School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK1
Author for correspondence: Simon F. Park. Tel: +44 1483 879024. Fax: +44 1483 300374. e-mail: s.park{at}surrey.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: LuxS, autoinducer-2, camplylobacters
Abbreviations: AI-1/2, autoinducer-1/2; HSL, homoserine lactone
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C. jejuni is associated with many domesticated animals and birds, and consequently four common sources, poultry, raw milk, water and pets, account for nearly all cases of human infection. Although the ability of campylobacters to survive in food and the environment is primarily important to their infective and contamination cycle, little is known of the adaptive survival responses that these organisms are able to initiate in response to exposure to environmental stress. Information contained within the recently completed genome sequence for C. jejuni NCTC 11168 (Parkhill et al., 2000 ), however, has provided some important insights into the physiology of this pathogen. Most notably, it has become apparent that, whilst C. jejuni can survive in a diverse range of environments, its capacity for regulating gene expression in response to environmental stress appears to be very limited compared to other bacteria, including Escherichia coli and Bacillus subtilis (Park, 2000
).
Bacteria differ in their response to environmental stress, but all have at least some capacity for monitoring the environment for changes, which require an adaptive response in order to survive. In the past decade, it has become apparent that many bacterial species are able to regulate a wide variety of traits in response to cell density. Cellcell communication, or quorum sensing, involves the synthesis, secretion and detection of extracellular signalling molecules termed autoinducers (Bassler, 1999 ). When these molecules reach a critical threshold concentration within a population, a signal transduction cascade is triggered, which forms the basis for alterations in gene expression.
Quorum sensing in many Gram-negative bacteria is based upon the signalling molecule homoserine lactone (HSL) which controls the expression of numerous traits including bioluminescence, antibiotic production and virulence factor production (Fuqua & Greenberg, 1998 ; De Kievit & Iglewski, 2000
; Bassler, 1999
). In Pseudomonas aeruginosa, for example, two HSLs control the expression of a number of extracellular virulence factors (Pearson et al., 1994
) and are also involved in biofilm differentiation (Davies et al., 1998
). P. aeruginosa has also been shown to produce two other types of signalling molecule, a quinolone signalling molecule that regulates the expression of the virulence gene lasB (Pesci et al., 1999
; McKnight et al., 2000
) and diketopiperazines, which at high concentrations are able to cross-activate HSL LuxR-based quorum-sensing systems (Holden et al., 1999
). Gram-positive bacteria generally secrete processed peptide signalling molecules, which act via a membrane-bound histidine protein kinase (Lazazzera & Grossman, 1998
; De Kievit & Iglewski, 2000
; Bassler, 1999
; Mayville et al., 1999
). In Vibrio harveyi, another quorum-sensing system has recently been identified that produces the signalling molecule autoinducer-2 (AI-2). This system is highly conserved in both Gram-positive (Kuroda et al., 2001
) and Gram-negative bacteria and thought to be used for interspecies communication (Bassler, 1999
). The chemical structure of the cognate signalling molecule AI-2 has recently been predicted as a furanone (Schauder et al., 2001
) and the luxS gene codes the final enzyme in the biosynthetic pathway for its production. AI-2 is detected by a sensory histidine kinase located within the cytoplasmic membrane (Surette et al., 1999
).
Several species, including E. coli (Sperandio et al., 1999 ), Salmonella typhimurium (Surette & Bassler, 1999
), Shigella flexneri (Day & Maurelli, 2001
), Helicobacter pylori (Forsyth & Cover, 2000
; Joyce et al., 2000
) and Vibrio vulnificus (McDougald et al., 2001
) have been shown to produce an AI-2-like activity. This study demonstrates luxS-dependent production of an extracellular signalling molecule by C. jejuni with AI-2-like activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of C. jejuni luxS mutant.
