Identification of a Quorum-Sensing Signal Molecule in the Facultative Intracellular Pathogen <em>Brucella melitensis</em>

Bernard Taminiau

Mavis Daykin

Simon Swift

Maria Boschiroli

Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et Microbiologie, Facultés Universitaires Notre-Dame de la Paix, 5000 Namur, Belgium,^{School of Pharmaceutical Sciences, University Park, University of Nottingham, Nottingham NG7 2RD,}^{Institute of Infections and Immunity, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom,}^{INSERM U431, Faculté de Médecine, 30900 Nîmes, France}⁴

^{Corresponding author. Mailing address: Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et Microbiologie Facultés, Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, 5000 Namur, Belgium. Phone: 3281724409. Fax: 3281724420. E-mail:}eb.ca.pdnuf@uainimaT.dranreB.

Received 2001 Nov 8; Revised 2002 Jan 15; Accepted 2002 Mar 21.

Abstract

Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

Abstract

Brucella is the causative agent of brucellosis, a widely distributed zoonosis affecting a broad range of mammals and causing a chronic undulant fever in humans (reviewed by Smith and Ficht [53]). Brucellosis remains a health problem and a source of major economic losses in developing countries, where it is endemic. Brucella are nonmotile gram-negative bacteria and facultative intracellular pathogens that are able to invade and replicate in macrophages and nonprofessional phagocytes (12). In HeLa cells, virulent Brucella cells alter the intracellular trafficking of membrane-bound intracellular compartments to create a novel vacuolar niche in which they replicate (41). The mechanisms and the factors involved in this process are not well understood, with knowledge about the interactions between Brucella and the mammalian cell being mainly descriptive.

Molecular genetic analyses have identified several types of factors that are important for Brucella-host cell interactions: structural components like lipopolysaccharide (1, 16, 32) and cyclic β(1-2) glucan (19), stress response proteins like DnaK (23) and Hfq, a homologue of Escherichia coli host factor I (HF-I) (43), and also a type IV secretion system (14, 34, 51). The VirB type IV secretion system is homologous to the VirB system of Agrobacterium tumefaciens and to the Ptl system of Bordetella pertussis (4). DNA sequence analysis of the Brucella suis virB operon has revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins encoded by the pTi plasmid of Agrobacterium and a 12th ORF encoding a putative lipoprotein (34). Independent mutants of virB5, virB9, or virB10 are highly attenuated in an in vitro infection model of human macrophages (34). Two recent signature-tagged mutagenesis studies have confirmed the requirement of virB2, virB4, and virB8 for survival within macrophages and HeLa cells (14) and of virB1 and virB10 in the early stages of chronic infection in BALB/c mice (18).

The process of infection requires a complex and multifactorial regulation of virulence factors. One of the most abundant types of virulence regulatory system is the two-component family of signal transducers (7). Two-component regulatory systems that have recently been described are involved in the virulence of Brucella: the BvrR/BvrS system is critical for the intracellular growth of Brucella abortus (54), and a second system, homologous to VsrB/VsrC of Ralstonia solanacearum, has been identified in Brucella melitensis in a signature-tagged mutagenesis screening in the mouse infection model (27). If two-component regulation systems allow each single bacterium within the population to react in face of environmental changes, other systems, such as the quorum sensing, respond to signals produced by the bacteria themselves and control virulence factors at the whole-population level.

Quorum sensing (QS) is a regulatory system for controlling gene expression in response to increasing cell density (reviewed in references 13, 15, 58, and 66). This cell-to-cell signaling is achieved through the production and release of a small signaling molecule or pheromone. With the growth of the bacterial population, there is an accumulation of this pheromone until a threshold concentration is reached, indicating that a quorum of bacteria is present. At this point, the pheromone activates a transcriptional regulator, leading to the activation or, in some cases, the repression of target genes (49, 55, 64, 65, 70). In a large number of gram-negative bacterial species, the signal molecule is an N-acyl homoserine lactone (acyl-HSL). In most cases, the acyl-HSL synthase is a member of the LuxI family and the response regulator is a member of the LuxR family (reviewed by Swift [59]). Here, the concentration of the signal molecule reflects the numbers of bacterial cells and the perception of a threshold level of the signal indicates a quorate population, ready to employ multicellular adaptive responses involved in, for example, virulence (21, 37, 46, 64), symbiosis (29, 63), and biofilm formation (10, 30) as well as individual survival strategies, such as the induction of stationary phase responses (25) and motility for colony escape (2, 42).

