1887

Abstract

Novel molecular tools have been constructed which allow for detection of -acyl homoserine lactone (AHL)-mediated quorum sensing in biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive, single-cell-level detection of quorum sensing in laboratory-based biofilms. It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga , is capable of interfering with AHL-mediated quorum sensing in . It is demonstrated that the furanone compound specifically represses expression of a P reporter fusion without affecting growth or protein synthesis. In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the quorum sensing circuit. The furanone was applied to biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.

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2002-01-01
2020-08-13
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References

  1. Albus A. M., Pesci E. C., Runyen-Janecky L. J., West S. E., Iglewski B. H.. 1997; Vfr controls quorum sensing in Pseudomonas aeruginosa . J Bacteriol179:3928–3935
    [Google Scholar]
  2. Andersen J. B., Sternberg C., Poulsen L. K., Bjorn S. P., Givskov M., Molin S.. 1998; New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol64:2240–2246
    [Google Scholar]
  3. Andersen J. B., Heydorn A., Hentzer M., Eberl L., Geisenberger O., Christensen B. B., Molin S., Givskov M.. 2001; gfp based N -acyl-homoserine-lactone monitors for detection of bacterial communication. Appl Environ Microbiol67:575–585[CrossRef]
    [Google Scholar]
  4. Anderson R. M., Zimprich C. A., Rust L.. 1999; A second operator is involved in Pseudomonas aeruginosa elastase ( lasB ) activation. J Bacteriol181:6264–6270
    [Google Scholar]
  5. Ayora S., Götz F.. 1994; Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus . Mol Gen Genet242:421–430
    [Google Scholar]
  6. Bertani G.. 1951; Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli . J Bacteriol62:293–300
    [Google Scholar]
  7. Bever R. A., Iglewski B. H.. 1988; Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol170:4309–4314
    [Google Scholar]
  8. Bloemberg G. V., O’Toole G. A., Lugtenberg B. J. J., Kolter R.. 1997; Green fluorescent protein as a marker for Pseudomonas spp. Appl Environ Microbiol63:4543–4551
    [Google Scholar]
  9. von Bodman S. B., Farrand S. K.. 1995; Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N -acylhomoserine lactone autoinducer. J Bacteriol177:5000–5008
    [Google Scholar]
  10. Brint J. M., Ohman D. E.. 1995; Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. J Bacteriol177:7155–7163
    [Google Scholar]
  11. Brumlik M. J., Storey D. G.. 1992; Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase. Mol Microbiol6:337–344[CrossRef]
    [Google Scholar]
  12. Brumlik M. J., Storey D. G.. 1998; Post-transcriptional control of Pseudomonas aeruginosa lasB expression involves the 5′ untranslated region of the mRNA. FEMS Microbiol Lett159:233–239
    [Google Scholar]
  13. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C.. 1994; Green fluorescent protein as a marker for gene expression. Science263:802–805[CrossRef]
    [Google Scholar]
  14. Charlton T. S., Netting A., Kumar N., Hentzer M., Givskov M., Kjelleberg S., de Nys R.. 2000; A novel and sensitive method for the quantification of N -3-oxo-acyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm. Environ Microbiol2:530–541[CrossRef]
    [Google Scholar]
  15. Christensen B. B., Sternberg C., Andersen J. B., Nielsen A. T., Givskov M., Molin S., Palmer R. J. Jr. 1999; Molecular tools for study of biofilm physiology. Methods Enzymol310:20–42
    [Google Scholar]
  16. Ciofu O., Giwercman B., Pedersen S. S., Hoiby N.. 1994; Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center. APMIS102:674–680[CrossRef]