Genomic DNA was isolated from C. jejuni using guanidium thiocyanate (Pitcher et al., 1989 ). The putative luxS from C. jejuni NCTC 11168 (accession no. AL111168; Parkhill et al., 2000
) was amplified from genomic DNA using PCR and the oligonucleotide primers LUX1 5'-AGGCAAAGCTCCTGGTAAGGCCAA-3' and LUX2 5'-GGATCCGTATAGGTAAGTTCATTTTTGCTCC-3'. The PCR reaction contained 0·5 µmol each primer, Taq DNA polymerase (Qiagen) and dNTPs (Roche; 200 µmol). PCR reactions were performed using a Perkin-Elmer GeneAmp PCR system 2400 thermal cycler. Following an initial denaturation for 3 min at 94 °C, there were 30 cycles of amplification comprising 30 s denaturation at 94 °C, 60 s annealing at 55 °C and a 60 s extension at 72 °C. There was a final extension for 10 min at 72 °C. The 1080 bp DNA fragment generated in this manner was cloned into pBAD-TOPO TA cloning vector (Invitrogen) and transformed into E. coli DH5
. The resulting plasmid was designated pKE25 and in this vector the putative luxS gene was orientated so that its transcription could be initiated from the inducible araB promoter.
The luxS gene was mutated using the inverse PCR protocol described by Wren et al. (1993) . This was used to introduce a unique BglII restriction site and to delete 50 bp of the gene. Oligonucleotide primers LUX3 5'-caacaAGATCTCCCGTGCGACAACCCATAGGTGAA-3' and LUX4 5'-caacaAGATCTCTTGGGAAGCAGCCATGAAAGATG-3' with unique restriction sites for BglII (underlined) and clamp sequences, not complementary to C. jejuni DNA (lower case) were used. In this PCR, a two-stage cycling programme was used to amplify longer DNA fragments. The first stage comprised 10 cycles, each including 10 s denaturation at 94 °C, 60 s annealing at 58 °C and a 5 min 15 s extension at 68 °C. The following stage comprised a further 25 cycles, each including 10 s denaturation at 94 °C, 60 s annealing at 58 °C and a 5 min 15 s extension at 68 °C with 10 s added for each cycle number. The resulting PCR fragment was digested with BglII, self-ligated and transformed into E. coli DH5
. The resulting plasmid, pKE27, was next digested with BglII and a kanamycin resistance cassette with BamHI ends, from pJMK30 (J. Ketley, University of Leicester), inserted into this site. This step generated the suicide plasmid pKE28, which was introduced into C. jejuni NCTC 11168 competent cells by electroporation at 2·5 kV, 200
and 25 µF. Three luxS mutants generated by a double homologous recombination event were designated CJLUXS0103.
DNADNA hybridizations.
These were carried out using a non-radioactive AlkPhos direct labelling and detection system (Amersham Pharmacia Biotech). Chromosomal DNA was isolated from kanamycin-resistant transformants and wild-type cells, and digested with the restriction endonuclease XmnI. The resulting fragments were separated on a 0·8% agarose gel. The DNA was depurinated (0·125 M HCl), denatured (1·5 M sodium chloride/0·5 M sodium hydroxide) and transferred to Hybond-N+ nitrocellulose membranes using an alkali transfer procedure (Amersham Pharmacia Biotech). A probe generated by PCR using primers LUX1 and LUX2 with genomic DNA as template was labelled with thermostable alkaline phosphatase and used to detect homology according to the manufacturers instructions.
Preparation of cell-free culture medium for AI-2 assays.
Cells of C. jejuni were grown to confluence on M-H agar. After 16 h growth, the cultures were harvested in 1 ml AB medium per plate and the OD600 of each cell suspension was adjusted to 0·4 with AB medium using a Helios Alpha spectrophotometer (Unicam) and cuvettes with a 1 cm path length. Cell-free culture medium was prepared from these suspensions by centrifugation for 15 min at 8000 g, followed by filtration of the supernatant through a Minisart 0·2 µm single-use filter unit (Sartorius). Cell-free culture media from V. harveyi strains, E. coli DH5 containing pBAD-TOPO, and E. coli DH5
expressing the C. jejuni luxS gene were prepared in the same manner, except that the cells were cultured aerobically in AB medium at 30 °C and 37 °C respectively.