Acyl-HSL signals are composed of a homoserine lactone ring linked via an amide bond to an acyl chain of variable length and with variable substitutions. In vitro and in vivo studies have demonstrated that acyl-HSL synthesis couples amino acid and fatty acid biosynthesis, with the HSL moiety derived from S-adenosyl methionine and the acyl moiety from acylated-acyl carrier protein (acyl-ACP) (20, 33, 35, 48, 61). Signals with shorter acyl chains (C4 and C6) are freely diffusible (22, 38); however, in Pseudomonas aeruginosa, the long acyl chain signal (3-oxo-C12-HSL) requires an active efflux pump for its release, although it may enter cells by diffusion (38).

Surprisingly, acyl-HSL signals with long acyl chains exert significant effects upon the cells and tissues of eukaryotic organisms. An immunosuppressive effect of 3-oxo-C12-HSL upon the host immune system has been observed by Telford et al. (60), an effect that will prejudice the host against antibacterial action and so favor bacteria. Additionally, Lawrence et al. have described a vasorelaxant activity for 3-oxo-C12-HSL, which may contribute to the ability of the bacteria to maintain the supply of key nutrients to a site of infection by increasing the local blood flow (26).

The advantages to an intracellular pathogen of using QS to regulate genes within the closed environment of the replication vacuole and also to be able to modulate the activity of the host's immune responses are clear. We therefore sought to determine whether Brucella employs acyl-HSL-mediated QS, and in this paper, we report the identification and purification of N-dodecanoyl-homoserine lactone (C12-HSL) and its role in the control of the transcription of the virB operon, thus suggesting a putative link between QS and virulence in Brucella.

Acknowledgments

We are grateful to J.-M. Verger (Laboratoire de Pathologie Infectieuse et d'Immunologie, Institut National de Recherche Agronomique, Nouzilly, France) for the B. melitensis 16M Nal^{nalidixic acid-resistant strain.}

Bernard Taminiau holds a specialization grant from the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (FRIA). This work was supported in part by a grant (to P. Williams) from the Biotechnology and Biological Sciences Research Council U.K. and in part by the Commission of the European Communities, contract no. QLK2-CT-1999-00014 (Anne Tibor), which are gratefully acknowledged.