    [Google Scholar]
  17. Clark D. J., Maaløe O.. 1967; DNA replication and the division cycle in Escherichia coli . J Mol Biol23:99–112[CrossRef]
    [Google Scholar]
  18. Costerton J. W., Lewandowski Z., Caldwell D. E., Korber D. R., Lappin-Scott H. M.. 1995; Microbial biofilms. Annu Rev Microbiol49:711–745[CrossRef]
    [Google Scholar]
  19. Costerton J. W., Stewart P. S., Greenberg E. P.. 1999; Bacterial biofilms: a common cause of persistent infections. Science284:1318–1322[CrossRef]
    [Google Scholar]
  20. 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. Science280:295–298[CrossRef]
    [Google Scholar]
  21. Diver J. M., Bryan L. E., Sokol P. A.. 1990; Transformation of Pseudomonas aeruginosa by electroporation. Anal Biochem189:75–79[CrossRef]
    [Google Scholar]
  22. Eberl L.. 1999; N -acyl homoserinelactone-mediated gene regulation in gram-negative bacteria. Syst Appl Microbiol22:493–506[CrossRef]
    [Google Scholar]
  23. 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 Microbiol20:127–136[CrossRef]
    [Google Scholar]
  24. Eberl L., Molin S., Givskov M.. 1999; Surface motility of Serratia liquefaciens MG1. J Bacteriol181:1703–1712
    [Google Scholar]
  25. Fukushima J., Ishiwata T., You Z.. 9 other authors 1997; Dissection of the promoter/operator region and evaluation of N -acylhomoserine lactone mediated transcriptional regulation of elastase expression in Pseudomonas aeruginosa . FEMS Microbiol Lett146:311–318[CrossRef]
    [Google Scholar]
  26. Fuqua C., Greenberg E. P.. 1999; Self perception in bacteria: quorum sensing with acylated homoserine lactones. Curr Opin Microbiol1:183–189
    [Google Scholar]
  27. Fuqua C., Winans S. C., Greenberg E. P.. 1997; Quorum sensing in bacteria: the LuxR–LuxI family of cell density-responsive transcriptional regulators. J Bacteriol176:269–275
    [Google Scholar]
  28. Gambello M. J., Iglewski B. H.. 1991; Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol173:3000–3009
    [Google Scholar]
  29. Gambello M. J., Kaye S., Iglewski B. H.. 1993; LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene ( apr ) and an enhancer of exotoxin A expression. Infect Immun61:1180–1184
    [Google Scholar]
  30. Givskov M., Manefield M., Gram L., Maximilien R., Eberl L., Molin S., Steinberg P. D., Kjelleberg S., de Nys R.. 1996; Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. J Bacteriol178:6618–6622
    [Google Scholar]
  31. Givskov M., Eberl L., Molin S.. 1997; Control of exoenzyme production, motility and cell differentiation in Serratia liquefaciens . FEMS Microbiol Lett148:115–122[CrossRef]
    [Google Scholar]
  32. Givskov M., Ostling J., Eberl L., Lindum P. W., Christensen A. B., Christiansen G., Molin S., Kjelleberg S.. 1998; Two separate regulatory systems participate in control of swarming motility of Serratia liquefaciens MG1. J Bacteriol180:742–745
    [Google Scholar]
  33. 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 Bacteriol181:1623–1629
    [Google Scholar]
  34. Gray K. M., Passador L., Iglewski B. H., Greenberg E. P.. 1994; Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa . J Bacteriol176:3076–3080
    [Google Scholar]
  35. Greenberg E. P.. 1997; Quorum sensing in gram-negative bacteria. ASM News63:371–377
    [Google Scholar]
  36. Guzman L. M., Belin D., Carson M. J., Beckwith J.. 1995; Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol177:4121–4130
    [Google Scholar]
  37. 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 Microbiol34:1082–1093[CrossRef]
    [Google Scholar]
  38. 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 Bacteriol172:6557–6567
    [Google Scholar]
