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Bioassays of Quorum Sensing Compounds Using Agrobacterium tumefaciens and Chromobacterium violaceum

  • Weihua Chu
  • Dhiraj A. Vattem
  • Vatsala Maitin
  • Mary B. Barnes
  • Robert J. C. McLean
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 692)

Abstract

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

Key words

Quorum sensing N-acyl homoserine lactone Violacein Chromobacterium violaceum Agrobacerium tumefaciens Violacein 

Notes

Acknowledgements

We are grateful to Clay Fuqua and Steve Winans for providing these strains and introducing us to quorum signaling. Work in RJCM’s laboratory is supported by a grant from the Norman Hackerman Advanced Research Program (003615-0037-2007).

References

  1. 1.
    Nealson, K. H., Platt, T., and Hastings, J. W. (1970) Cellular control of the synthesis and activity of the bacterial luminescent system, J. Bacteriol. 104, 313–22.PubMedGoogle Scholar
  2. 2.
    Ng, W. L. and Bassler, B. L. (2009) Bacterial quorum-sensing network architectures, Annu. Rev. Genet. 43, 197–222.PubMedCrossRefGoogle Scholar
  3. 3.
    Whiteley, M., Lee, K. M., and Greenberg, E. P. (1999) Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa, Proc. Natl. Acad. Sci. USA. 96, 13904–9.PubMedCrossRefGoogle Scholar
  4. 4.
    Schertzer, J. W., Boulette, M. L., and Whiteley, M. (2009) More than a signal: non-signaling properties of quorum sensing molecules, Trends Microbiol. 17, 189–95.PubMedCrossRefGoogle Scholar
  5. 5.
    Fuqua, C., Winans, S. C., and Greenberg, E. P. (1996) Census and consensus in bacterial ecosystems: the LuxR–LuxI family of quorum-sensing transcriptional regulators, Annu. Rev. Microbiol. 50, 727–51.PubMedCrossRefGoogle Scholar
  6. 6.
    Fuqua, C., Parsek, M. R., and Greenberg, E. P. (2001) Regulation of gene expression by cell-to-cell communication, Annu. Rev. Genet. 35, 439–68.PubMedCrossRefGoogle Scholar
  7. 7.
    Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H., and Oppenheimer, N. J. (1981) Structural identification of autoinducer of Photobac terium fischeri luciferase, Biochemistry. 20, 2444–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Pearson, J. P., Passador, L., Iglewski, B. H., and Greenberg, E. P. (1995) A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa, Proc. Natl. Acad. Sci. USA. 92, 1490–4.PubMedCrossRefGoogle Scholar
  9. 9.
    Zhang, L., Murphy, P. J., Kerr, A., and Tate, M. E. (1993) Agrobacterium conjugation and gene regulation by N-acyl-l-homoserine lactones, Nature. 362, 446–8.PubMedCrossRefGoogle Scholar
  10. 10.
    McClean, K. H., Winson, M. K., Fish, L., Taylor, A., Chhabra, S. R., Camara, M., Daykin, M., Lamb, J. H., Swift, S., Bycroft, B. W., Stewart, G. S. A. B., and Williams, P. (1997) Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones, Microbiology. 143, 3703–11.PubMedCrossRefGoogle Scholar
  11. 11.
    Williams, P. (2007) Quorum sensing, communication and cross-kingdom signalling in the bacterial world, Microbiology 153, 3923–38.PubMedCrossRefGoogle Scholar
  12. 12.
    Rumbaugh, K. P., Griswold, J. A., Iglewski, B. H., and Hamood, A. N. (1999) Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections, Infect. Immun. 67, 5854–62.PubMedGoogle Scholar
  13. 13.
    Wu, H., Song, Z., Givskov, M., Döring, G., Worlitzsch, D., Mathee, K., Rygaard, J., and Hoiby, N. (2001) Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection, Microbiology. 147, 1105–13.PubMedGoogle Scholar
  14. 14.
