1887

Abstract

Denitrification and arginine fermentation are major parts of the anaerobic metabolism of , which is important for biofilm formation and infection. The two-component regulatory system NarX-NarL is part of the underlying network and is required for denitrifying growth. All target promoters identified so far are activated by NarL. In this study the effect of NarL on arginine fermentation was investigated using proteome, Northern blot and reporter gene analyses. NarL-dependent repression of the operon was observed and the corresponding NarL-binding site in the promoter region was functionally localized at −60 bp upstream of the transcriptional start site using site-directed promoter mutagenesis and reporter gene fusion experiments. The results clearly show that in the presence of nitrate NarL represses the arginine-dependent activation of the operon mediated by ArgR. It does not influence the oxygen-tension-dependent activation via Anr. Thus, the anaerobic energy metabolism of is coordinated via NarX-NarL activity. In the presence of nitrate the highly efficient denitrification is preferred over the less attractive arginine fermentation.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2008/018929-0
2008-10-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/10/3053.html?itemId=/content/journal/micro/10.1099/mic.0.2008/018929-0&mimeType=html&fmt=ahah

References

  1. Arai, H., Kodama, T. & Igarashi, Y. ( 1997; ). Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa. Mol Microbiol 25, 1141–1148.[CrossRef]
    [Google Scholar]
  2. Arai, H., Kodama, T. & Igarashi, Y. ( 1999; ). Effect of nitrogen oxides on expression of the nir and nor genes for denitrification in Pseudomonas aeruginosa. FEMS Microbiol Lett 170, 19–24.[CrossRef]
    [Google Scholar]
  3. Arai, H., Mizutani, M. & Igarashi, Y. ( 2003; ). Transcriptional regulation of the nos genes for nitrous oxide reductase in Pseudomonas aeruginosa. Microbiology 149, 29–36.[CrossRef]
    [Google Scholar]
  4. Barraud, N., Hassett, D. J., Hwang, S. H., Rice, S. A., Kjelleberg, S. & Webb, J. S. ( 2006; ). Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 188, 7344–7353.[CrossRef]
    [Google Scholar]
  5. Becher, A. & Schweizer, H. P. ( 2000; ). Integration-proficient Pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ and lux gene fusions. Biotechniques 29, 948–950, 952.
    [Google Scholar]
  6. Boes, N., Schreiber, K., Härtig, E., Jaensch, L. & Schobert, M. ( 2006; ). The Pseudomonas aeruginosa universal stress protein PA4352 is essential for surviving anaerobic energy stress. J Bacteriol 188, 6529–6538.[CrossRef]
    [Google Scholar]
  7. Carlson, C. A. & Ingraham, J. L. ( 1983; ). Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Appl Environ Microbiol 45, 1247–1253.
    [Google Scholar]
  8. Chiang, R. C., Cavicchioli, R. & Gunsalus, R. P. ( 1992; ). Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli. Mol Microbiol 6, 1913–1923.[CrossRef]
    [Google Scholar]
  9. Constantinidou, C., Hobman, J. L., Griffiths, L., Patel, M. D., Penn, C. W., Cole, J. A. & Overton, T. W. ( 2006; ). A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth. J Biol Chem 281, 4802–4815.[CrossRef]
    [Google Scholar]
  10. de Lorenzo, V. & Timmis, K. N. ( 1994; ). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235, 386–405.
    [Google Scholar]
  11. Dunn, N. W. & Holloway, B. W. ( 1971; ). Pleiotrophy of p-fluorophenylalanine-resistant and antibiotic hypersensitive mutants of Pseudomonas aeruginosa. Genet Res 18, 185–197.[CrossRef]
    [Google Scholar]
  12. Filiatrault, M. J., Picardo, K. F., Ngai, H., Passador, L. & Iglewski, B. H. ( 2006; ). Identification of Pseudomonas aeruginosa genes involved in virulence and anaerobic growth. Infect Immun 74, 4237–4245.[CrossRef]
    [Google Scholar]
  13. Galimand, M., Gamper, M., Zimmermann, A. & Haas, D. ( 1991; ). Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa. J Bacteriol 173, 1598–1606.
