcgccagca tgacgcgcgg caagccgctg caggagggga tccgggtgaa
gctgccggcc 300 ggcaccagct gggaggaact ggccaggctg agccccgagg
aaatccgcaa gcaggggctg 360 ttccccggtg gcttcctgcc gctgccgcac
cccaaccatg ccgaaggcgg gatggtcttt 420 cccaagttcc tcatcgacga
gatcaagcgc caggaaagcc gcgacctgac ccgtttcgac 480 ctcgactacg
acctgccgga ccacttcctg ccggaattcc cggcaccgat gttccttacc 540
acccggcctg acctgggcga tgtgtccaag ggcaagctgg tgaccatcga caactatttc
600 gagttgttca acgggattct caatcccaag cagctggaag ggctgcgcct
gctgctaacg 660 gcctttccgc agcagcagtt caacctcacc gacgatcgcc
gtagcgagca tccgagccgc 720 ggcgtagcct gcttcgactg ccatgcgaac
ggccacacca atgccgctac tcacctggcc 780 ggcgatgtgc gcccgcagcc
gttccgccac cgcatcgaca caccgacgct gcgcggggtg 840 aacatccagc
ggttgttcgg ctcgcagagg gcgctgaaga ccgtcgagga cttcaccgag 900
ttcgagcagc gcgccgccta cttcgacggt gatccggtaa tcgccaccaa gaagggggtg
960 aacgtgctcg agcgtggcag tcaagtgcat ttcatgggtg agttccaggc
gctgctggac 1020 ttccccccgg caccgaagct ggatgtggag gggcggctcg
atccgggcaa ggccagcgag 1080 caggaattgc gtggcgaaaa gctgttctac
ggcaaggcgg cctgcgccgg gtgccatgcg 1140 ccgccttact tcaccgacaa
cctgatgcac aacctgaagg tggagcgctt ctacgatccg 1200 aaactggtca
atggcgtgat ggcgtccgcc gacgggccga tcaagacctt cccgttgcgc 1260
gggatcaagg attcgccgcc gtacctgcac gacgaccgcc tgctgaccct ggaggacacc
1320 gtggagttct tcaacctggt actggagcgc aagctgtccg cggaagagaa
gggcgacctg 1380 gtggcctacc tgcgtaccct gtga 1404 28 1386 DNA
Pseudomonas aeruginosa 28 atgctcacgg tgtgtgcgaa ccccaagggt
atttcccgac cagctccccc gcggtcggga 60 tttttttttg cctgtcgctc
agcgcttcgg gtcgaagggc gaatagcccc gccggcgcag 120 gctcgccagg
ggcgcgaaca gcggctccgg cagatcggcc caatcgaacc aggcccagcc 180
gtcgcacttg tccggctcca tgaggcgcgc ctcggcatcc tccgcgcaac cggccaggat
240 gaacgcggtg aggtagtggc gcccctcgaa gacgtcattg ctgaacgggc
cgtggcgcag 300 ttcgctcagc gccaggtcgg tctcttccag ggcttcgcgc
agggcgcagt cctccaccgc 360 ctcgccgaac tcgagatggc cgccgggcgc
cgaccagcag ccagcgccat gactgccctt 420 gcggcgcccc agcaacacct
tgccgtcccg caagatcagg acgcccacgc ctacctgcgg 480 tgccggcatc
gtcgtactcc tgcttcggga tcagagatgg agcgtaccgc tcatgtacaa 540
cgccgccttg ccggagatga ccacccgctc gccgcgcacg tcgcattcca ggcgcccctt
600 gcgcgccccg ccctgctcgg cgctcagccg ggtcttgccc aggcgctgcg
cccagtacgg 660 cgccagggag gtatgcgcgg agccggtcac cgggtcttcg
ttgacgccga cgttgggccc 720 gaaccagcgc gagacgaaat cgaagcgctg
gctgcgcgcg gtcaccgcca ccccgcggca 780 cggcaagccc ttcagccggg
cgaagtcagg cgccagggcg gcgatcgtct tttcgtcgtc 840 gaccaccacg
aggtaatcgt cggtcttcag cacttccgcc tcggcaatac ccagcgcctc 900
cagcagtccg tccggtgtcg cgcaaggctc cggacgcttg gccgggaagt ccatcgccag
960 cgagtcgccc tcgcgccgca cgctcagctc accgctacgg gtagcgaaac
gcagtaccgg 1020 ggaagcgtcg tcgagcttgt ggatcagtac ccaggccgtc
gccagggtcg catgaccgca 1080 caggtccacc tcgacctgcg gcgtgaacca
gcgcaatcga tagtcgccgt cgcggccgac 1140 gacaaaggcg gtttccgaaa
gattgttctc ttccgcgatg gcctgcaggc gctcgtcgtc 1200 cagccaggca
tcgagggggc agaccgccgc cggattgccc tggaagggac tgtcggcgaa 1260
tgcgtctacc tggaagatcg tcagttccat gttccggact cctgtatcga tgggctgcgc
1320 accttagcag ccggaccgag accaggacaa tgccgcgccc cgcgcaggcg
cctcgctcag 1380 atctga 1386 29 1104 DNA Pseudomonas aeruginosa 29
atgaaaaaag tttgtgcact ggcgttatcg atcctgacga cgatcggtgc gacagcggcg
60 gacagtgcat gggctgcgca aaccagcgtc catctttaca actggtatga
cttcatcgcc 120 ccggaaacgc ccaaggcttt ccagaaggaa accggcaccc
gtgtcgtcct cgacaccttc 180 gacagcgccg agaccgcgca gggcaagctg
atggtcggcc gctccggcta cgacgtggtg 240 gtgatcacct ccaacatcct
gcccgggctg atcaaggcgg gcgtcctcca ggaactcgac 300 cgcgaccggc
tcccccactg gaagaacctc gacgcggaca tcctcgggaa gcttcaggcc 360
aacgatcccg gcaatcgcta tgccgtacct tatctctggg gaaccaccgg gatcgcctac
420 gatgtggaca aggtccgcaa gctgctcggc cccgacgcgc cggtcgactc
ctgggacctg 480 gtcttcaagg aggagaacat ctcccgcctc agccagtgcg
gcgtggccac gctggactcc 540 tccaccgagc tggtgtccat cgccctcaac
tacctgggcc tgccgcacaa cagccagaat 600 cccgaggact accagaaagc
ccaggaactg ttgctgaagg ttcgccccta cattcgctat 660 ttcgactcct
ccagagtcga caccgatctc tccaacggca acgtctgcgt ggtggtcggc 720
tggcagggca cggcctacat ggcccaggtc aacaacgaac aggccgggaa cggtcgccat
780 atcgcctaca gcattccccg ggaaggctcg ctggtctggg ccgagaacat
ggtgctgctc 840 aaggatgcac cgcatccgca gcagggttat gcgctgatcg
actacctgct gcgtccggag 900 gtcatcgcca ggacctccaa ctacgtgggc