AI-2 bioluminescence assay.
The AI-2 reporter strain, V. harveyi BB170, was grown for 16 h with aeration (175 r.p.m.) at 30 °C in AB medium, and then diluted 1:5000 in fresh AB medium. Cell-free preparations were then added to the diluted V. harveyi culture at a 10% (v/v) final concentration. The flasks were incubated at 30 °C with aeration (175 r.p.m.) and aliquots of 100 µl were removed hourly for total luminescence measurements using Lumac/3M biocounter 2010A. Growth of the reporter strain was also measured by OD600 and by viable cell counts with appropriate dilution.
Growth and survival of C. jejuni.
Bacterial suspensions of C. jejuni NCTC 11168 and CJLUXS01 were prepared in M-H broth from confluent lawns and adjusted to an OD600 of 0·3. Flasks containing 50 ml M-H broth were inoculated with 100 µl cell suspension and incubated at 37 °C microaerobically and aerobically at 125 and 250 r.p.m., respectively. Growth and survival of the cultures was monitored by viable counts.
Motility assays and resistance to oxidative stress.
Motility assays were performed at 37 °C on 0·4% agar plates containing M-H broth. The motility haloes were measured after 24 h. Growth inhibition of C. jejuni NCTC 11168 and CJLUXS01 due to paraquat (Sigma) and hydrogen peroxide (Sigma) was monitored using well diffusion susceptibility assays. Cell suspensions (100 µl) were spread onto M-H plates and 6 mm wells cut into the agar were filled with 30 µl of different concentrations of paraquat (140 mM) and hydrogen peroxide (0·30·015%, v/v). Plates were incubated microaerobically at 37 °C for 24 h. Zones of inhibition were then measured.
Production of C. jejuni signalling molecule at different growth phases.
To examine the kinetics of autoinducer production in C. jejuni NCTC 11168 and CJLUXS01, cell-free culture media were prepared at time points reflecting all phases of growth from broth cultures. Cultures were grown microaerobically at 37 °C at 125 r.p.m. At 5, 18, 28 and 42 h, 4 ml samples were removed from the flasks and used to prepare cell-free culture media (described above) and assessed for viable counts. The AI-2 bioassay was carried out as described above.
Mammalian cell culture.
Stock cultures of Caco-2 cells were obtained from the European Collection of Cell Cultures (ECACC), Porton Down, UK. They were grown as monolayers in Minimal Essential Medium Eagle (MEM; Sigma) with 10% (v/v) foetal bovine serum (Gibco-BRL), 1% (v/v) 200 mM glutamine (Gibco-BRL) and 1 % (v/v) non-essential amino acids (Sigma). No antibiotics were added. The cultures were incubated in a CO2 5% (v/v) incubator at 37 °C. For experimental assays, cultures were harvested by trypsinization and seeded into 6-well tissue culture trays at 3·8x105 cells per well and incubated 24 h in a CO2 incubator at 37 °C.
Adherence and invasion assay.