    [Google Scholar]
  14. Gamper, M., Zimmermann, A. & Haas, D. ( 1991; ). Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa. J Bacteriol 173, 4742–4750.
    [Google Scholar]
  15. Gamper, M., Ganter, B., Polito, M. R. & Haas, D. ( 1992; ). RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa. J Mol Biol 226, 943–957.[CrossRef]
    [Google Scholar]
  16. Härtig, E., Schiek, U., Vollack, K. U. & Zumft, W. G. ( 1999; ). Nitrate and nitrite control of respiratory nitrate reduction in denitrifying Pseudomonas stutzeri by a two-component regulatory system homologous to NarXL of Escherichia coli. J Bacteriol 181, 3658–3665.
    [Google Scholar]
  17. Hassett, D. J., Cuppoletti, J., Trapnell, B., Lymar, S. V., Rowe, J. J., Yoon, S. S., Hilliard, G. M., Parvatiyar, K., Kamani, M. C. & other authors ( 2002; ). Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets. Adv Drug Deliv Rev 54, 1425–1443.[CrossRef]
    [Google Scholar]
  18. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. & Molin, S. ( 2000; ). Quantification of biofilm structures by the novel computer program comstat. Microbiology 146, 2395–2407.
    [Google Scholar]
  19. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  20. 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]
  21. Krieger, R., Rompf, A., Schobert, M. & Jahn, D. ( 2002; ). The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor. Mol Genet Genomics 267, 409–417.[CrossRef]
    [Google Scholar]
  22. Lu, C. D., Winteler, H., Abdelal, A. & Haas, D. ( 1999; ). The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa. J Bacteriol 181, 2459–2464.
    [Google Scholar]
  23. Lu, C. D., Yang, Z. & Li, W. ( 2004; ). Transcriptome analysis of the ArgR regulon in Pseudomonas aeruginosa. J Bacteriol 186, 3855–3861.[CrossRef]
    [Google Scholar]
  24. Luthi, E., Baur, H., Gamper, M., Brunner, F., Villeval, D., Mercenier, A. & Haas, D. ( 1990; ). The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein. Gene 87, 37–43.[CrossRef]
    [Google Scholar]
  25. Mercenier, A., Simon, J. P., Vander Wauven, C., Haas, D. & Stalon, V. ( 1980; ). Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa. J Bacteriol 144, 159–163.
    [Google Scholar]
  26. Münch, R., Hiller, K., Grote, A., Scheer, M., Klein, J., Schobert, M. & Jahn, D. ( 2005; ). Virtual Footprint and PRODORIC: an integrative framework for regulon prediction in prokaryotes. Bioinformatics 21, 4187–4189.[CrossRef]
    [Google Scholar]
  27. Palmer, K. L., Brown, S. A. & Whiteley, M. ( 2007; ). Membrane-bound nitrate reductase is required for anaerobic growth in cystic fibrosis sputum. J Bacteriol 189, 4449–4455.[CrossRef]
    [Google Scholar]
  28. Platt, M. D., Schurr, M. J., Sauer, K., Vazquez, G., Kukavica-Ibrulj, I., Potvin, E., Levesque, R. C., Fedynak, A., Brinkman, F. S. & other authors ( 2008; ). Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions. J Bacteriol 190, 2739–2758.[CrossRef]
    [Google Scholar]
  29. Rabin, R. S. & Stewart, V. ( 1993; ). Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12. J Bacteriol 175, 3259–3268.