    Bjarnsholt, T., Jensen, P. O., Burmolle, M., Hentzer, M., Haagensen, J. A. J., Hougen, H. P., Calum, H., Madsen, K. G., Moser, C., Molin, S., Hoiby, N., and Givskov, M. (2005) Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent, Microbiology. 151, 373–83.PubMedCrossRefGoogle Scholar
  15. 15.
    Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W., and Greenberg, E. P. (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm, Science. 280, 295–8.PubMedCrossRefGoogle Scholar
  16. 16.
    Shrout, J. D., Chopp, D. L., Just, C. L., Hentzer, M., Givskov, M., and Parsek, M. R. (2006) The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional, Mol. Microbiol. 62, 1264–77.PubMedCrossRefGoogle Scholar
  17. 17.
    de Nys, R., Givskov, M., Kumar, N., Kjelleberg, S., and Steinberg, P. D. (2006) Furanones, Prog. Mol. Subcell. Biol. 42, 55–86.PubMedGoogle Scholar
  18. 18.
    Adonizio, A. L., Downum, K., Bennett, B. C., and Mathee, K. (2006) Anti-quorum sensing activity of medicinal plants in southern Florida, J. Ethnopharmacol. 105, 427–35.PubMedCrossRefGoogle Scholar
  19. 19.
    Defoirdt, T., Miyamoto, C. M., Wood, T. K., Meighen, E. A., Sorgeloos, P., Verstraete, W., and Bossier, P. (2007) The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional regulator protein luxR, Environ. Microbiol. 9, 2486–95.PubMedCrossRefGoogle Scholar
  20. 20.
    Rasmussen, T. B. and Givskov, M. (2006) Quorum-sensing inhibitors as anti-pathogenic drugs, Int. J. Med. Microbiol. 296, 149–61.PubMedCrossRefGoogle Scholar
  21. 21.
    Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., Molin, S., Steinberg, P. D., and Kjelleberg, S. (1996) Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling, J. Bacteriol. 178, 6618–22.PubMedGoogle Scholar
  22. 22.
    Moré, M. I., Finger, L. D., Stryker, J. L., Fuqua, C., Eberhard, A., and Winans, S. C. (1996) Enzymatic synthesis of a quorum-sensing autoinducer through the use of defined substrates, Science. 272, 1655–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Makemson, J., Eberhard, A., and Mathee, K. (2006) Simple electrospray mass spectrometry detection of acylhomoserine lactones, Luminescence. 21, 1–6.PubMedCrossRefGoogle Scholar
  24. 24.
    Eberhard, A. and Schineller, J. B. (2000) Chemical synthesis of bacterial autoinducers and analogs, Methods Enzymol. 305, 301–15.PubMedCrossRefGoogle Scholar
  25. 25.
    Charlton, T. S., de Nys, R., Netting, A., Kumar, N., Hentzer, M., Givskov, M., and Kjelleberg, S. (2000) A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm, Environ. Microbiol. 2, 530–41.PubMedCrossRefGoogle Scholar
  26. 26.
    Steindler, L. and Venturi, V. (2007) Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors, FEMS Microbiol. Lett. 266, 1–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Fuqua, C. and Winans, S. C. (1996) Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes, J. Bacteriol. 178, 435–40.PubMedGoogle Scholar
  28. 28.
    Zhu, J., Chai, Y., Zhong, Z., Li, S., and Winans, S. C. (2003) Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii, Appl. Environ. Microbiol. 69, 6949–53.PubMedCrossRefGoogle Scholar
  29. 29.
    Glansdorp, F. G., Thomas, G. L., Lee, J. K., Dutton, J. M., Salmond, G. P. C., Welch, M., and Spring, D. R. (2004) Synthesis and stability of small molecule probes for Pseudomonas aeruginosa quorum sensing modulation, Org. Biomol. Chem. 2, 3329–36.PubMedCrossRefGoogle Scholar
  30. 30.
    Wood, D. W. and Pierson, L. S. (1996) The phzI gene of Pseudomonas aureofaciens 30–84 is responsible for the production of a diffusible signal required for phenazine antibiotic production, Gene. 168, 49–53.PubMedCrossRefGoogle Scholar
