1887

Microbiology Society logo

Volume 149, Issue 12

Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Free

Abstract

In at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, -galactosidase activity from a plasmid carrying the gene fused to the promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the gene produced only very low levels of -galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic -galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by NO. Additions of nitrate or nitroprusside lowered -galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): FF site, FNR-binding site , FNR, fumarate and nitrate reductase regulator (E. coli) , FnrP, NarR, nitrate reductase regulators (P. denitrificans) , NAR, nitrate reductase , NIR, nitrite reductase (cytochrome cd1) , NNR, nitrite and nitric oxide reductases regulator (P. denitrificans) , NOR, membrane-bound nitric oxide reductase (cytochrome bc) and SNP, sodium nitroprusside
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26546-0
2003-12-01
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/12/mic1493405.html?itemId=/content/journal/micro/10.1099/mic.0.26546-0&mimeType=html&fmt=ahah

References

  1. Aida T., Hata S., Kusunoki H. 1986; Temporary low oxygen conditions for the formation of nitrate reductase and nitrous oxide reductase by denitrifying Pseudomonas sp. G59. Can J Microbiol 32:543–547
     [Google Scholar]
  2. 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
     [Google Scholar]
  3. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1995 Short Protocols in Molecular Biology New York: Wiley;
  4. Baker S. C., Ferguson S. J., Ludwig B., Page M. D., Richter O.-M. H., van Spanning R. J. M. 1998; Molecular genetics of the genus Paracoccus : metabolically versatile bacteria with bioenergetic flexibility. Microbiol Mol Biol Rev 62:1046–1078
     [Google Scholar]
  5. Baumann B., Snozzi M., Zehnder A. J. B., van der Meer J. R. 1996; Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes. J Bacteriol 178:4367–4374
     [Google Scholar]
  6. Boublíková P., Kučera I., Dadák V. 1985; The effect of oxygen and nitrate on the biosynthesis of denitrification enzymes in Paracoccus denitrificans . Biologia (Bratislava) 40:357–363
     [Google Scholar]
  7. Craske A., Ferguson S. J. 1986; The respiratory nitrate reductase from Paracoccus denitrificans . Molecular characterization and kinetic properties. Eur J Biochem 158:429–436
     [Google Scholar]
  8. Cruz-Ramos H., Crack J., Wu G., Hughes M. N., Scott C., Thomson A. J., Green J., Poole R. K. 2002; NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. EMBO J 21:3235–3244
     [Google Scholar]
  9. de Boer A. P., Reijnders W. N., Kuenen J. G., Stouthamer A. H., van Spanning R. J. 1994; Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans . Antonie Van Leeuwenhoek 66:111–127
     [Google Scholar]
  10. de Vries G. E., Harms N., Hoogendijk J., Stouthamer A. H. 1989; Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn)-DNA-modifying property. Arch Microbiol 152:52–57
     [Google Scholar]
  11. 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]
  12. Green J., Bennett B., Jordan P., Ralph E. T., Thomson A. J., Guest J. R. 1996; Reconstitution of the [4Fe–4S] cluster in FNR and demonstration of the aerobic–anaerobic transcription switch in vitro . Biochem J 316:887–892
     [Google Scholar]
  13. Härtig E., Zumft W. G. 1999; Kinetics of nirS expression (cytochrome cd 1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification: evidence for a denitrification-specific nitrate- and nitrite-responsive regulatory system. J Bacteriol 181:161–166
     [Google Scholar]
  14. Hasegawa N., Arai H., Igarashi Y. 1998; Activation of a consensus FNR-dependent promoter by DNR of Pseudomonas aeruginosa in response to nitrite. FEMS Microbiol Lett 166:213–217
     [Google Scholar]
  15. Hutchings M. I., Spiro S. 2000; The nitric oxide regulated nor promoter of Paracoccus denitrificans . Microbiology 146:2635–2641
     [Google Scholar]
  16. Hutchings M. I., Shearer N., Wastell S., van Spanning R. J. M., Spiro S. 2000; Heterologous NNR-mediated nitric oxide signalling in Escherichia coli . J Bacteriol 182:6434–6439
     [Google Scholar]
  17. Hutchings M. I., Crack J. C., Shearer N., Thompson B. J., Thompson A. J., Spiro S. 2002; Transcription factor FnrP from Paracoccus denitrificans contains an iron–sulfur cluster and is activated by anoxia: identification of essential cysteine residues. J Bacteriol 184:503–508
     [Google Scholar]
  18. Jordan P. A., Thomson A. J., Ralph E. T., Guest J. R., Green J. 1997; FNR is a direct oxygen sensor having a biphasic response curve. FEBS Lett 416:349–352
     [Google Scholar]
  19. Khoroshilova N., Beinert H., Kiley P. 1995; Association of a polynuclear iron–sulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A 92:2499–2503
     [Google Scholar]