  39. Hoiby N.. 1977; Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey. Acta Pathol Microbiol Scand Suppl1–96 .
  40. Hoiby N.. 2000; Prospects for the prevention and control of pseudomonal infection in children with cystic fibrosis. Paediatr Drugs2:451–463[CrossRef]
    [Google Scholar]
  41. Hoiby N., Koch C.. 1990; Pseudomonas aeruginosa infection in cystic fibrosis and its management. Thorax45:881–884[CrossRef]
    [Google Scholar]
  42. Holloway B. W.. 1955; Genetic recombination in Pseudomonas aeruginosa . J Gen Microbiol13:572–581[CrossRef]
    [Google Scholar]
  43. Kessler B., Timmis K. N., de Lorenzo V.. 1992; A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet233:293–301[CrossRef]
    [Google Scholar]
  44. de Kievit T. R., Iglewski B. H.. 2000; Bacterial quorum sensing in pathogenic relationships. Infect Immun68:4839–4849[CrossRef]
    [Google Scholar]
  45. de Kievit T., Seed P. C., Nezezon J., Passador L., Iglewski B. H.. 1999; RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa . J Bacteriol181:2175–2184
    [Google Scholar]
  46. Kjelleberg S., Steinberg P. D., Givskov M., Gram L., Manefield M., de Nys R.. 1999; Do marine natural products interfere with prokaryotic AHL regulatory systems?. Aquat Microb Ecol13:85–93
    [Google Scholar]
  47. Kleerebezem M., Quadri L. E., Kuipers O. P., de Vos W. M.. 1997; Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol Microbiol24:895–904[CrossRef]
    [Google Scholar]
  48. Kline T., Bowman J., Iglewski B. H., Kakai Y., Passador L., de Kievit T.. 1999; Novel synthetic analogs of the Pseudomonas autoinducer. Bioorg Med Chem Lett9:3447–3452[CrossRef]
    [Google Scholar]
  49. Koch C., Hoiby N.. 1993; Pathogenesis of cystic fibrosis. Lancet341:1065–1069[CrossRef]
    [Google Scholar]
  50. Lam J., Chan R., Lam K., Costerton J. W.. 1980; Production of mucoid microcolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immun28:546–556
    [Google Scholar]
  51. Latifi A., Foglino M., Tanaka K., Williams P., Lazdunski A.. 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 Microbiol21:1137–1146[CrossRef]
    [Google Scholar]
  52. 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 Bacteriol180:6384–6388
    [Google Scholar]
  53. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N.. 1990; Mini-Tn 5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol172:6568–6572
    [Google Scholar]
  54. Manefield M., Kumar N., Read R., Givskov M., Steinberg P. D., Kjelleberg S., de Nys R.. 1999; Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein. Microbiology145:283–291[CrossRef]
    [Google Scholar]
  55. Manefield M., Harris L., Rice S. A., Kjelleberg S., de Nys R.. 2000; Inhibition of luminescence and virulence in the black tiger prawn ( Penaeus monodon ) pathogen Vibrio harveyi by intercellular signal antagonists. Appl Environ Microbiol66:2079–2084[CrossRef]
    [Google Scholar]
  56. Manny A. J., Kjelleberg S., Kumar N., Read R., Steinberg P., de Nys R.. 1997; Reinvestigation of the sulfuric acid-catalysed cyclisation of brominated 2-alkyllevulinic acids to 3-alkyl-5-methylene-2(5h)-furanones. Tetrahedron53:15813–15826[CrossRef]
    [Google Scholar]
  57. de Nys R., Wright A. D., Sticher O., König G. M.. 1993; New halogenated furanones from the marine alga Delisea pulchra . Tetrahedron49:11213–11220[CrossRef]
    [Google Scholar]
  58. de Nys R., Steinberg P. D., Willemsen P., Dworjanyn S. A., Gabelish C. L., King R. J.. 1995; Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays. Biofouling8:259–271[CrossRef]
    [Google Scholar]
  59. Ochsner U. A., Reiser J.. 1995; Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa . Proc Natl Acad Sci USA92:6424–6428[CrossRef]
    [Google Scholar]
  60. O’Toole G. A., Kolter R.. 1998; Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol30:295–304[CrossRef]
    [Google Scholar]
  61. 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 USA97:8789–8793[CrossRef]
    [Google Scholar]
  62. Passador L., Iglewski B. H.. 1995; Quorum sensing and virulence gene regulation in Pseudomonas aeruginosa . In Virulence Mechanisms of Bacterial Pathogens pp65–78 Edited by Roth J. A.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  63. Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H.. 1993; Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science260:1127–1130[CrossRef]
    [Google Scholar]
  64. Passador L., Tucker K. D., Guertin K. R., Journet M. P., Kende A. S., Iglewski B. H.. 1996; Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. J Bacteriol178:5995–6000
    [Google Scholar]
  65. 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 USA91:197–201[CrossRef]
