1887

Abstract

and often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn insertion mutants of H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces -acylhomoserine lactones (AHLs) due to an inactivation of the gene. and the AHL synthase gene together constitute the quorum-sensing system of . By using a gene replacement method, two defined mutants, H111-I and H111-R, were constructed in which and , respectively, had been inactivated. These mutants were used to demonstrate that biofilm formation by H111 requires a functional quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment, but rather controls the maturation of the biofilm. Furthermore, it is shown that is capable of swarming motility, a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of is quorum-sensing-regulated, possibly through the control of biosurfactant production. Complementation of the mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility is not essential for biofilm formation on abiotic surfaces.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-9-2517
2001-09-01
2019-09-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/9/1472517a.html?itemId=/content/journal/micro/10.1099/00221287-147-9-2517&mimeType=html&fmt=ahah

References

  1. Ahimou, F., Jacques, P. & Deleu, M. ( 2000; ). Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity. Enzyme Microb Technol 27, 749-754.[CrossRef]
    [Google Scholar]
  2. Allison, C. & Hughes, C. ( 1991; ). Bacterial swarming: an example of prokaryotic differentiation and multicellular behaviour. Sci Prog 75, 403-422.
    [Google Scholar]
  3. Andersen, J. B., Sternberg, C., Poulsen, L. K., Petersen Bjørn, S., Givskov, M. & Molin, S. ( 1998; ). New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64, 2240-2246.
    [Google Scholar]
  4. Atkinson, S., Throup, J. P., Stewart, G. S. & Williams, P. ( 1999; ). A hierarchical quorum-sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping. Mol Microbiol 33, 1267-1277.
    [Google Scholar]
  5. Belas, M. R. & Colwell, R. R. ( 1982; ). Adsorption kinetics of laterally and polarly flagellated Vibrio. J Bacteriol 151, 1568-1580.
    [Google Scholar]
  6. Christensen, B. B., Sternberg, C., Andersen, J. B., Palmer, R. J., Nielsen, A. T., Givskov, M. & Molin, S. ( 1999; ). Molecular tools for the study of biofilm physiology. Methods Enzymol 310, 20-42.
    [Google Scholar]
  7. Clark, J. D. & Maaløe, O. ( 1967; ). DNA replication and the division cycle in Escherichia coli. J Mol Biol 23, 99-112.[CrossRef]
    [Google Scholar]
  8. Connell., T. D., Metzger, D. J., Lynch, J. & Folster, J. P. ( 1998; ). Endochitinase is transported to the extracellular milieu by the eps-encoded general secretory pathway of Vibrio cholerae. J Bacteriol 180, 5591-5600.
    [Google Scholar]
  9. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. & Lappin-Scott, H. M. ( 1995; ). Microbial biofilms. Annu Rev Microbiol 49, 711-745.[CrossRef]
    [Google Scholar]
  10. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. ( 1999; ). Bacterial biofilms: a common cause of persistent infections. Science 284, 1318-1322.[CrossRef]
    [Google Scholar]
  11. Danese, P. N., Pratt, L. A., Dove, S. L. & Kolter, R. ( 2000; ). The outer membrane protein, antigen 43, mediates cell-to-cell interactions within Escherichia coli biofilms. Mol Microbiol 37, 424-432.[CrossRef]
    [Google Scholar]
  12. Darling, P., Chan, M., Cox, A. D. & Sokol, P. A. ( 1998; ). Siderophore production by cystic fibrosis isolates of Burkholderia cepacia. Infect Immun 66, 874-877.
    [Google Scholar]
  13. Davey, M. E. & O’Toole, G. A. ( 2000; ). Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64, 847-867.[CrossRef]
    [Google Scholar]
  14. 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.[CrossRef]
    [Google Scholar]
  15. Eberl, L., Winson, M. K., Sternberg, C. & 7 other authors ( 1996; ). Involvement of N-acyl-l-homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens. Mol Microbiol 20, 127–136.[CrossRef]
    [Google Scholar]
  16. Eberl, L., Molin, S. & Givskov, M. ( 1999; ). Surface motility of Serratia liquefaciens MG1. J Bacteriol 181, 1703-1712.
    [Google Scholar]
  17. Geisenberger, O., Givskov, M., Riedel, K., Høiby, N., Tümmler, B. & Eberl, L. ( 2000; ). Production of N-acyl-l-homoserine lactones by P. aeruginosa isolates from chronic lung infections associated with cystic fibrosis. FEMS Microbiol Lett 184, 273-278.
    [Google Scholar]
  18. Gessner, A. R. & Mortensen, J. E. ( 1990; ). Pathogenic factors of Pseudomonas cepacia isolates from patients with cystic fibrosis. J Med Microbiol 33, 115-120.[CrossRef]
    [Google Scholar]
  19. Glessner, A., Smith, R. S., Iglewski, B. H. & Robinson, J. B. ( 1999; ). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. J Bacteriol 181, 1623-1629.
