1887

Abstract

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the mutant. As already described, the mutant could not grow in LB medium, unless high numbers of cells (>10 ml) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of -acylhomoserine lactones or the quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the gene was in single copy, while pyocyanin levels were increased when was present in a multicopy vector. Conversely, plating efficiency was increased only when the gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of , and expression in the mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.031971-0
2010-03-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/3/678.html?itemId=/content/journal/micro/10.1099/mic.0.031971-0&mimeType=html&fmt=ahah

References

  1. Bollinger N., Hassett D. J., Iglewski B. H., Costerton J. W., McDermott T. R. 2001; Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation. J Bacteriol 183:1990–1996
    [Google Scholar]
  2. Caiazza N. C., Shanks R. M., O'Toole G. A. 2005; Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa. J Bacteriol 187:7351–7361
    [Google Scholar]
  3. Choi K. H., Schweizer H. P. 2006; Mini-Tn 7 insertion in bacteria with single attTn 7 sites: example Pseudomonas aeruginosa. Nat Protoc 1:153–161
    [Google Scholar]
  4. Choi K. H., Kumar A., Schweizer H. P. 2006; A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397
    [Google Scholar]
  5. Cornelis P., Bouia A., Belarbi A., Guyonvarch A., Kammerer B., Hannaert V., Hubert J. C. 1989; Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa. Mol Microbiol 3:421–428
    [Google Scholar]
  6. Danese P. N., Pratt L. A., Kolter R. 2000; Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J Bacteriol 182:3593–3596
    [Google Scholar]
  7. Davey M. E., Caiazza N. C., O'Toole G. A. 2003; Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 185:1027–1036
    [Google Scholar]
  8. Dietrich L. E., Price-Whelan A., Petersen A., Whiteley M., Newman D. K. 2006; The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol 61:1308–1321
    [Google Scholar]
  9. Dietrich L. E., Teal T. K., Price-Whelan A., Newman D. K. 2008; Redox-active antibiotics control gene expression and community behavior in divergent bacteria. Science 321:1203–1206
    [Google Scholar]
  10. Elzer P. H., Kovach M. E., Phillips R. W., Robertson G. T., Peterson K. M., Roop R. M. II 1995; In vivo and in vitro stability of the broad-host-range cloning vector pBBR1MCS in six Brucella species. Plasmid 33:51–57
    [Google Scholar]
  11. Fletcher M. P., Diggle S. P., Crusz S. A., Chhabra S. R., Camara M., Williams P. 2007; A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules. Environ Microbiol 9:2683–2693
    [Google Scholar]
  12. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
    [Google Scholar]
  13. Hassett D. J., Charniga L., Bean K., Ohman D. E., Cohen M. S. 1992; Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. Infect Immun 60:328–336
    [Google Scholar]
  14. Hassett D. J., Ma J. F., Elkins J. G., McDermott T. R., Ochsner U. A., West S. E., Huang C. T., Fredericks J., Burnett S. 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 34:1082–1093
    [Google Scholar]
  15. Hassett D. J., Alsabbagh E., Parvatiyar K., Howell M. L., Wilmott R. W., Ochsner U. A. 2000; A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J Bacteriol 182:4557–4563
    [Google Scholar]
  16. Hentzer M., Riedel K., Rasmussen T. B., Heydorn A., Andersen J. B., Parsek M. R., Rice S. A., Eberl L., Molin S. other authors 2002; Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148:87–102
    [Google Scholar]
  17. 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]
  18. Kim E. J., Wang W., Deckwer W. D., Zeng A. P. 2005; Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density. Microbiology 151:1127–1138
    [Google Scholar]
  19. Kohler T., Curty L. K., Barja F., van Delden C., Pechere J. C. 2000; Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182:5990–5996
    [Google Scholar]
  20. Kovach M. E., Phillips R. W., Elzer P. H., Roop R. M., Peterson K. M. 1994; pBBR1MCS, a broad-host-range cloning vector. Biotechniques 16:800–801
    [Google Scholar]
  21. Lau G. W., Britigan B. E., Hassett D. J. 2005; Pseudomonas aeruginosa OxyR is required for full virulence in rodent and insect models of infection and for resistance to human neutrophils. Infect Immun 73:2550–2553
    [Google Scholar]
  22. Livak K. J., Schmittgen T. D. 2001; Analysis of relative gene expression data using real-time quantitative PCR and the method. Methods 25:402–408
    [Google Scholar]
  23. Maddocks S. E., Oyston P. C. 2008; Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 154:3609–3623
    [Google Scholar]
  24. Mavrodi D. V., Bonsall R. F., Delaney S. M., Soule M. J., Phillips G., Thomashow L. S. 2001; Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 183:6454–6465
    [Google Scholar]
  25. McClean K. H., Winson M. K., Fish L., Taylor A., Chhabra S. R., Camara M., Daykin M., Lamb J. H., Swift S. other authors 1997; Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711
    [Google Scholar]
  26. McMichael J. C. 1992; Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish. J Gen Microbiol 138:2687–2695
    [Google Scholar]
  27. Ochsner U. A., Vasil M. L., Alsabbagh E., Parvatiyar K., Hassett D. J. 2000; Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB- ankB, ahpB, and ahpC- ahpF. J Bacteriol 182:4533–4544
    [Google Scholar]
  28. Panmanee W., Gomez F., Witte D., Pancholi V., Britigan B. E., Hassett D. J. 2008; The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture. J Bacteriol 190:3658–3669
    [Google Scholar]
  29. Price-Whelan A., Dietrich L. E., Newman D. K. 2006; Rethinking ‘secondary' metabolism: physiological roles for phenazine antibiotics. Nat Chem Biol 2:71–78
    [Google Scholar]
  30. Price-Whelan A., Dietrich L. E., Newman D. K. 2007; Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14. J Bacteriol 189:6372–6381
    [Google Scholar]
  31. Reszka K. J., O'Malley Y., McCormick M. L., Denning G. M., Britigan B. E. 2004; Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase11 and hydrogen peroxide. Free Radic Biol Med 36:1448–1459
    [Google Scholar]
  32. Shaw P. D., Ping G., Daly S. L., Cha C., Cronan J. E. Jr, Rinehart K. L., Farrand S. K. 1997; Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci U S A 94:6036–6041
    [Google Scholar]
  33. Siegmund I., Wagner F. 1991; New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnol Tech 5:265–268
    [Google Scholar]
  34. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791
    [Google Scholar]
  35. Storz G., Imlay J. A. 1999; Oxidative stress. Curr Opin Microbiol 2:188–194
    [Google Scholar]
  36. Vinckx T., Matthijs S., Cornelis P. 2008; Loss of the oxidative stress regulator OxyR in Pseudomonas aeruginosa PAO1 impairs growth under iron-limited conditions. FEMS Microbiol Lett 288:258–265
    [Google Scholar]
  37. Wangt Y., Newman D. K. 2008; Redox reactions of phenazine antibiotics with ferric (hydr)oxides and molecular oxygen. Environ Sci Technol 42:2380–2386
    [Google Scholar]
  38. Williams P. 2007; Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology 153:3923–3938
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.031971-0
Loading
/content/journal/micro/10.1099/mic.0.031971-0
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