Acknowledgments

Notes

Editor: V. J. DiRita

Notes

Editor: V. J. DiRita

REFERENCES

References

1. Allen, C. A., L. G. Adams, and T. A. Ficht. 1998. Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect. Immun.66:1008-1016.
2. Atkinson, S., J. P. Throup, G. S. Stewart, and P. Williams. 1999. A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping. Mol. Microbiol.33:1267-1277. [[PubMed]
3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1997. Short protocols in molecular biology, 3rd ed. John Wiley & Sons, Inc., New York, N.Y.
4. Boschiroli, M. L., S. Ouahrani-Bettache, V. Foulongne, S. Michaux-Charachon, G. Bourg, A. Allardet-Servent, C. Cazevieille, J. P. Liautard, M. Ramuz, and D. O’Callagghan. 2002. The Brucella suis virB operon is induced intracellularly in macrophages. Proc. Natl. Acad. Sci. USA92:1544-1549.
5. Burns, DL. 1999. Biochemistry of type IV secretion. Curr. Opin. Microbiol.2:25-29. [[PubMed][Google Scholar]
6. Chhabra, S. R., P. Stead, N. J. Bainton, G. P. Salmond, G. S. Stewart, P. Williams, and B. W. Bycroft. 1993. Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone. J. Antibiot. (Tokyo)46:441-454. [[PubMed]
7. Coers, J., C. Monahan, and C. R. Roy. 1999. Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth. Nat. Cell. Biol.1:451-453. [[PubMed]
8. Cotter, P. A., and J. F. Miller. 1998. In vivo and ex vivo regulation of bacterial virulence gene expression. Curr. Opin. Microbiol.1:17-26. [[PubMed]
9. Cubo, M. T., A. Economou, G. Murphy, A. W. Johnston, and J. A. Downie. 1992. Molecular characterization and regulation of the rhizosphere-expressed genes rhiABCR that can influence nodulation by Rhizobium leguminosarum biovar viciae. J. Bacteriol.174:4026-4035.
10. Cui, Y., A. Chatterjee, Y. Liu, C. K. Dumenyo, and A. K. Chatterjee. 1995. Identification of a global repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that controls extracellular enzymes, N-(3-oxohexanoyl)-l-homoserine lactone, and pathogenicity in soft-rotting Erwinia spp. J. Bacteriol.177:5108-5115.
11. Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science280:295-298. [[PubMed]
12. Delrue, R. M., M. Martinez-Lorenzo, P. Lestrate, I. Danese, V. Bielarz, P. Mertens, X. De Bolle, A. Tibor, J. P. Gorvel, and J. J. Letesson. 2001. Identification of Brucella spp. genes involved in intracellular trafficking. Cell. Microbiol.3:487-497. [[PubMed]
13. Detilleux, P. G., B. L. Deyoe, and N. F. Cheville. 1990. Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro. Infect. Immun.58:2320-2328.
14. Dunny, G. M., and S. C. Winans. 1999. Bacterial life: neither lonely nor boring, p. 1-5. In G. M. Dunny and S. C. Winans (ed.), Cell-cell signaling in bacteria. ASM Press, Washington, D.C.
15. Foulongne, V., G. Bourg, C. Cazevieille, S. Michaux-Charachon, and D. O'Callaghan. 2000. Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposon mutagenesis. Infect. Immun.68:1297-1303.
16. Fuqua, W. C., S. C. Winans, and E. P. Greenberg. 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density- responsive transcriptional regulators. J. Bacteriol.176:269-275.
17. Godfroid, F., B. Taminiau, I. Danese, P. Denoel, A. Tibor, V. Weynants, A. Cloeckaert, J. Godfroid, and J. J. Letesson. 1998. Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages. Infect. Immun.66:5485-5493.
18. Holden, M. T., S. Ram Chhabra, R. de Nys, P. Stead, N. J. Bainton, P. J. Hill, M. Manefield, N. Kumar, M. Labatte, D. England, S. Rice, M. Givskov, G. P. Salmond, G. S. Stewart, B. W. Bycroft, S. Kjelleberg, and P. Williams. 1999. Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria. Mol. Microbiol.33:1254-1266. [[PubMed]
19. Hong, P. C., R. M. Tsolis, and T. A. Ficht. 2000. Identification of genes required for chronic persistence of Brucella abortus in mice. Infect. Immun.68:4102-4107.
20. Inon de Iannino, N., G. Briones, M. Tolmasky, and R. A. Ugalde. 1998. Molecular cloning and characterization of cgs, the Brucella abortus cyclic beta(1-2) glucan synthetase gene: genetic complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB mutants. J. Bacteriol.180:4392-4400.
21. Jiang, Y., M. Camara, S. R. Chhabra, K. R. Hardie, B. W. Bycroft, A. Lazdunski, G. P. Salmond, G. S. Stewart, and P. Williams. 1998. In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone. Mol. Microbiol.28:193-203. [[PubMed]
22. Jones, S., B. Yu, N. J. Bainton, M. Birdsall, B. W. Bycroft, S. R. Chhabra, A. J. Cox, P. Golby, P. J. Reeves, S. Stephens, et al. 1993. The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J.12:2477-2482.
23. Kaplan, H. B., and E. P. Greenberg. 1985. Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J. Bacteriol.163:1210-1214.