tatccgaatg gcaaccaggc ggcgctgccg 960 ctggtagagc ggaaactgcg
ggaaaacccg gcggtttacc tgagcaagga aaccatggcg 1020 accctcttcc
cgctggaaac cctgccactg aaggtcgaga gaatccgtac ccgggtctgg 1080
agccgggtca agaccgggag ctga 1104 30 1251 DNA Pseudomonas aeruginosa
30 gtgggctgtc cggggcggct aggatggaca ttttcatcgt ctcgggcagg
cctgtcgcga 60 ccgcgcgaag tcgcgacgga tgccgctgct aaggagcaac
ggatgaccgt tcttatccag 120 ggggccggga tcgccggcct ggcgctggcg
cgcgaattca ccaaggcagg catcgactgg 180 ctgctggtcg agcgggccag
cgagatcagg cccatcggta ccggcatcac cctggcgagc 240 aatgcgttga
cggcgttgtc cagcaccctg gatctcgacc ggctgttccg ccgtggcatg 300
ccgttggccg gcatcaacgt atacgcccac gacggttcga tgctgatgtc gatgccttcc
360 agtctgggtg ggaattcccg cggcggcctg gcgttgcagc gccacgaact
gcatgcggcg 420 ctactggagg ggctggatga gtcgcgcatt cgggtcgggg
tctccatcgt gcagatcctc 480 gacggactcg accacgaacg cgtgaccctg
agcgacggca ctgtccacga ctgttcgctg 540 gtggtcggtg cggatggcat
tcgttcgagc gtgcgacgtt atgtctggcc ggaggcgacc 600 ttgcgtcatt
ccggcgaaac ctgctggcgc ctggtcgttc cccatcggct ggaggacgcc 660
gagctggcgg gagaggtctg ggggcacggc aagcgcctcg gcttcatcca
gatcagcccg
720 cgcgagatgt atgtctacgc gaccctgaag gtgcgccggg aggagcccga
ggacgaggag 780 ggcttcgtaa ccccgcaacg gctggccgcc cactacgcgg
acttcgacgg catcggcgcg 840 agcatcgccc ggctcatacc gagcgccacc
acgctggtgc acaacgacct cgaggagttg 900 gccggcgcct cctggtgccg
cggacgggta gtgctgatcg gtgacgccgc acacgccatg 960 acgccgaacc
tggggcaggg cgcggccatg gccctggagg acgccttcct gctggcgcgc 1020
ctgtggtgcc tggcgccgcg cgccgagacg ctgatcctgt tccagcagca acgcgaggcg
1080 cggatcgagt tcatcaggaa gcaatcctgg atcgtcggcc gccttggtca
gtgggaatcg 1140 ccctggagcg tctggctgag gaataccctc gttcgcctgg
tgccgaatgc cagtcgcagg 1200 cgcctccacc agcgtctttt caccggtgtc
ggtgagatgg ccgcacagta g 1251 31 1754 DNA Pseudomonas aeruginosa 31
atgatggacg ccttcgaact tcccaccacc ctggtccagg ccctgcgtcg ccgcgctgtc
60 caggagcccg agcgcctggc gctgcgcttc ctcgccgagg acgatggcga
aggcgtggtc 120 ctcagctatc gcgatctcga cctgcgcgcg cggagcatcg
ccgcggccct gcaggcccat 180 gcgcagctgg gcgatcgcgc ggtactgctg
tttcccagcg gccccgacta cgtcgcggcg 240 ttcttcggtt gcctgtatgc
cggggtcatc gcggtgccgg cctacccgcc ggaatcggcg 300 cgccgccatc
accaggaacg cctgttgtcg atcatcgccg acgccgagcc gcgcctggtc 360
ctgaccaccg ctgacctgcg cgagccattg ctgcagatga acgcgcaact gtccgccgcc
420 aacgccccgc aactgctctg cgtcgaccag ttggacccgg ccgttgccga
ggcctgggac 480 gagccgcaag tgcgtcccga gcacatcgcc ttcctccagt
acacctccgg ttcaaccgca 540 ttgcccaagg gcgtgcaggt cagccatggc
aacctggtcg ccaacgaggt gctgatccgc 600 cgaggcttcg gcatcggtgc
cgacgacgtg atcgtcagct ggctgccgct gtaccacgac 660 atgggcctga
tcggcggcct gctgcaaccg atcttcagcg gcgtaccctg cgtgctgatg 720
tcgccgcgct acttcctcga acgtccggtg cgctggctgg aagccatcag ccagtacggc
780 ggcaccgtca gcggcggtcc cgatttcgcc taccggctgt gcagcgagcg
ggtcgccgag 840 tcggccctgc agcgtctcga cctgagcggt tggcgggtag
ccttctccgg ttccgagccg 900 atccgccagg acagcctgga acgcttcgcc
gagaaattcg ccgccagccg cttcgacgcg 960 tccagtttct tcgcctgcta
cggcctcgcc gaggcgaccc tgttcgtcac cggcggccag 1020 cgcggccagg
gcattcccgc cctggcggtg gatggcgagg cgctggcgcg caaccgcatc 1080
gccgaaggcg aaggcagcgt gctgatgtgc tgcggccgca gccagccgga acacgccgtg
1140 ctgatcgtcg acgcggcgag cggcgaggtc ctcggcgacg acaacgtcgg
cgagatctgg 1200 gccgccgggc cgagcatcgc ccacggctac tggcgcaacc
cggaagcttc ggcgaaggcc 1260 ttcgtcgagc gtgacgggcg cacctggctg
cgcaccggcg acctcggctt cctccgcgac 1320 ggcgaactgt tcgtcaccgg
gcgcctgaag gacatgctca tcgtccgcgg ccacaacctc 1380 tatccgcagg
acatcgaacg caccgtcgag agcgaggtgc cgtcggcgcg caagggcagg 1440
gtcgcggcct tcgcggtcac ggtcgatggc gaggaaggca tcggcatcgc cgccgagatc
1500 ggtcgcggcg tccagaaatc ggtgccggcc caggagctga tcgactcgat
ccgccaggcg 1560 gtggccgagg cctaccagga agcgccgaag gtggtggcgc
tgctcaatcc cggcgccttg 1620 ccgaagacgt ccagcggcaa gctgcaacgt
tccgcctgcc gcctgcgcct ggaagacggc 1680 agcctggaca gctatgcgct
gtttcccggc ctccaggccg tgcaggaggc gcagccgccg 1740 gcaggcgacg acga
1754 32 7335 DNA Pseudomonas aeruginosa 32 gtgttggtca tcacccagca
ccatatcgtg tccgacggtt ggtcgatgca ggtgatggtc 60 gacgaactgc
tccaggccta tgccgcggcg cgccgcggcg aacaaccgac gctggcgcca 120
ttgacgctgc agtacgccga ctatgctgcc tggcatcgcg cctggctgga cagcggcgag
180 ggcgcgcggc agctggatta ctggcgtgag cgcctgggcg ccgagcagcc
ggtcctggaa 240 ctgcccgccg accgggtgcg cccggcccag gccagcggac
gcgggcagcg tctggacatg 300 gcgctgccgg tgtcattatc ggaggagctg