    [Google Scholar]
  20. Gotschlich, A., Huber, B., Geisenberger, O. & 11 other authors ( 2001; ). Synthesis of multiple N-acyl-homoserine lactones is wide-spread among the members of the Burkholderia cepacia complex. Syst Appl Microbiol 24, 1–14.[CrossRef]
    [Google Scholar]
  21. Govan, J. R. W. & Deretic, V. ( 1996; ). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60, 539-574.
    [Google Scholar]
  22. Harshey, R. M. ( 1994; ). Bees aren’t the only ones: swarming in gram-negative bacteria. Mol Microbiol 13, 389-394.[CrossRef]
    [Google Scholar]
  23. Herrero, M., de Lorenzo, V. & Timmis, K. N. ( 1990; ). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172, 6557-6567.
    [Google Scholar]
  24. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersbøll, B. K. & Molin, S. ( 2000; ). Quantification of biofilm structures by the novel computer program comstat. Microbiology 146, 2395-2407.
    [Google Scholar]
  25. Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. ( 1998; ). A broad-host range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77-86.[CrossRef]
    [Google Scholar]
  26. Hutchison, M. L., Poxton, I. R. & Govan, J. R. W. ( 1998; ). Burkholderia cepacia produces a hemolysin that is capable of inducing apoptosis and degranulation of mammalian phagocytes. Infect Immun 66, 2033-2039.
    [Google Scholar]
  27. Isles, A., Maclusky, I., Corey, M., Gold, R., Prober, C., Fleming, P. & Levison, H. ( 1984; ). Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 104, 206-210.[CrossRef]
    [Google Scholar]
  28. de Kievit, T. R. & Iglewski, B. H. ( 2000; ). Bacterial quorum sensing in pathogenic relationships. Infect Immun 68, 4839-4849.[CrossRef]
    [Google Scholar]
  29. Koch, C. & Høiby, N. ( 1993; ). Pathogenesis of cystic fibrosis. Lancet 341, 1065-1069.[CrossRef]
    [Google Scholar]
  30. Köhler, T., Curty, L. K., Barja, F., van Delden, C. & Pechère, J. C. ( 2000; ). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182, 5990-5996.[CrossRef]
    [Google Scholar]
  31. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. & Peterson, K. M. ( 1995; ). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176.[CrossRef]
    [Google Scholar]
  32. Lam, J., Chan, R., Lam, K. & Costerton, J. W. ( 1980; ). Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immun 28, 546-556.
    [Google Scholar]
  33. Lewenza, S., Conway, B., Greenberg, E. P. & Sokol, P. A. ( 1999; ). Quorum sensing in Burkholderia cepacia: identification of the LuxRI homologs CepRI. J Bacteriol 181, 748-765.
    [Google Scholar]
  34. Lindum, P. W., Anthoni, U., Christophersen, C., Eberl, L., Molin, S. & Givskov, M. ( 1998; ). N-Acyl-l-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1. J Bacteriol 180, 6384-6388.
    [Google Scholar]
  35. Lonon, M. K., Woods, D. E. & Straus, D. C. ( 1988; ). Production of lipase by clinical isolates of Pseudomonas cepacia. J Clin Microbiol 26, 979-984.
    [Google Scholar]
  36. Loo, C. Y., Corliss, D. A. & Ganeshkumar, N. ( 2000; ). Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J Bacteriol 182, 1374-1382.[CrossRef]
    [Google Scholar]
  37. de Lorenzo, V. & Timmis, K. N. ( 1994; ). Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5- and Tn10-derived mini-transposons. Methods Enzymol 235, 386-405.
    [Google Scholar]
  38. McKenney, D., Brown, K. E. & Allison, D. G. ( 1995; ). Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J Bacteriol 177, 6989-6992.
    [Google Scholar]
  39. McKevitt, A. I., Bajaksouzian, S., Klinger, J. D. & Woods, D. E. ( 1989; ). Purification and characterization of an extracellular protease from Pseudomonas cepacia. Infect Immun 57, 771-778.