    [Google Scholar]
  66. Pearson J. P., Passador L., Iglewski B. H., Greenberg E. P.. 1995; A second N -acylhomoserine lactone signal produced by Pseudomonas aeruginosa . Proc Natl Acad Sci USA92:1490–1494[CrossRef]
    [Google Scholar]
  67. Pearson J. P., Pesci E. C., Iglewski B. H.. 1997; Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol179:5756–5767
    [Google Scholar]
  68. Pedersen S. S., Hoiby N., Espersen F., Koch C.. 1992; Role of alginate in infection with mucoid Pseudomonas aeruginosa in cystic fibrosis. Thorax47:6–13[CrossRef]
    [Google Scholar]
  69. Pesci E. C., Iglewski B. H.. 1997; The chain of command in Pseudomonas quorum sensing. Trends Microbiol5:132–134[CrossRef]
    [Google Scholar]
  70. Pesci E. C., Milbank J. B., 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 USA96:11229–11234[CrossRef]
    [Google Scholar]
  71. Piper K. R., Farrand S. K., von Bodman S. B.. 1993; Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature362:448–450[CrossRef]
    [Google Scholar]
  72. Pollack M.. 1990; Pseudomonas aeruginosa . In Principles and Practice of Infectious Diseases pp1673–1691 Edited by Mandell G. L.. Douglas R. G., Bennett J. E.. New York: Churchhill Livingstone;
    [Google Scholar]
  73. Rasmussen T. B., Manefield M., Andersen J. B., Eberl L., Anthoni U., Christophersen C., Steinberg P., Kjelleberg S., Givskov M.. 2000; How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1. Microbiology146:3237–3244
    [Google Scholar]
  74. Reichelt J. L., Borowitzka M. A.. 1984; Antimicrobial activity from marine algae: results of a large-scale screening programme. Hydrobiology116/117:158–168[CrossRef]
    [Google Scholar]
  75. Reimmann C., Beyeler M., Latifi A., Winteler H., Foglino M., Lazdunski A., Haas D.. 1997; The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N -butyryl-homoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol Microbiol24:309–319[CrossRef]
    [Google Scholar]
  76. Rust L., Pesci E. C., Iglewski B. H.. 1996; Analysis of the Pseudomonas aeruginosa elastase ( lasB ) regulatory region. J Bacteriol178:1134–1140
    [Google Scholar]
  77. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  78. Schlictman D., Kubo M., Shankar S., Chakrabarty A. M.. 1995; Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa : roles of algR2 and algH . J Bacteriol177:2469–2474
    [Google Scholar]
  79. Schweizer H. P.. 1991; Improved broad-host-range lac -based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa . Gene103:87–92[CrossRef]
    [Google Scholar]
  80. Seed P. C., Passador L., Iglewski B. H.. 1995; Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J Bacteriol177:654–659
    [Google Scholar]
  81. 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. Nature407:762–764[CrossRef]
    [Google Scholar]
  82. Swift S., Throup J. P., Williams P., Salmond G. P., Stewart G. S.. 1996; Quorum sensing: a population-density component in the determination of bacterial phenotype. Trends Biochem Sci21:214–219[CrossRef]
    [Google Scholar]
  83. Toder D. S., Gambello M. J., Iglewski B. H.. 1991; Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR . Mol Microbiol5:2003–2010[CrossRef]
    [Google Scholar]
  84. Toder D. S., Ferrell S. J., Nezezon J. L., Rust L., Iglewski B. H.. 1994; lasA and lasB genes of Pseudomonas aeruginosa : analysis of transcription and gene product activity. Infect Immun62:1320–1327
    [Google Scholar]
  85. Van Delden C., Iglewski B. H.. 1998; Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Dis4:551–560[CrossRef]
    [Google Scholar]
  86. West S. E. H., Schweizer H. P., Dall C., Sample A. K., Runyenjanecky L. J.. 1994; Construction of improved Escherichia Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa . Gene148:81–86[CrossRef]
    [Google Scholar]
  87. Whiteley M., Lee K. M., Greenberg E. P.. 1999; Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa . Proc Natl Acad Sci USA96:13904–13909[CrossRef]
    [Google Scholar]
  88. Whiteley M., Parsek M. R., Greenberg E. P.. 2000; Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa . J Bacteriol182:4356–4360[CrossRef]
    [Google Scholar]
  89. 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 Sci355:667–680[CrossRef]
    [Google Scholar]
  90. Winson M. K., Camara M., Latifi A.. 10 other authors 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[CrossRef]
    [Google Scholar]
  91. Wu H., Song Z., Hentzer M.. 8 other authors 2000; Detection of N -acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa . Microbiology146:2481–2493
    [Google Scholar]
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