ctggcctgcg cccggcggga gggtgtcacc 360 ccgttcatgc ttctattggc
ctcgttccag gtgctgttga agcgctatag cgggcagtcg 420 gacattcgcg
tcggggtacc tatcgccaac cgcaaccgcg ccgaggtcga gcgcctgatc 480
ggcttcttcg tcaataccca ggtgctgcgt tgccaggtcg atgctggcct ggctttccgc
540 gatctactgg gccgcgtgcg cgaggcggcg ctgggcgcgc aggcgcacca
ggatctgccg 600 ttcgagcaat tggtcgatgc cttgcagccc gaacgcaatc
tcagccacag cccgttgttc 660 caggtgatgt ataaccacca gagcggcgag
cggcaggatg cccaagtcga tggtttgcac 720 atcgagagtt ttgcctggga
tggtgctgcc gcacagttcg atcttgccct cgatacctgg 780 gaaaccccgg
acggccttgg ggcggcgctg acctacgcga ccgacctgtt cgaggcgcgg 840
accgtcgagc gcatggcgcg gcattggcag aacctgctgc gcggcatgct ggaaaacccg
900 caggccagcg tcgactcgct gccgatgctc gatgccgagg agcgtggcca
gttgctggaa 960 ggctggaacg ccactgccgc cgagtacccg ctgcaacgcg
gcgtgcaccg gttgttcgag 1020 gagcaggtcg agcgcacgcc gacggcgccg
gcgctggcct tcggcgagga acgcctggac 1080 tacgccgagc tgaaccgccg
ggccaaccgc ctggcgcatg ccctgatcga gcgcggggtc 1140 ggtgcggacc
gcctggtggg cgtggccatg gagcgttcca tcgagatggt cgtggccctg 1200
atggcgatcc tcaaggccgg cggcgcctac gtgccggtgg acccggagta ccccgaggag
1260 cgccaggcct acatgctgga ggacagcggc gtgcagctgc tgctcagcca
gtcgcacctg 1320 aagctgccgc tggcgcaagg cgtgcagcgg atcgacctgg
accaggccga tgcctggctg 1380 gaaaaccatg ccgagaacaa tccggggatc
gagctgaacg gcgagaatct tgcctatgtc 1440 atctacacct ccggctccac
cggcaagccc aagggtgccg gcaaccgcca ttcggcgctg 1500 agcaaccgct
tgtgctggat gcagcaggcc tacggcctgg gcgtcggcga cacggtgttg 1560
cagaagaccc cgttcagctt cgacgtgtcg gtctgggagt tcttctggcc gctgatgagt
1620 ggggcacgtt tggtggtggc cgcgccgggt gaccatcgcg acccggcgaa
gctggtggcg 1680 ctgatcaacc gcgaaggggt cgacacgctg cacttcgtgc
cgtcgatgct gcaggccttc 1740 ctgcaggacg aagacgtcgt ctcctgcacc
agcctgaaac gcatcgtttg cagcggcgag 1800 gcgctgtcgg cggacgccca
gcagcaggtg ttcgccaagc tgccgcaggc cggcctctat 1860 aacctctatg
gcccgaccga ggcggccatc gacgtcaccc actggagctg cgtggaggag 1920
ggcaaggacg cggtgccgat cggccggccg atcgccaacc tgggctgcta catcctcgat
1980 ggcgacctgg agccggtgcc ggtgggcgtg ctcggcgagc tgtacctggc
cggtcggggc 2040 ctggctcgtg gctaccacca gcgtccgggg ctgactgccg
agcgtttcgt cgccagcccg 2100 ttcgtggctg gggagcggat gtaccgcacc
ggcgacctgg cgcgctaccg cgccgatggg 2160 gtgatcgagt acgccgggcg
gatcgaccac caggtgaagc tgcgcggcct gcgcatcgag 2220 ctgggcgaga
tcgaggcgcg cctgctggag catccgtggg tgcgcgaggc ggcggtgctg 2280
gcggtggaca gcaggcagtt ggtcggctac gtggtgctgg agagcgaggg cggcgactgg
2340 cgcgaagcgc tggccgcgca cctggcgaca agcctgccgg aatacatggt
gccggcgcag 2400 tggctggcgc tggagcggat gccgctgagt ccgaacggca
agctggatcg caaggcgctg 2460 ccgcgaccgc aagctgctgc ggggcagacg
catgttgcgc cgcagaatga aatggagcga 2520 cgtatcgcgg ccgtctgggc
ggacgtgctg aagctggagg aggtgggcgc caccgacaac 2580 ttctttgccc
tgggtggcga ttccatcgtt tcgatccagg tggtgagtcg atgccgtgcg 2640
gcgggcatcc agttcactcc gaaggacctg ttccaacaac agaccgtaca ggggctggcg
2700 cgagtcgccc gcgtaggggc tgcggtgcaa atggagcagg ggcctgtgag
cggcgagacg 2760 gtgttgttgc cgttccagcg gttgttcttc gaacagccga
ttcccaatcg ccagcactgg 2820 aaccagtcat tgctgttgaa gccgcgcgag
gccctgaatg cgaaggcact cgaagcggcc 2880 ttgcaggccc tggttgaaca
tcacgacgca ttgcgtctgc gcttccatga aacggacgga 2940 acctggcatg
ccgaacatgc cgaagcaacg ctgggcggtg cgctgctctg gcgtgccgag 3000
gcggtggacc gacaagcgct ggagtcgctc tgcgaggagt cgcagcgcag cctggacctg
3060 gccgacggcc cactgttgcg gagcctgttg gtggatatgg ccgacggcgg
ccagcgtctg 3120 ttgttggtga tccaccatct ggtggtggac ggggtgtcct
ggcgcattct gctggaggat 3180 ttgcaaaggg cttaccagca gagcctccgt
ggagaagctc cgcggctgcc tggcaagacc 3240 agcccgttca aggcctgggc
cggccgagtg agcgagcatg cccgtggtga gtcgatgaag 3300 gcgcaattgc
agttttggcg cgagctgctg gaaggtgcgc cggccgagct tccgtgcgag 3360
catccgcaag gcgctctgga gcagcgtttc gctacctccg tgcagagtcg cttcgaccgc
3420 agcttgaccg aacgcttgct gaagcaggcg ccggcagcct accggaccca
ggtcaacgat 3480 cttctgctga ccgccctggc gcgagtggtc tgccgttgga
gcggcgcctc ttcaagcctg 3540 gtacagctgg aagggcatgg gcgcgaggag
ctgttcgccg atatcgacct gagtcgcacc 3600 gtgggttggt tcaccagttt
gttcccggtg cgcctgagcc cggtcgcgga tcttggcgag 3660 tccctgaagg
cgatcaagga acagttgcgt gcgattcccg acaagggcct gggttatggc 3720
ttgctgcgct atctggctgg agaggaaagt gcccgggtcc tggcggggtt gccgcaggcg
3780 cggatcactt tcaattacct gggccagttc gacgctcagt tcgacgagat