Bacterial suspensions of C. jejuni NCTC 11168 and CJLUXS01 were prepared in M-H broth from confluent plates and adjusted to OD600 0·3. The suspensions were centrifuged at 3000 r.p.m. for 15 min at 15 °C. The pellet was resuspended in MEM and viable counts were carried out on M-H agar. Caco-2 cell cultures in 6-well plates were inoculated with 7·8x108 c.f.u. C. jejuni NCTC 11168 or CJLUXS01 in 0·5 ml; 12 wells in total were inoculated for each strain. The plates were incubated at 37 °C in 5% CO2 for 3 h to allow the bacteria to adhere to and invade the epithelial cells. The monolayers were washed three times with pre-warmed Hanks Balanced Salt Solution (HBSS; Sigma) and then six of the wells for each strain were incubated for 1·5 h in 2 ml pre-warmed MEM with 100 µg gentamicin sulphate ml-1 (Sigma). In the remaining wells only MEM (2 ml) was added. C. jejuni NCTC 11168 and CJLUXS01 were shown to be sensitive to gentamicin with no viable cells recovered after 15 min exposure to the antibiotic (data not shown). The monolayers were washed three times with HBSS and lysed with 0·1% (v/v) Triton X-100 in PBS (Sigma). The lysed monolayer suspensions were serially diluted and spread on M-H agar for viable counts of C. jejuni.
Statistical analysis.
Comparisons between C. jejuni NCTC 11168 and CJLUXS01 in adherence and invasion of Caco-2 cells and the motility assays were analysed using the unpaired MannWhitney two sample test using Minitab. A P value of <0·05 was considered to be statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Production of the C. jejuni signalling molecule at different growth phases
The V. harveyi BB170 reporter was used to monitor production of the signalling molecule at different stages of growth of C. jejuni (Fig. 4). Maximal autoinducer activity was detected in cell-free conditioned medium after 18 h growth and remained at this level for at least 42 h. As expected, cell-free conditioned medium taken from CJLUXS01 did not stimulate luminescence in the reporter system for any growth phase (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pattern of AI-2 production with respect to growth phase and changes in environment may give some insight into the function of this signalling system. In this context, the growth-phase-dependent production of this molecule appears to vary with species. Accordingly, whilst the concentration of this signalling molecule is maximal in late-log or early-stationary-phase cultures in Sal. typhimurium, E. coli (Surette & Bassler, 1998 ; Surette et al., 1999
), V. vulnificus (McDougald et al., 2001
) and Shi. flexneri (Day & Maurelli, 2001
), in H. pylori maximal production was shown in the early-log to mid-exponential phase and activity was diminished in stationary phase (Forsyth & Cover, 2000
; Joyce et al., 2000
). AI-2 production is also responsive to perturbations in the metabolic activity of cells brought about by stress (DeLisa et al., 2001
). In C. jejuni NCTC 11168, maximal AI-2 production was induced in early-stationary-phase cultures and this activity remained at this elevated level for at least 42 h, suggesting that the C. jejuni signalling molecule is not degraded up to this point.
The systems used by bacteria to signal and sense their population density as well as the population density of other bacteria appear highly conserved. The responses to these environmental cues, however, are varied. Phenotypic responses not directly associated with pathogenesis include induction of bioluminescence in V. harveyi (Surette et al., 1999 ), competence and sporulation in B. subtilis (Lazazzera & Grossman, 1998
), biofilm formation by P. aeruginosa (Davies et al., 1998
; De Kievit et al., 2001
) and starvation survival in V. vulnificus (McDougald et al., 2001
). In pathogenesis, quorum sensing has been shown to regulate virulence factors (Parsek & Greenberg, 2000
; De Kievit & Iglewski, 2000
). Whilst the production of an AI-2 signalling molecule has been experimentally determined in a number of bacteria (Surette et al., 1999
; Joyce et al., 2000
; Day & Maurelli, 2001
; McDougald et al., 2001
), and now C. jejuni, many features of the AI-2 family of quorum-sensing systems are not yet completely understood (Surette et al., 1999
). The structure of the AI-2 molecule has recently been predicted as a furanone and is produced by three enzymic steps from S-adenosylmethionine, the final step being catalysed by the luxS gene product to give 4,5-dihydroxy-2,3-pentanedione which cyclizes into a furanone ring (Schauder et al., 2001
).