    [Google Scholar]
  40. McLean, R. J., Whiteley, M., Stickler, D. J. & Fuqua, W. C. ( 1997; ). Evidence of autoinducer activity in naturally occurring biofilms. FEMS Microbiol Lett 154, 259-263.[CrossRef]
    [Google Scholar]
  41. Ochsner, U. A. & Reiser, J. ( 1995; ). Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 92, 6424-6428.[CrossRef]
    [Google Scholar]
  42. Oka, A., Sugisaki, H. & Takanami, M. ( 1981; ). Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147, 217-226.[CrossRef]
    [Google Scholar]
  43. O’Toole, G. A. & Kolter, R. ( 1998a; ). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 28, 449-461.[CrossRef]
    [Google Scholar]
  44. O’Toole, G. A. & Kolter, R. ( 1998b; ). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295-304.[CrossRef]
    [Google Scholar]
  45. O’Toole, G., Kaplan, H. B. & Kolter, R. ( 2000; ). Biofilm formation as microbial development. Annu Rev Microbiol 54, 49-79.[CrossRef]
    [Google Scholar]
  46. Parsek, M. R. & Greenberg, E. P. ( 2000; ). Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc Natl Acad Sci USA 97, 8789-8793.[CrossRef]
    [Google Scholar]
  47. Pratt, L. A. & Kolter, R. ( 1998; ). Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30, 285-293.[CrossRef]
    [Google Scholar]
  48. Pratt, L. A. & Kolter, R. ( 1999; ). Genetic analyses of bacterial biofilm formation. Curr Opin Microbiol 2, 598-603.[CrossRef]
    [Google Scholar]
  49. Römling, U., Fiedler, B., Bosshammer, J., Grothues, D., Greipel, J., von der Hardt, H. & Tümmler, B. ( 1994; ). Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis. J Infect Dis 170, 1616-1621.[CrossRef]
    [Google Scholar]
  50. Rosenberg, E. & Ron, E. Z. ( 1999; ). High- and low-molecular mass microbial surfactants. Appl Microbiol Biotechnol 52, 154-162.[CrossRef]
    [Google Scholar]
  51. Saiman, L., Cacalano, G. & Prince, A. ( 1990; ). Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa. Infect Immun 58, 2578-2584.
    [Google Scholar]
  52. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
  53. Sanchez-Romero, J. M., Diaz-Orejas, R. & de Lorenzo, V. ( 1998; ). Resistance to tellurite as a selection marker for genetic manipulations of Pseudomonas strains. Appl Environ Microbiol 64, 4040-4046.
    [Google Scholar]
  54. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  55. Schierholz, J. M., Beuth, J., König, D., Nürnberger, A. & Pulverer, G. ( 1999; ). Antimicrobial substances and effects on sessile bacteria. Zentbl Bakteriol 289, 165-177.[CrossRef]
    [Google Scholar]
  56. Schwyn, B. & Neilands, J. B. ( 1987; ). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47-56.[CrossRef]
    [Google Scholar]
  57. Shaw, P. D., Ping, G., Daly, S. L., Cha, C., Cronan, J. E., Rinchart, K. L. & Farrand, S. K. ( 1997; ). Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci USA 94, 6036-6041.[CrossRef]
    [Google Scholar]
  58. Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J. & Greenberg, E. P. ( 2000; ). Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407, 762-764.[CrossRef]
    [Google Scholar]
  59. Stickler, D. J., Morris, N. S., McLean, R. J. & Fuqua, C. ( 1998; ). Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl Environ Microbiol 64, 3486-3490.
    [Google Scholar]
  60. Tolker-Nielsen, T. & Molin, S. ( 2000; ). Spatial organization of microbial biofilm communities. Microb Ecol 40, 75-84.
    [Google Scholar]
  61. Tümmler, B. & Kiewitz, C. ( 1999; ). Cystic fibrosis: an inherited susceptibility to bacterial respiratory infections. Mol Med Today 5, 351-358.[CrossRef]
    [Google Scholar]
  62. Watnick, P. I. & Kolter, R. ( 1999; ). Steps in the development of a Vibrio cholerae El Tor biofilm. Mol Microbiol 34, 586-595.[CrossRef]
    [Google Scholar]
  63. Watnick, P. & Kolter, R. ( 2000; ). Biofilm, city of microbes. J Bacteriol 182, 2675-2679.[CrossRef]
    [Google Scholar]
  64. Williams, P., Camara, M., Hardman, A. & 7 other authors ( 2000; ). Quorum sensing and the population-dependent control of virulence. Philos Trans R Soc Lond B Biol Sci 355, 667–680.[CrossRef]
    [Google Scholar]
  65. Winson, M. K., Swift, S., Hill, P. J., Sims, C. M., Griesmayr, G., Bycroft, B. W., Williams, P. & Stewart, G. S. A. B. ( 1998a; ). Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini Tn5 constructs. FEMS Microbiol Lett 163, 193-202.[CrossRef]
    [Google Scholar]
  66. Winson, M. K., Swift, S., Fish, L., Throup, J. P., Jørgensen, F., Chhabra, S. R., Bycroft, B. W., Williams, P. & Stewart, G. S. A. B. ( 1998b; ). Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163, 185-192.[CrossRef]
    [Google Scholar]
  67. Xu, K. D., McFeter, G. A. & Stewart, P. S. ( 2000; ). Biofilm resistance to antimicrobial agents. Microbiology 146, 547-549.
    [Google Scholar]
  68. Yohalem, D. S. & Lorbeer, J. W. ( 1994; ). Intraspecific metabolic diversity among strains of Burkholderia cepacia isolated from decayed onions, soils, and the clinical environment. Antonie Leeuwenhoek 65, 111-131.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-9-2517
Loading
/content/journal/micro/10.1099/00221287-147-9-2517
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error