ggctctgctg 3840 gacccggctg gcgaaagcgc gggggcagag atggaccccg
gcgctccgct ggacaactgg 3900 ctgagtctca atggccgggt gttcgacggt
gaactgagta tcgactggag cttcagctcg 3960 cagatgttcg gcgaggacca
ggtgcgtcgc ctggccgatg actatgtggc tgagctgacg 4020 gcgctggtcg
acttctgctg cgattcgcca cggcatggcg cgacgccttc cgatttcccg 4080
ctggcggggt tggaccaggc gcgtctggat gccctgccgg tcgcgctgga agaggtcgag
4140 gacatctatc cgctgtcacc catgcagcag ggcatgctgt tccattcgct
gtacgagcag 4200 gcatcgagcg actacatcaa tcagatgcgt gtggatgtgt
ccggcctcga tctcccgcgc 4260 ttccgcgcag cctggcagtc cgccctggac
cggcacgcga tcctgcgcag tggtttcgcc 4320 tggcaggggg agctgcagca
gcccttgcag atcgtctatc gacagcgcca gttgcccttc 4380 gccgaagagg
acctgagcca ggcggcgaat cgggacgccg cgctgctcgc gctggctgcg 4440
gccgagcgcg aacgcggttt cgaactgcag cgtgcgccac tgttgcggct gctgttggtg
4500 aagactgccg aaggtgagca tcacctgatc tacacccatc atcacatcct
gctggacgga 4560 tggagcaatg cccagttgct cagcgaggtg ctggagtcct
atgccggacg ctcgccggag 4620 cagctccggg atggccgcta tagcgactac
atcgcctggt tgcagcggca ggacgcggca 4680 gctaccgagg cattctggcg
cgagcagatg gcggctctgg acgagccgac gcgattggtc 4740 gaggcactgg
ctcagccggg actgacatcg gccaacggcg tcggagagca cctgcgtgag 4800
gtggacgcaa cggctaccgc gcggctccgg gatttcgccc ggcgccacca ggtcactctc
4860 aataccctgg tccaggcggg ctgggcgctg ctcctgcaac gctataccgg
acaacacacc 4920 gtggtcttcg gcgccaccgt ctccgggcgc cctgccgatc
tgccgggtgt cgagaaccag 4980 gtcgggttgt tcatcaatac cttgccggtg
gtggtaacgc tggctccaca gatgaccctc 5040 gacgaactgc tgcaagggct
gcaacggcag aacctggcgt tgcgcgaaca ggagcacacg 5100 cctctgttcg
agctgcagcg ctgggcgggg ttcggcggcg aggcggtttt cgacaacctg 5160
ttggtgttcg aaaactaccc ggtggacgag gtgctcgaac ggtcctccgc tggaggcgtg
5220 cgtttcggtg ccgtagcgat gcacgagcag accaactatc cgctggccct
ggcgctgggt 5280 ggcggggata gcttgtcact gcaattcagc tacgatcgcg
gactgttccc ggccgctacg 5340 atcgagcgcc tgggtcgcca cctgacgact
ctgctggagg cattcgccga acatccgcag 5400 cgacgtctgg tcgatctgca
gatgctcgag aaggcggagc ttagcgctat cggcgctatc 5460 tggaaccgca
gcgattcggg ctatccggca acgccgctgg tacaccagcg agtggccgag 5520
cgggcgcgta tggcgccgga tgcggtggcg gtgatcttcg acgaggaaaa gctcacctac
5580 gccgagctgg atagccgggc caaccgcctg gcacatgcgt tgatcgcccg
aggcgtcggc 5640 cccgaagtgc gtgtggcgat cgccatgcag cgcagcgcgg
agatcatggt ggcgttcctg 5700 gcggtactga aggccggcgg cgcctacgtg
ccgctggaca tcgaataccc gcgcgagcgc 5760 ctgctgtaca tgatgcagga
cagtcgcgcg cacctgctgc tgacccatag ccacctgctg 5820 gagcgtctgc
cgatccccga ggggttgtcc tgcctgtccg tggatcgcga ggaggagtgg 5880
gccggcttcc ccgcccatga tccagaggtg gcgctgcacg gcgacaacct ggcctatgtg
5940 atctacacct ccggctccac cggcatgccc aagggcgtgg cggtgtccca
cggtccgttg 6000 atcgcccata tcgtggctac cggcgagcgc tacgagatga
ccccggagga ctgcgagctg 6060 cacttcatgt cgttcgcctt cgacggttcc
cacgaaggct ggatgcaccc gttgatcaac 6120 ggcgcgcggg tgctgatccg
cgacgacagc ctgtggctgc cggaacggac ctacgccgag 6180 atgcatcgcc
acggggtaac ggtgggggtg ttcccgccgg tgtacctgca gcaactggcc 6240
gagcatgccg agcgcgacgg caatccgccg ccggtacggg tctattgctt cggcggcgac
6300 gcggtggcgc aggccagcta tgacctggcg tggcgggcgc tgaagccgaa
gtacctgttc 6360 aacggctacg gcccgaccga gacggtggtg acgccgctgc
tgtggaaagc acgggcgggc 6420 gatgcctgcg gcgcggccta catgccgatc
ggtacgctgc tgggcaaccg tagcggctac 6480 atcctcgacg ggcagttgaa
cctgctgccg gtaggcgtgg cgggcgaact gtacctgggc 6540 ggggaagggg
tggcgcgcgg ctacctggag cgtccggcgc tgaccgccga gcgtttcgtg 6600
ccggatccct ttggcgcgcc gggcagccgg ctgtaccgca gcggcgacct gacccgtggg
6660 cgtgcggatg gggtggtgga ctacctcgga cgggtggacc accaggtgaa
gatccgaggc 6720 ttccgcatcg aactgggaga gatcgaggcg cgcctgcgcg
agcatccgtc ggtgcgcgag 6780 gcggtggtgg tggcccagcc gggcgcggtg
ggccagcagt tggtgggcta cgtggtggcg 6840 caggcgccag cggtcgcgga
ttcgccggaa gcgcaggcgg agtgccgggc gcagttgaag 6900 acggcgctgc
gcgagcgcct gccggaatac atggtgccgt cgcacctgtt gttcctggcg 6960
cggatgccgc tgacgccgaa cggcaagctg gaccgcaagg gcctgccaca gccggatgcg
7020 agcctgttgc agcaggtcta cgtggcgccg cgaagcgatc tggagcaaca
ggtcgcgggg 7080 atctgggcgg aggtcctgca attgcaacag gtcgggctcg
acgacaactt cttcgagctt 7140 ggcggccact cgttgctggc gatccaggtg
actgcccgga tgcagagcga ggtcggcgtg 7200 gagctgccgc tggcggcgct
gttccagacc gagtcgctgc aagcctatgc cgagcttgcc 7260 gcggcgcaga
cttccagcaa tgacaccgat ttcgatgacc ttcgtgaatt catgagcgaa 7320
ctagaggcga tctga 7335 33 2556 DNA Pseudomonas aeruginosa 33