  31. 31.
    McLean, R. J. C., Pierson, L. S., and Fuqua, C. (2004) A simple screening protocol for the identification of quorum signal antagonists, J. Microbiol. Methods. 58, 351–60.PubMedCrossRefGoogle Scholar
  32. 32.
    Fuqua, W. C. and Winans, S. C. (1994) A luxR-luxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite, J. Bacteriol. 176, 2796–806.PubMedGoogle Scholar
  33. 33.
    Zhu, J., Beaber, J. W., Moré, M. I., Fuqua, C., Eberhard, A., and Winans, S. C. (1998) Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens, J. Bacteriol. 180, 5398–405.PubMedGoogle Scholar
  34. 34.
    Shaw, P. D., Ping, G., Daly, S. L., Cha, C., Cronan, J. E., Jr., Rinehart, K. L., and Farrand, S. K. (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin layer chromatography, Proc. Natl. Acad. Sci. USA. 94, 6036–41.PubMedCrossRefGoogle Scholar
  35. 35.
    Blosser, R. S. and Gray, K. M. (2000) Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers, J. Microbiol. Methods. 40, 47–55.PubMedCrossRefGoogle Scholar
  36. 36.
    McLean, R. J. C., Whiteley, M., Stickler, D. J., and Fuqua, W. C. (1997) Evidence of autoinducer activity in naturally-occurring biofilms, FEMS Microbiol. Lett. 154, 259–63.PubMedCrossRefGoogle Scholar
  37. 37.
    Stickler, D. J., Morris, N. S., McLean, R. J. C., and Fuqua, C. (1998) Biofilms on indwelling urinary catheters produce quorum-sensing signal molecules in situ and in vitro, Appl. Environ. Microbiol. 64, 3486–90.PubMedGoogle Scholar
  38. 38.
    Bates, C. L., Forstner, M. R. J., Barnes, M. B., Whiteley, M., and McLean, R. J. C. (2006) Identification of heterotrophic limestone-adherent biofilm isolates from the Edwards Aquifer, Texas, Southwest. Nat. 51, 299–309.CrossRefGoogle Scholar
  39. 39.
    Vattem, D. A., Mihalik, K., Crixell, S. H., and McLean, R. J. C. (2007) Dietary phytochemicals as quorum sensing inhibitors, Fitoterapia. 78, 302–10.PubMedCrossRefGoogle Scholar
  40. 40.
    McLean, R. J. C., Bryant, S. A., Vattem, D. A., Givskov, M., Rasmussen, T. B., and Balaban, N. (2008) Detection in vitro of quorum-sensing molecules and their inhibitors, in The Control of Biofilm Infections by Signal Manipulation (Balaban, N., Ed.) pp. 39–50, Springer-Verlag, Heidelberg.CrossRefGoogle Scholar
  41. 41.
    Fuqua, C., Burbea, M., and Winans, S. C. (1995) Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene, J. Bacteriol. 177, 1367–73.PubMedGoogle Scholar
  42. 42.
    McLean, R. J. C., Barnes, M. B., Windham, M. K., Merchant, M. M., Forstner, M. R. J., and Fuqua, C. (2005) Cell–cell influences on bacterial community development in aquatic biofilms, Appl. Environ. Microbiol. 71, 8987–90.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Weihua Chu
    • 1
    • 2
  • Dhiraj A. Vattem
    • 3
  • Vatsala Maitin
    • 4
  • Mary B. Barnes
    • 5
  • Robert J. C. McLean
    • 1
  1. 1.Department of BiologyTexas State University-San MarcosSan MarcosUSA
  2. 2.Department of Microbiology, School of Life Science and TechnologyChina Pharmaceutical UniversityNanjingP. R. China
  3. 3.Molecular and Cellular Nutrition Program, Department of Family and Consumer ScienceTexas State University-San MarcosSan MarcosUSA
  4. 4.Department of Family and Consumer Science, Molecular and Cellular Nutrition ProgramTexas State University-San MarcosSan MarcosUSA
  5. 5.Tulane National Primate Research CenterCovingtonUSA

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