    [Google Scholar]
  30. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. & Davies, D. G. ( 2002; ). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184, 1140–1154.[CrossRef]
    [Google Scholar]
  31. Schreiber, K., Boes, N., Eschbach, M., Jaensch, L., Wehland, J., Bjarnsholt, T., Givskov, M., Hentzer, M. & Schobert, M. ( 2006; ). Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein. J Bacteriol 188, 659–668.[CrossRef]
    [Google Scholar]
  32. Schreiber, K., Krieger, R., Benkert, B., Eschbach, M., Arai, H., Schobert, M. & Jahn, D. ( 2007; ). The anaerobic regulatory network required for Pseudomonas aeruginosa nitrate respiration. J Bacteriol 189, 4310–4314.[CrossRef]
    [Google Scholar]
  33. Sharma, V., Noriega, C. E. & Rowe, J. J. ( 2006; ). Involvement of NarK1 and NarK2 proteins in transport of nitrate and nitrite in the denitrifying bacterium Pseudomonas aeruginosa PAO1. Appl Environ Microbiol 72, 695–701.[CrossRef]
    [Google Scholar]
  34. Spiro, S. & Guest, J. R. ( 1990; ). FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol Rev 6, 399–428.
    [Google Scholar]
  35. Stewart, V. ( 2003; ). Biochemical Society Special Lecture. Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria. Biochem Soc Trans 31, 1–10.
    [Google Scholar]
  36. Stewart, V. & Rabin, R. S. ( 1995; ). Dual sensors and dual response regulators interact to control nitrate- and nitrite-responsive gene expression in Escherichia coli. In Two-Component Signal Transduction, pp. 233–252. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC: American Society for Microbiology.
  37. Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J. & other authors ( 2000; ). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef]
    [Google Scholar]
  38. Torrents, E., Poplawski, A. & Sjoberg, B. M. ( 2005; ). Two proteins mediate class II ribonucleotide reductase activity in Pseudomonas aeruginosa: expression and transcriptional analysis of the aerobic enzymes. J Biol Chem 280, 16571–16578.[CrossRef]
    [Google Scholar]
  39. Tyson, K. L., Bell, A. I., Cole, J. A. & Busby, S. J. ( 1993; ). Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL. Mol Microbiol 7, 151–157.[CrossRef]
    [Google Scholar]
  40. Van Alst, N. E., Picardo, K. F., Iglewski, B. H. & Haidaris, C. G. ( 2007; ). Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa. Infect Immun 75, 3780–3790.[CrossRef]
    [Google Scholar]
  41. Vander Wauven, C., Pierard, A., Kley-Raymann, M. & Haas, D. ( 1984; ). Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J Bacteriol 160, 928–934.
    [Google Scholar]
  42. Vollack, K. U., Härtig, E., Korner, H. & Zumft, W. G. ( 1999; ). Multiple transcription factors of the FNR family in denitrifying Pseudomonas stutzeri: characterization of four fnr-like genes, regulatory responses and cognate metabolic processes. Mol Microbiol 31, 1681–1694.[CrossRef]
    [Google Scholar]
  43. Worlitzsch, D., Tarran, R., Ulrich, M., Schwab, U., Cekici, A., Meyer, K. C., Birrer, P., Bellon, G., Berger, J. & other authors ( 2002; ). Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J Clin Invest 109, 317–325.[CrossRef]
    [Google Scholar]
  44. Xu, K. D., Stewart, P. S., Xia, F., Huang, C. T. & McFeters, G. A. ( 1998; ). Spatial physiological heterogeneity in Pseudomonas aeruginosa biofilm is determined by oxygen availability. Appl Environ Microbiol 64, 4035–4039.
    [Google Scholar]
  45. Yamano, Y., Nishikawa, T. & Komatsu, Y. ( 1993; ). Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1. Mol Microbiol 8, 993–1004.[CrossRef]
    [Google Scholar]
  46. Ye, R. W., Haas, D., Ka, J. O., Krishnapillai, V., Zimmermann, A., Baird, C. & Tiedje, J. M. ( 1995; ). Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J Bacteriol 177, 3606–3609.
    [Google Scholar]
  47. Zumft, W. G. ( 1997; ). Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61, 533–616.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2008/018929-0
Loading
/content/journal/micro/10.1099/mic.0.2008/018929-0
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