Whilst the function of the LuxS signalling systems remains unclear, some insights into its function have recently become apparent. In this context, LuxS has been shown to regulate the locus of enterocyte effacement (LEE) operon in enterohaemorrhagic E. coli O157:H7 and enterpathogenic E. coli (Sperandio et al., 1999 ) and AI-2 activity has also been shown to be responsible for the late-exponential-phase peak of expression of VirB, a transcription factor essential for the expression of invasion loci in Shigella flexneri (Day & Maurelli, 2001
). Inactivation of LuxS in the Gram-positive pathogen Streptococcus pyogenes and analysis of resulting mutants also suggested a role for LuxS in modulation of virulence during infection (Lyon et al., 2001
). However, a luxS-deficient mutant of H. pylori had comparable growth, motility, urease and vacuolating cytotoxin activity in comparison to the wild-type (Forsyth & Cover, 2000
; Joyce et al., 2000
). Quorum sensing in P. aeruginosa has been shown to contribute to the regulation of genes for relieving oxidative stress (Hassett et al., 1999
). P. aeruginosa mutants devoid of one or both HSL signalling molecules were more sensitive to hydrogen peroxide than the parental strain. The luxS mutant of C. jejuni NCTC 11168 showed comparable growth rate, resistance to oxidative stress and ability to invade Caco-2 cell monolayers to the parental strain. The invasiveness of C. jejuni NCTC 11168 in Caco-2 cell monolayers was comparable to published data for clinical C. jejuni isolates (Everest et al., 1992
; Harvey et al., 1999
). The CJLUXS01 mutant did, however, consistently give rise to smaller motility haloes than the parental strain, suggesting a role for quorum sensing in the regulation of motility. A similar role has been proposed for E. coli O157 since luxS mutants of this strain also showed smaller motility haloes (Sperandio et al., 2001
).
C. jejuni clearly uses a AI-2-based cell-signalling system but the function of this system is not yet fully known. This study has shown that it plays a role in motility; however, it is likely that this system also serves as a global regulatory mechanism for basic physiological functions and possibly virulence factors, as is the case in other bacterial pathogens.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bassler, B. (1999). How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol 2, 582-587.[Medline]
Bassler, B. L., Wright, M. & Silverman, M. R. (1994). Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13, 273-286.[Medline]
Bassler, B. L., Greenberg, E. P. & Stevens, A. M. (1997). Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179, 4043-4045.[Abstract]
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295-298.
Day, W. A. & Maurelli, A. T. (2001). Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence. Infect Immun 69, 15-23.
De Kievit, T. R. & Iglewski, B. H. (2000). Bacterial quorum sensing in pathogenic relationships. Infect Immun 68, 4839-4849.
De Kievit, T. R., Gillis, R., Marx, S., Brown, C. & Iglewski, B. H. (2001). Quorum-sensing in Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl Environ Microbiol 67, 1865-1873.
DeLisa, M. P., Valdes, J. J. & Bentley, W. E. (2001). Mapping stress-induced changes in autoinducer AI-2 production in chemostat-cultivated Escherichia coli K-12. J Bacteriol 183, 2918-2928.
Everest, P. H., Goossens, H., Butzler, J.-P., Lloyd, D., Knutton, S., Ketley, J. M. & Williams, P. H. (1992). Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli. J Med Microbiol 37, 319-325.[Abstract]
Forsyth, M. H. & Cover, T. L. (2000). Intercellular communication in Helicobacter pylori: LuxS is essential for the production of an extracellular signaling molecule. Infect Immun 68, 3193-3199.
Fuqua, C. & Greenberg, E. P. (1998). Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr Opin Microbiol 1, 183-189.[Medline]
Greenberg, E. P., Hastings, J. W. & Ulitzur, S. (1979). Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria. Arch Microbiol 120, 87-91.