24. Kohler, S., J. Teyssier, A. Cloeckaert, B. Rouot, and J. P. Liautard. 1996. Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes. Mol. Microbiol.20:701-712. [[PubMed]
25. Kulakov, Y. K., P. G. Guigue-Talet, M. R. Ramuz, and D. O'Callaghan. 1997. Response of Brucella suis 1330 and B. canis RM6/66 to growth at acid pH and induction of an adaptive acid tolerance response. Res. Microbiol.148:145-151. [[PubMed]
26. Latifi, A., M. Foglino, K. Tanaka, P. Williams, and A. Lazdunski. 1996. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol. Microbiol.21:1137-1146. [[PubMed]
27. Lawrence, R. N., W. R. Dunn, B. Bycroft, M. Camara, S. R. Chhabra, P. Williams, and V. G. Wilson. 1999. The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone, inhibits porcine arterial smooth muscle contraction. Br. J. Pharmacol.128: 845-848.
28. Lestrate, P., R. M. Delrue, I. Danese, C. Didembourg, B. Taminiau, P. Mertens, X. De Bolle, A. Tibor, C. M. Tang, and J. J. Letesson. 2000. Identification and characterization of in vivo attenuated mutants of Brucella melitensis. Mol. Microbiol.38:543-551. [[PubMed]
29. Lewenza, S., B. Conway, E. P. Greenberg, and P. A. Sokol. 1999. Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI. J. Bacteriol.181:748-756.
30. Lithgow, J. K., A. Wilkinson, A. Hardman, B. Rodelas, F. Wisniewski-Dye, P. Williams, and J. A. Downie. 2000. The regulatory locus cinRI in Rhizobium leguminosarum controls a network of quorum-sensing loci. Mol. Microbiol.37:81-97. [[PubMed]
31. Lynch, M. J., S. Swift, D. F. Kirke, C. E. R. Dodd, C. W. Keevil, G. S. A. B. Stewart, and P. Williams. 1999. Investigation of quorum sensing in Aeromonas hydrophila biofilms formed on stainless steel, p. 209-221. In J. Wimpenny, P. Gilbert, J. Walker, M. Brading, and R. Bayston (ed.), Biofilms: the good, the bad and the ugly. BioLine, Cardiff, United Kingdom.
32. McClean, K. H., M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M. Camara, M. Daykin, J. H. Lamb, S. Swift, B. W. Bycroft, G. S. Stewart, and P. Williams. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology143(Pt. 12):3703-3711. [[PubMed]
33. McQuiston, J. R., R. Vemulapalli, T. J. Inzana, G. G. Schurig, N. Sriranganathan, D. Fritzinger, T. L. Hadfield, R. A. Warren, N. Snellings, D. Hoover, S. M. Halling, and S. M. Boyle. 1999. Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence. Infect. Immun.67:3830-3835.
34. More, M. I., L. D. Finger, J. L. Stryker, C. Fuqua, A. Eberhard, and S. C. Winans. 1996. Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates. Science272:1655-1658. [[PubMed]
35. O'Callaghan, D., C. Cazevieille, A. Allardet-Servent, M. L. Boschiroli, G. Bourg, V. Foulongne, P. Frutos, Y. Kulakov, and M. Ramuz. 1999. A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis. Mol. Microbiol.33:1210-1220. [[PubMed]
36. Parsek, M. R., D. L. Val, B. L. Hanzelka, J. E. Cronan, Jr., and E. P. Greenberg. 1999. Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA96:4360-4365.
37. Passador, L., J. M. Cook, M. J. Gambello, L. Rust, and B. H. Iglewski. 1993. Expression of Pseudomonas aeruginosa virulence genes requires cell-to- cell communication. Science260:1127-1130. [[PubMed]
38. Pearson, J. P., M. Feldman, B. H. Iglewski, and A. Prince. 2000. Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect. Immun.68:4331-4334.
39. Pearson, J. P., C. Van Delden, and B. H. Iglewski. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol.181:1203-1210.
40. Piper, K. R., S. Beck Von Bodman, I. Hwang, and S. K. Farrand. 1999. Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium. Mol. Microbiol.32:1077-1089. [[PubMed]
41. Pirhonen, M., D. Flego, R. Heikinheimo, and E. T. Palva. 1993. A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. EMBO J.12:2467-2476.
42. Pizarro-Cerda, J., S. Meresse, R. G. Parton, G. van der Goot, A. Sola-Landa, I. Lopez-Goni, E. Moreno, and J. P. Gorvel. 1998. Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes. Infect. Immun.66:5711-5724.
43. Puskas, A., E. P. Greenberg, S. Kaplan, and A. L. Schaefer. 1997. A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides. J. Bacteriol.179:7530-7537.
44. Robertson, G. T., and R. Roop. 1999. The Brucella abortus host factor I (HF-I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice. Mol. Microbiol.34:690-700. [[PubMed]
45. Rodelas, B., J. K. Lithgow, F. Wisniewski-Dye, A. Hardman, A. Wilkinson, A. Economou, P. Williams, and J. A. Downie. 1999. Analysis of quorum-sensing-dependent control of rhizosphere-expressed (rhi) genes in Rhizobium leguminosarum bv. viciae. J. Bacteriol.181:3816-3823.
46. Roy, C. R., K. H. Berger, and R. R. Isberg. 1998. Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol.28:663-674. [[PubMed]