atgctttcca atccaaacct ggacctcgtg tcccgcttcg ttcgcctgcc tctggcgcag
60 cagaaattgt tctatcagcg tgtccaggcc aagggcatga gcttcgcccg
cctgccgatc 120 ccgcagactc gccaggagat ggacaacctg ccgctgtcct
atgcccaaga gcggcagtgg 180 ttcctctggc agctggagcc ggagagttcc
gcctaccaca ttcctaccgc cctgcgcctg 240 cgcggcaggt tggacattgc
gtccttgcag cgcagcttcg cggcgctcgt cgagcggcac 300 gaaagcctgc
gcacgcggat cgcgcggatg ggtgatgaat gggtgcaggt cgtctccgcc 360
gacgtctcgc tggcgctcga agtcgaagtg caacggggac tcgacgaaca gcgattgctg
420 gagcgggtcg aggcggagat cgcacgaccc ttcgatctcg aacagggacc
gttactgcgg 480 gtgactttgc tggaggtgga cgccgacgag catgtgctgg
tcatggtcca gcaccatatc 540 gtctccgacg gttggtcgat gcaattgatg
gtcgaggaac tggtccagct ctatgccgcc 600 tatagccaag ggctcgacgt
ggtgttgccg gccctgccga tccagtacgc ggactacgcc 660 ctgtggcagc
gcagctggat ggaggcgggg gaaaaggagc gccagttggc gtactggacc 720
ggcctgctgg gcggcgagca gccggtgatc gagttgcccc tcgatcaccc gcggcagccg
780 ctgcgcagct atcgtggagc gcaattggac ctggagctgg agccacacct
ggcccttgcc 840 ttgaaacagc tggttcagcg caagggtgtg accatgttca
tgctgttgct ggcttccttc 900 caggcgctgt tgcatcgcta tagcggacag
gcggatatcc gtgtcggcgt gcctatcgcc 960 aaccgtaacc gggttgaaac
cgagcggctg atcggtttct tcgtcaacac ccaggtgctc 1020 aaggccgaca
tcaatggccg gatgggtttc gacgagttgc tggcccaggc ccgccagcgc 1080
gcgctggagg cacaggctca ccaggacctg ccgttcgagc aactggtgga ggctttgcag
1140 ccggaacgca gcctcggcca caacccgttg ttccaggtca tgttcaatca
ccaggccgac 1200 tctcgttcgg ccaaccaggg cgtgcaactg ccaggcctgt
cgctggagcg gatggagtgg 1260 cggagcagct ccgtggcctt cgacctgacg
ctggacgtgc acgaggccga ggacggtatc 1320 tgggcatcgt tcggctatgc
cacggatctg ttcgaggcct cgaccgtcga gcgcctggct 1380 cggcactggc
agaatctcct gcgcggcatc gtggccgaac cgggccggcc ggtcgccgag 1440
ttgccgctgt tgctggacga ggagcgcgat tgcctgtcgc gggcctgggc agagaacgcc
1500 gacgagggtg ggttgccgcc cctggtccag ttgcagatcc aggagcaggc
ccgtctgcgt 1560 ccgcaggcgc aagcactggc gctggagggg caggccttga
gctacgccga gctcaacgcc 1620 cgcgccaatc gtctggctca ctgcctgata
gcgcgtggcg tcggtcccga tgtgctggtg 1680 ggaatcgccg tcgagcgctc
gctggacatg gtggtcggtc tgctggcgat cctcaaggcc 1740 ggtggtgcct
atgtgccgct ggacccgacc tatccgcagg accgtttgcg tcacatgctc 1800
gaggacagcg ccgtcggcct gttgctcagc caggagcatt tgctgcccgg gctgcctttg
1860 cacgaagggc tggaggtgct ctccatcgac cgcctggaac gggacgcatc
ggtgtctacg 1920 gatgatccgg tggtgaacct gcggccggag aacctggcct
atgtgatcta cacctccggc 1980 tccaccggaa aacccaaggg cgtggccatc
agccatgcgg cgcttgcgca gttctcgcgt 2040 atcgccagtg gttattccgc
gctcaccccg gaggatcgga tattgcagtt cgccaccctg 2100 agcttcgacg
gcttcgtcga acagctctat ccggcgctga cccgtggtgc ctgcgtggtc 2160
ctgcgtggcg gcgacctctg ggataccggt gagctgtatc ggcagatagt cgagcagggc
2220 gtgacccttg ccgacctgcc cacggcgtac tggaacctgt tcctgctcga
tgccctggcc 2280 gagccacggc gttcctacgg tgccttgcgg cagatccaca
tcggtggcga agccatgcca 2340 ctggaggggc cgaagctctg gcggcaagcc
ggcatgggcc gggtgaggtt gctcaatacc 2400 tatggaccga ccgaggccac
ggtggtgtcc agcgtcttcg attgttccgc cgagaacgcc 2460 cgggtgggca
atgccagtcc tatcggccag gcgctacccg gccgtacgtt gctggtgctg 2520
gatgaacatc tcggcctact gcccgtaggg cggtag 2556 34 2334 DNA
Pseudomonas aeruginosa 34 atgtcccggc cgttccggcc accactttgc
agagaaacga catcgatggg gatgcgtacc 60 gtactgaccg gcctggccgg
catgctgttg ggttcgatga tgccggtcca ggccgatatg 120 ccgcggccga
ccgggctggc cgcggatatc cgctggaccg cctatggcgt gccgcacatc 180
cgggccaagg atgagcgcgg cctgggctat ggcatcggct acgcctacgc gcgcgacaac
240 gcctgcctgc tggccgagga gatcgtcacc gcgcgcggcg agcgggcgcg
ctatttcggc 300 agcgagggca agtcgtcggc cgagctggac aacctgccgt
ccgacatctt ctacgcctgg 360 ctcaaccaac ccgaggcgct gcaagccttc
tggcaggcgc agacgcccgc ggtacgccag 420 ttgctcgaag gctacgccgc
cggtttcaac cgcttcctcc gcgaggccga cggcaagacc 480 accagttgcc
ttggccagcc ctggctgcgg gccatcgcga ccgatgacct gctgcgcctg 540
acccggcgcc tgctggtcga aggcggggtc ggccagttcg ccgacgcgct ggtggccgcc
600 gcgccgcccg gagcggagaa ggtcgccttg agcggcgagc aggcgttcca
ggtcgccgag 660 cagcggcgcc agcgcttccg cctggagcgc ggcagcaacg
ccattgccgt tggcagcgaa 720 cgttcggcgg acggcaaggg catgctcctg
gccaacccgc acttcccctg gaacggcgcg 780 atgcgtttct accagatgca
cctgaccatt cccggccggc tcgacgtgat gggggcctcg 840 ctgcccggcc
tgccggtggt caacatcggc ttcagccgcc acctggcctg gacccacacg 900
gtggatacct ccagccactt caccctgtat cgcctggcgc tggacccgaa ggacccgcgg
960 cgctacctgg tcgacggtcg ttcgctgccg ctggaggaga agtccgtcgc