Harvey, P., Battle, T. & Leach, S. (1999). Different invasion phenotypes of Campylobacter isolates in Caco-2 cell monolayers. J Med Microbiol 48, 461-469.[Abstract]
Hassett, D. J., Ma, J. F., Elkins, J. G. & 10 other authors (1999). Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol Microbiol 34, 10821093.[Medline]
Holden, M. T. G., Chhabra, S. R., de Nys, R. & 14 other authors (1999). Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria. Mol Microbiol 33, 12541266.[Medline]
Joyce, E. A., Bassler, B. L. & Wright, A. (2000). Evidence for a signalling system in Helicobacter pylori: detection of a luxS-encoded autoinducer. J Bacteriol 182, 3638-3643.
Kuroda, M., Ohta, T., Uchiyama, I. & 34 other authors (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 12251240.
Lazazzera, B. A. & Grossman, A. D. (1998). The ins and outs of peptide signalling. Trends Microbiol 6, 288-294.[Medline]
Lyon, W. R., Madden, J. C., Levin, J. C., Stein, J. L. & Caparon, M. G. (2001). Mutation of luxS affects growth and virulence factor expression in Streptococcus pyogenes. Mol Microbiol 42, 145-157.[Medline]
Mayville, P., Ji, G., Beavis, R., Yang, H., Goger, M., Novick, R. P. & Muir, T. W. (1999). Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc Natl Acad Sci USA 96, 1218-1223.
McDougald, D., Rice, S. A. & Kjelleberg, S. (2001). SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus. J Bacteriol 183, 758-763.
McKnight, S. L., Iglewski, B. H. & Pesci, E. C. (2000). The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. J Bacteriol 182, 2702-2708.
Nachamkin, I., Allos, B. M. & Ho, T. (1998). Campylobacter species and Guillain-Barré syndrome. Clin Microbiol Rev 11, 555-567.
Park, S. F. (2000). Environmental regulatory genes. In Campylobacter , pp. 423-440. Edited by I. Nachamkin & M. J. Blaser. Washington, DC:American Society for Microbiology.
Parkhill, J., Wren, B. W., Mungall, K. & 18 other authors (2000). The genome sequence of the foodborne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665668.[Medline]
Parsek, M. R. & Greenberg, E. P. (2000). Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: a signalling mechanism involved in associations with higher organisms. Proc Natl Acad Sci USA 97, 8789-8793.
Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H. & Greenberg, E. P. (1994). Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91, 197-201.[Abstract]
Pesci, E. C., Milbank, J. B. J., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P. & Iglewski, B. H. (1999). Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 96, 11229-11234.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151-156.
Schauder, S., Shokat, K., Surette, M. G. & Bassler, B. (2001). The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol Microbiol 41, 463-476.[Medline]
Sperandio, V., Mellies, J. L., Nguyen, W., Shin, S. & Kaper, J. B. (1999). Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli. Proc Natl Acad Sci USA 96, 15196-15201.
Sperandio, V., Torres, A. G., Girón, J. A. & Kaper, J. B. (2001). Quorum sensing is a global regulatory mechanism in enterohemorrhagic Escherichia coli O157:H7. J Bacteriol 183, 5187-5197.
Surette, M. G. & Bassler, B. L. (1998). Quorum sensing in Escherichia coli and Salmonella typhimurium. Proc Natl Acad Sci USA 95, 7046-7050.
Surette, M. G. & Bassler, B. L. (1999). Regulation of autoinducer production in Salmonella typhimurium. Mol Microbiol 31, 585-595.[Medline]
Surette, M. G., Miller, M. B. & Bassler, B. L. (1999). Quorum sensing in Escherichia coli, Salmonella typhimurium and Vibrio harveyi: a new family of genes responsible for autoinducer production. Proc Natl Acad Sci USA 96, 1639-1644.
Wren, B. W., Henderson, J. & Ketley, J. M. (1993). A PCR-based strategy for the rapid construction of defined bacterial deletion mutants. Biotechniques 16, 994-996.
Received 3 January 2002;
accepted 16 January 2002.