  20. Khoroshilova N., Popescu C., Münck E., Beinert H., Kiley P. 1997; Iron–sulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe–4S] to [2Fe–2S] conversion with loss of biological activity. Proc Natl Acad Sci U S A 94:6087–6092
     [Google Scholar]
  21. Kučera I. 1992; Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans . Biochem J 286:111–116
     [Google Scholar]
  22. Kučera I., Kaplan P. 1996; A study on the transport and dissimilatory reduction of nitrate in Paracoccus denitrificans using viologen dyes as electron donors. Biochim Biophys Acta 1276:203–209
     [Google Scholar]
  23. Kučera I., Mat'chová I. 1997; Iron as a possible mediator of the oxic-to-anoxic transition in Paracoccus denitrificans . Biochem Mol Biol Int 43:305–310
     [Google Scholar]
  24. Kučera I., Boublíková P., Dadák V. 1984; Function of terminal acceptors in the biosynthesis of denitrification pathway components in Paracoccus denitrificans . Folia Microbiol 29:108–114
     [Google Scholar]
  25. Kučera I., Mat'chová I., Dadák V. 1990; Respiratory rate as a regulatory factor in the biosynthesis of the denitrification pathway of the bacterium Paracoccus denitrificans . Biocatalysis 4:29–37
     [Google Scholar]
  26. Kučera I., Mat'chová I., Spiro S. 1994; Respiratory inhibitors activate an FNR-like regulatory protein in Paracoccus denitrificans : implications for the regulation of the denitrification pathway. Biochem Mol Biol Int 32:245–250
     [Google Scholar]
  27. Kwiatkowski A. V., Laratta W. P., Toffanin A., Shapleigh J. P. 1997; Analysis of the role of the nnR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide. J Bacteriol 179:5618–5620
     [Google Scholar]
  28. Lodge J., Williams R., Bell A., Chan B., Busby S. 1990; Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa : use of a new broad-host-range promoter-probe plasmid. FEMS Microbiol Lett 67:221–226
     [Google Scholar]
  29. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  30. Nedoma J. 1983; Determination of nitrates in waters using the salicylate method and chromotropic acid. Chem Listy 77:638–641 in Czech
    [Google Scholar]
  31. Parsonage D., Greenfield A. J., Ferguson S. J. 1985; The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells. Biochim Biophys Acta 807:81–95
     [Google Scholar]
  32. Payne W. J., Riley P. S., Cox C. D. Jr 1971; Separate nitrite, nitric oxide, and nitrous oxide reducing fractions from Pseudomonas perfectomarinus . J Bacteriol 106:356–361
     [Google Scholar]
  33. Saunders N. F. W., Houben E. N. G., Koefoed S., de Weert S., Reijnders W. N. M., Westerhoff H. V., de Boer A. P. N., van Spanning R. J. M. 1999; Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein. Mol Microbiol 34:24–36
     [Google Scholar]
  34. Sears H. J., Ferguson S. J., Richardson S. J., Spiro S. 1993; The identification of a periplasmic nitrate reductase in Paracoccus denitrificans . FEMS Microbiol Lett 113:107–112
     [Google Scholar]
  35. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:1–6
     [Google Scholar]
  36. Spiro S. 1992; An FNR-dependent promoter from Escherichia coli is active and anaerobically inducible in Paracoccus denitrificans . FEMS Microbiol Lett 98:145–148
     [Google Scholar]
  37. Tosques I. E., Shi J., Shapleigh J. P. 1996; Cloning and characterization of nnr , whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J Bacteriol 178:4958–4964
     [Google Scholar]
  38. van Spanning R. J. M., de Boer A. P. N., Reijnders W. N. M., Spiro S., Westerhoff H. V., Stouthamer A. H., van der Oost J. 1995; Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators. FEBS Lett 360:151–154
     [Google Scholar]
  39. van Spanning R. J. M., de Boer A. P. N., Reijnders W. N. M., Westerhoff H. V., Stouthamer A. H., van der Oost J. 1997; FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation. Mol Microbiol 23:893–907
     [Google Scholar]
  40. van Spanning R. J. M., Houben E., Reijnders W. N. M., Spiro S., Westerhoff H. V., Saunders N. 1999; Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans . J Bacteriol 181:4129–4132
     [Google Scholar]
  41. Wood N. J., Alizadeh T., Bennett S., Pearce J., Ferguson S. J., Richardson D. J., Moir J. W. B. 2001; Maximal expression of membrane-bound nitrate reductase in Paracoccus is induced by nitrate via a third FNR-like regulator named NarR. J Bacteriol 183:3606–3613
     [Google Scholar]
  42. Wu G., Cruz-Ramos H., Hill S., Green J., Sawers G., Poole R. K. 2000; Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr). Sensitivity to oxygen, reactive oxygen species, and nitric oxide. J Biol Chem 275:4679–4686
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26546-0
Loading
Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans
Microbiology 149, 3405 (2003); https://doi.org/10.1099/mic.0.26546-0
/content/journal/micro/10.1099/mic.0.26546-0
/content/journal/micro/10.1099/mic.0.26546-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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