gatcgaggtg 1020 cgcggcgccg acggcaagct gtcgcgcgtc gagcacaagg
tctaccagtc gatctacggc 1080 ccgctggtgg tctggcccgg caagctggac
tggaaccgca gcgaggccta tgcgctgcgt 1140 gacgccaacc tggagaacac
ccgggtactg caacagtggt actcgatcaa ccaggccagc 1200 gacgtcgccg
acctgcgccg gcgcgtcgag gcgctacagg ggatcccctg ggtcaacacc 1260
ctggccgcgg acgagcaggg caacgccctg tacatgaacc agtcggtggt gccctacctg
1320 aagccggaac tgattcccgc ctgcgccatt ccgcaactgg tcgccgaagg
cctgccggcc 1380 ctccaggggc aggacagccg ctgtgcctgg agtcgcgacc
cggccgcggc ccaggctggc 1440 atcaccccgg cggcgcaact gccggtgctg
ttgcgcaggg acttcgtgca gaactccaac 1500 gacagcgcct ggctgaccaa
cccggcgagc ccgctgcagg gcttctcgcc cctggtcagc 1560 caggagaagc
ccatcggtcc gcgggcgcgc tacgccctga gccggctaca gggcaagcag 1620
ccgctggagg cgaagacgct cgaggagatg gtcaccgcca accatgtctt cagcgccgac
1680 caggtgctgc cggacctgct ccgcctgtgc cgcgacaacc agggcgagaa
gtcccttgcc 1740 cgcgcctgcg cggccctggc gcagtgggac cgtggcgcca
acctcgacag cggcagcggc 1800 ttcgtctact tccagcgctt catgcaacgc
ttcgccgaac tcgacggcgc gtggaaggaa 1860 ccgttcgatg cgcaacgtcc
cctggatacg ccgcaaggca tcgccctcga ccggccgcag 1920 gtggcgaccc
aggtgcgcca ggcgctggcg gacgcggcgg cggaggtgga gaagagcggg 1980
attcccgacg gcgcgcgctg gggcgacctg caagtgagca cccgtggcca ggaacgcatc
2040 gcgattcccg gcggcgatgg ccatttcggg gtctacaacg cgatccagag
cgtccgcaag 2100 ggcgaccacc tggaggtggt cggcggcact agctacatcc
agctggtgac cttccccgag 2160 gaagggccca aggctcgcgg gttgctggct
ttctcccagt ccagcgatcc gcgctcgccg 2220 cactaccgcg accagaccga
gctgttttcc cgccagcaat ggcagacctt gccgttcagc 2280 gacaggcaga
tcgacgccga cccgcaactg caacggctaa gcattcgcga atga 2334 35 6390 DNA
Pseudomonas aeruginosa 35 gtgcgaggga tagccatgag tgcgtcagaa
gacctgcaat ccgctgtgca accggccgcg 60 agcgaagcgc tcgaaggatt
cccgctgtct cccttgcaga cccgcgcctg gcgccgccat 120 gccgagcggc
cggaaaatac ggttgtcggc gtgcgcctgc acgccccggc cgatcccgtg 180
gcgacgctgg agcggctgcg ccgggcgctg gacggcgagg cgcaactgcg cgtggcctac
240
cggacgatgc cgggcatgag cctgccggtg caggtactgg atgggcgcgc ggccgatctg
300 ctggtcgagc gcctgccggg agacggcgac tgggccggac gcttcgcgcg
cgaaagcgcg 360 cgtctcgccg cttcgcccct gggcggggaa ggccagccgg
tactggcgct cggcctgctg 420 ctggacgccg ccggagagac gctccagggg
ctgttgctgg cggcgccggc gttcgtcgtc 480 gatgcggcca gcctggtggc
gctgctgcgc cgcggcctgg ggccggccgg ccaggcgagc 540 gcggacgagg
gagacgaggc gctgctgttc cagcatttct ccgagtgggc caacgaggcg 600
ctggccggcg aagacggcga aagcgccagc ggttactggc gagagcaggc ggccgttgcg
660 gcggagagtc cgctggcgct ggcggacgac ctgggcgaag gcgagtggac
ggcgcggcgc 720 ctgctgccgc gcgcgctgct cgaacgcctg gccgccaacg
gcttgccgga ggcggccgcc 780 ctgctggcct ggacccaggt cgccgggcag
ttccagggcg acgagggcct cccgctggaa 840 atggcgcgac tggtctcggg
gcgcctgttc aacgagttcg ccgagctggc cggaccgttc 900 gccggggtcg
cgccgctgtg cctggagaat gtccgcgcgg gcagcgtcgg cgagcggctc 960
gacgccctcc aggcggcgat cctcgcccag gaggaggcag cggccctgcg cgatcccttt
1020 gcccccgact ggccgctcgc cgagttgggc ttcgcctggc tggcgggcga
actggatggc 1080 gccggggtgg ccgagctgga ttgccgtcag ccgccgctgg
gcgggttcct cgagttgcag 1140 gtgctgcccc acggcgaagg caggctggcc
agcctgcggg tccgtcgcga ccatgacgga 1200 acgctggccg ggcgcttgct
cgacgcctgg gtcgaatgcc tggaaagcat cgccgccgac 1260 aggcaactgc
cactggccgg gctgccgttg atcggcgcgg ccgagcgcga gcgctaccag 1320
gcctggcagg gcgagcgcgt ggagcccgcg ccggtggaat ccctggtggc cgcgttcgat
1380 ctgcgcgccg ccctgcagcc gcaggcgccg gcgttgctgg atgcccacgg
cagcctggat 1440 ttcgccacgc tgcgcgcgcg cagcgaagcg gtcgccgaag
cgctgctggc tgccggcgtg 1500 cggcccggcc aggcggtggc ggtgatgacc
gggcgcaacc gcgaggcgat cgtcgccctg 1560 ctcggggtga tgcgcgcggc
ggcggtgtac accccggtca atccggagtt tccggcggcg 1620 cgggtggagc
ggatgcgcga agcgggcggg atcgtcttcg cccttgccga tgccgagtgc 1680
gccgggcgcg cccgcgaggc cttcgccggg gcctgcctgg acctgtcgac gctgccgctt
1740 gccggcagcg gcatgagcct gccggcgccg ggcgggcgcg atgcggccta
catgatcttc 1800 acctcgggca ccagcggcca gcccaaaggc gtggtggtcg
agcacgccag cgcgctcaac 1860 ctgtcccagg ccctggcgcg cacggtatac
gcgaacgtgg tgggcgaggg cctgcgggtg 1920 acggtcaacg cgccgttctc
cttcgactcc tcgatcaagc agattctcca gttgctctcc 1980 ggccattgcc
tggtcctggt gccgcaggag gtgcgcagcg atccgcagcg gatgctgggg 2040
ttcctcgaag aacggcgcat cgacgtgctc gactgcaccc cgtcgctgtt ccgcctgctg
2100 ctccaggccg gcctcgacga tgcccacccg gcgctgcccg ggcgcatcct