47. Rumbaugh, K. P., J. A. Griswold, B. H. Iglewski, and A. N. Hamood. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections. Infect. Immun.67:5854-5862.
48. Saleh, A., C. Figarella, W. Kammouni, S. Marchand-Pinatel, A. Lazdunski, A. Tubul, P. Brun, and M. D. Merten. 1999. Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-l-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells. Infect. Immun.67:5076-5082.
49. Schaefer, A. L., D. L. Val, B. L. Hanzelka, J. E. Cronan, Jr., and E. P. Greenberg. 1996. Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc. Natl. Acad. Sci. USA93:9505-9509.
50. Shadel, G. S., and T. O. Baldwin. 1992. Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription. J. Biol. Chem.267:7696-7702. [[PubMed]
51. Shaw, P. D., G. Ping, S. L. Daly, C. Cha, J. E. Cronan, Jr., K. L. Rinehart, and S. K. Farrand. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA94:6036-6041.
52. Sieira, R., D. J. Comerci, D. O. Sanchez, and R. A. Ugalde. 2000. A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication. J. Bacteriol.182:4849-4855.
53. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology1:784-791. [PubMed]
54. Smith, L. D., and T. A. Ficht. 1990. Pathogenesis of Brucella. Crit. Rev. Microbiol.17:209-230. [[PubMed]
55. Sola-Landa, A., J. Pizarro-Cerda, M. J. Grillo, E. Moreno, I. Moriyon, J. M. Blasco, J. P. Gorvel, and I. Lopez-Goni. 1998. A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol. Microbiol.29:125-138. [[PubMed]
56. Stevens, A. M., and E. P. Greenberg. 1997. Quorum sensing in Vibrio fischeri: essential elements for activation of the luminescence genes. J. Bacteriol.179:557-562.
57. Swift, S., A. V. Karlyshev, L. Fish, E. L. Durant, M. K. Winson, S. R. Chhabra, P. Williams, S. Macintyre, and G. S. Stewart. 1997. Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J. Bacteriol.179:5271-5281.
58. Swift, S., M. J. Lynch, L. Fish, D. F. Kirke, J. M. Tomas, G. S. Stewart, and P. Williams. 1999. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila. Infect. Immun.67:5192-5199.
59. Swift, S., G. S. Stewart, and P. Williams. 1996. The inner workings of a quorum sensing signal generator. Trends Microbiol.4:463-466. [[PubMed]
60. Swift, S., P. Williams, and G. S. A. B. Stewart. 1999. N-acylhomoserine lactones and quorum sensing in Proteobacteria, p. 291-314. In G. M. Dunny and S. C. Winans (ed.), Cell-cell signaling in bacteria. ASM Press, Washington, D.C.
61. Telford, G., D. Wheeler, P. Williams, P. T. Tomkins, P. Appleby, H. Sewell, G. S. Stewart, B. W. Bycroft, and D. I. Pritchard. 1998. The Pseudomonas aeruginosa quorum-sensing signal molecule N-(3- oxododecanoyl)-l-homoserine lactone has immunomodulatory activity. Infect. Immun.66:36-42.
62. Val, D. L., and J. E. Cronan, Jr. 1998. In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases. J. Bacteriol.180:2644-2651.
63. Verger, J. M., M. Grayon, E. Chaslus-Dancla, M. Meurisse, and J. P. Lafont. 1993. Conjugative transfer and in vitro/in vivo stability of the broad-host-range IncP R751 plasmid in Brucella spp. Plasmid29:142-146. [[PubMed]
64. Visick, K. L., J. Foster, J. Doino, M. McFall-Ngai, and E. G. Ruby. 2000. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J. Bacteriol.182:4578-4586.
65. von Bodman, S. B., D. R. Majerczak, and D. L. Coplin. 1998. A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii. Proc. Natl. Acad. Sci. USA95:7687-7692.
66. Welch, M., D. E. Todd, N. A. Whitehead, S. J. McGowan, B. W. Bycroft, and G. P. Salmond. 2000. N-acyl homoserine lactone binding to the CarR receptor determines quorum-sensing specificity in Erwinia. EMBO J.19:631-641.
67. Williams, P., M. Camara, A. Hardman, S. Swift, D. Milton, V. J. Hope, K. Winzer, B. Middleton, D. I. Pritchard, and B. W. Bycroft. 2000. Quorum sensing and the population-dependent control of virulence. Philos. Trans. R. Soc. Lond. B. Biol. Sci.355:667-680.
68. Winson, M. K., M. Camara, A. Latifi, M. Foglino, S. R. Chhabra, M. Daykin, M. Bally, V. Chapon, G. P. Salmond, B. W. Bycroft, et al. 1995. Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA92:9427-9431.
69. Winson, M. K., S. Swift, L. Fish, J. P. Throup, F. Jorgensen, S. R. Chhabra, B. W. Bycroft, P. Williams, and G. S. Stewart. 1998. Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol. Lett.163:185-192. [[PubMed]
70. Zhan, Y., A. Kelso, and C. Cheers. 1995. Differential activation of Brucella-reactive CD4^{T cells by}Brucella infection or immunization with antigenic extracts. Infect. Immun.63:969-975.
71. Zhu, J., and S. C. Winans. 1999. Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells. Proc. Natl. Acad. Sci. USA96:4832-4837.