ggtagggggc 2160 gagcgcttcg acgaagcgtc ctgggaggtc gccgccggct
ggcgccgctg ccaggtgttc 2220 aatctctacg gtcctaccga agccacggtg
aacgccagcc tggcgcgggt cgccgagcat 2280 gcgcggccga ccatcggccg
cgccctggcc aacgtcgatc tgcatgtggt cgatggcctc 2340 ggtcgtcgca
agacccgtgg cgccagcggc gaactgtgga tcggcggcgc cggggtggcg 2400
cgcggctatg ccggcgacgc cggcgaggcg gccgggcgct tcgtcgagga gggctggccg
2460 ggcagcggcc gcctgtaccg cagcggcgac ctggtgcgct ggcgcgccga
cggttgcctg 2520 gagttcctcg ggcggatcga cgaacaggtg aagatcaacg
gctaccgcat cgaactgggc 2580 gagatccgca gcgcgttgct ggaacacccg
gcggtgggcg aggcggcggt actcaccgac 2640 gaggccgatg cggccgaacc
gggcgcggat cgccggatcg tcgccttcgt caccgccgcc 2700 gaggagaccg
cggacgagtc ctggctggaa gtcgacctgc ccagcgggca ccgggtcgcc 2760
ggactgaacc tcaacgaaac cgagtacgtc taccaggaaa tcttcgtcga cgaggtctac
2820 agccgcgacg gcatcgtcct gccgccggac gcggtggtcc tcgacgtcgg
tgccaacatc 2880 ggcctgttct cgctgtacat cgccagccgc gcgccgcgcg
cgcgagtggt cgccttcgag 2940 ccgctggcac cgatccgccg gcgcctggag
gccaacctcg gacgctacgc accgcaggtc 3000 gaggtattcg gcatcggtct
gtccgacgcc gagcgtgagg aaaccttcac ctactatccg 3060 ggctactcga
ccttctccgg gatcgccgag tacgccgacg ccagcggcga acgcgacgtc 3120
atccgacgct acctgagcaa ccagggcgag gagggcgggg ccaacctgct gctggacaac
3180 atcgacgaaa tcctcgacga ccgcctgcgc gccgaagccc accgctgccg
cctgcgccgc 3240 ctcgaccagg tgatcggcga actgggcctg gagcgtatcg
acctgctgaa gatcgacgtg 3300 cagcgcgcgg aaatggatgt gctgctcggt
ctcgacgatg cggcgctggc caaggtccgg 3360 cagatcgtcc tggaggtcca
tgacaagcgc gacggtgcca ccgccggccg cgccgatgcc 3420 ttgagcgacc
tgctgcgccg ccatggcttc gaggtgagca tccgtcagga cgcgctgctg 3480
gagggtaccg accgttacaa ctgctacgcg gtgcgcccgg gctatgccga gtcgctggct
3540 gagcgcatcg actggcgcgc gctcgcgccg cgccccgccg cggccctcgg
cggcgagctg 3600 agcgagcagg ccctgcgtgg cttcctcgag gcgcgcctgc
cggcctacat gctgccgagc 3660 cggatcgccc gggtcgaacg cctgccgctg
accgccgaag gcaagctcga ccgtcgcgcg 3720 ctgttggcgg cgctggccgc
cgaggcggcc gcgcagaccc tggaagcgcc ggccaatgcc 3780 accgaggcgg
ccctgctgga gatctggaag agcgtgctga aacgcccggc gatcggcgtc 3840
agcgacaatt tcttccaggt cggcggcgac tccatccgcc tgatccagat gcaggtcatg
3900 gcgcgcgagg cggggcttgc ctttaccctg cgcgacgtgt tcaaccacca
gagcatccgc 3960 gaactggcgc gcctgctggc cgctccggcg agtccggcgg
atgcgctcgg gacctcggcg 4020 ccgcagtcgc tggagccgtt cgccctgttg
tcggcggcgg aacgcaagcg cctgccggag 4080 gggctcgacg acgcctatcc
gatgaccagc ctgcaacagg gcatgctcct gcaaagcgag 4140 gccagcggcg
atccacggct gttgcacaac gtcgtcctgc acgaggtgca tggacgcctg 4200
gacggcgagt tgctggcgcg cgcctgggcg atcctgatcg gccgccacgc gatcctgcgt
4260 accggcttcg atctgcacgg tggccaggtt cccctgcaat gggtccaccc
ggccacggcg 4320 gtcgccgccg aggtgccggt gcacgacctg tgtggcctcg
atggggaaac acggcgcctg 4380 cgcctgcgtg cctggatcga ggaagagcag
gccaccccgt tcgactggag ccgcccaccg 4440 ctggtgcgcc tcgccgcgct
ggcgctggac gagcggcgct tcgccctggg cgtcgccgaa 4500 caccatagcg
tgctggacgg ctggagcctg caaagcctgg tggacgagct gctggcggtc 4560
tacgccgacc ttctcgccgg tgtcgtcgcg cgggaagcgg aagcgcccgc ggtaggcttc
4620 cgcgactacg tggcgctgga gcgtgaggcc gaggccaacg ccgcctcggc
gctgttctgg 4680 ctcgactacc tggccggcgc ccgctaccgg ccgttgcccg
gcctggcgga ggagggaccc 4740 cggcgcatgg cggcggtccg cgtggacgtg
ccggccgaca gcctgtcgcg cttgcgcgcc 4800 ctggccgaac gcagcggctt
gcccttgcgt tcgttgttgc tggcggcgca tggccgagcg 4860 ttgtgccgct
tcagcgatgc cgatgaagta gtcaccggct tcgtcagcca cgggcgcccc 4920
gaggagccgg gagcggaccg cctgctcggc ctgttcctga acaccctgcc gtgccggctg
4980 tcggcttccg tcgatctgct cgacagcgcc cgtcgcgcat tcgactacga
gcgcgcgagc 5040 ctggaacatc ggcgccatcc gctggcggcg attcgcaggc
gcaaccgcga gttgcgcctg 5100 gacagcctgt tcaacttcgt cgacttccac
caggacgacg ccgcgccggc gggagtaagg 5160 cacggcggca tcctcgacca
ggtggtggtg gacgtcgacg tgccgctggc ggtggacttc 5220 gaggtggccg
gcgagcgcct cgaggtgggc ttccagtatg ccgccggacg tttccccgcc 5280
gagcgcgccg aggcactggc cggcgcctac cgcgaggcgt tgctggcgct gctcggagac
5340 ccggtgcagc cgcccgcggc ggcccaggcc gaggacagcg tggagctgcg
gcgggtgctc 5400 aaggtgttgt cccgggtgct cggccggccg ctggcggccg
accagggctt cgccagcgcc 5460 ggcgggcatt cgctgctggg cgtgcaggcg
atcgccgaat tgcgccggct gaccggcagg 5520 caactgagcc tggggctgtt
gcagggcgat ccggatgccc gcgaagtggt gcgccgctgc 5580 catgccgccg
acgcgccgcc gttgccgccc gccaccgagc gcgcccgggc cctgtggttg 5640
cagcgcagcg ggagcgcgca gccgcgcctg cgcctgatcg cgctgccgcc cgcgggcggc
5700 aacgccggca ctttccgtgg ttgggacgcg cgcctgccgg cggacgtgga
gctgctggcg 5760 atccagtatc cggggcgcca ggaacgccag gacgagccat
tcgtcaccga tgtagaggcc 5820 atgctctgtg ccatcgacga cgcgctcctg
ccattgctcg accgtccgtt cgccctgatc 5880 ggcgccagcc tcggcggcat
gctcgcctac gaactggcgg cacgcctgga aagcctgcac 5940 ggcctgcgcg
ccaggcagtt gttcgtgatc agcagccgcg ctccggggcc ggacctggaa 6000
tacccgcgct tccatgcgat gggcgacgcc gagttgctgc gaaccctgcg cgagtacgac
6060 gtgctgccgc tggaagtgct cgacgacccg gagctgcgcg agatcagcct
ggccaccctg 6120 cgcgccgatt cgcgcctggc cgccgactat cgctaccgcc
cgcgcgagcc gctggccata 6180 ccgatcaccg cgatcctcgg cgagcaggac
ccgggcgtct ccagggtggc catcgacggc 6240 tggcggcggc acgccagccg
ctacgagctg gagaccctgg ccggcggcca cggcctggtg 6300 gtgacggcgg
cggaggaggt ctgcgcgatc ctgcggcagc gcctggcgcc cgatgtgcct 6360
ggcggcgtgc cggcgaacct ggcaacctga 6390 36 1395 DNA Pseudomonas
aeruginosa 36 atgaacctgc gcccggtgat cgtcggcggc ggctcggccg
gcatggccgc agccatcgag 60 ctggccaggc gcggggtccc ctgcgtcctc
ttcgacgagg cctcgcgtcc cggcggggtg 120 gtctatcgcg gccccttgcg
ggccggcgtc gatccggcct acctcggcgc gcgctacacc 180 cggatgctgg
aaaaactgcg gcgcgatttc tccgcctgcg ccgggcacat cgacctgcgc 240
ctgaacagcc gcgtggtcgg tggcgacggc cagcgcctga tggtcctcga cgaggcggaa
300 cggctgcacg aggtggagta ttcgcacctg ctcctggcca ccggctgcca
tgagcgcagc 360 gtgccgtttc ccggctggac cctgcccggg gtgatgctcc
tcggcggcct gcaattgcag 420 atcaagagcg gcgtggtgaa gcccctgggc
gataccctga tcgccggcag cggcccgctg 480 ctgccactgg tggcctgcca
gctgcatgcg gccggggtac gtgtcgccgg ggtctacgag 540 gcctgcgcgt
tcggccgcat ggccagggaa agcctggcgc tgctgaacaa gccgcaactg 600
ttcctcgacg gcctgagcat gctcggctat ctcaagctca acggcattcc gctgcactat
660 ggctggggcg tggtggaggc cagcggcgat ggggaactga cggaagtgac
ggtagcgccc 720 tacgacgaag agtggcggcc cgacctggaa aacgcgcgac
cggtgaaggc cagcaccctg 780 gcggtcggct atggcttcat cccgcgcacc
cagctcagcc agcagttggg tctggagcac 840 ggcttcagcg acgacggata
cctgcgcgcg gaatgcaacg tctggcagca gagcagccaa 900 ccgcacatcc
acctggccgg cgacatggcg ggtatccgcg gcggcgaggc ggcgatgatc 960
ggcgggcgca tcgcggcctt gtcgatcctc ctgcaacgcg aggccatcgc gcccgccgag
1020 gccatcgaac gccgagaatc ccatctcgcc cgcctggagg cgatcaagcg
cttccgcgcc 1080 ggagtcgagc gctacaccca gcgcggcgcc cgccaggtcg
aactggcgcg ggccgatacg 1140 gtgatctgcc gctgcgaaca ggtcacccgt
ggcgacatcg agcgcgcgct cgaacagggc 1200 gtgcaggaca tcgccgggct
gaagatgcgc acccgcgccg gcatgggcga ctgccagggg 1260 cggatgtgca
tcggctactg cagcgatcgc ctgcgccgtg ccaccggacg ccacgacgtc 1320
ggctggctgc ggccgcgttt cccgatcgat ccgatcccgt tttccgcatt ccagaacctc
1380 ggtacggaag cctga 1395 37 801 DNA Pseudomonas aeruginosa 37
atggcgtccg cccctaagga agaggagata aatatgattt attacttgat cggagtggcg
60 ctattcatct tcatgctgga acagttggtt cccggctgga aattgcccaa
ggtgagcacc 120 tgggtggccc gggtgatctt cctcaacatc gtccaggtgt
cgatcgccct gctcgccggc 180 atcacctgga acaaatggat gatggggcac
agcctgctgc acacctcgga tgccctgcca 240 ccactgctgg ccggcttcgc
cgcctacttc gtcaacacct tcgtcaccta ctggtggcat 300 cgcgcgcgcc
acgccaacga cacgctctgg cggctgttcc accagttgca ccacgcgccg 360
caacgcatcg aggtattcac ctccttctac aagcatccga ccgagatggt cttcaactcg
420 ctgctgggca gcttcgtcgc ctacgtggtg atgggcatca gcatcgaggc
cggcgcctac 480 tacatcatgt tcgccgcgct cggcgagatg ttctaccact
cgaacctgcg caccccgcac 540 gtcctcggct acctgttcca gcgcccggag
atgcaccgca tccaccacca gcgcgaccgt 600 cacgagtgca actacagcga
cttcccgatc tgggacatgt tgttcggcac ctacgagaac 660 ccccgccgca
tcgacgagcc gcagggcttc gccggcgaca aggagcagca gttcgtcgac 720
atgctgctgt tccgcgacgt gcacagcctc cccggaaaaa cccagcccgc tcccgtcctg
780 gtcaagcccg acgtcaggtg a 801 38 20 DNA Pseudomonas aeruginosa 38
acctgcccgg aagggcaggt 20 39 468 DNA Pseudomonas aeruginosa 39
ggggtacctg gcacctacca gatcgtgtag ttgagccggt acgagcgttc tgtgttttat
60 gcaatccaca tcagcgacca gggatgctgg ctatttgaaa cacttcacgg
aatgacgctg 120 aaagtcttcg cgacctcgtc tgtcgcacct taacgaaagc
attgcgaatc cattaccgac 180 aggtttccaa aagaaacccg ggatgaaact
cctattgcct ttcgaaaatt ggaaacgaca 240 ggcgaacata tgtaacgcga
aatttcaccc tacgtataaa caatgcgccc agcgaatatc 300 gctcccttac
cgagcgacga actcctgcgc gccagcgaat aaccgatgcc gcgagggaaa 360
agtttctccg gcatacctgg agagccctct cggaggcggc gcatgaacgg tcagcggtac
420 agggaaacac ccctcgacat cgagccgtct gcggcgcctt ctagagca 468
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References