1887

Abstract

A3(2) synthesizes three membrane-associated respiratory nitrate reductases (Nars). During aerobic growth in liquid medium the bacterium was able to reduce 50 mM nitrate stoichiometrically to nitrite. Construction and analysis of a mutant in which all three operons were deleted showed that it failed to reduce nitrate. Deletion of the gene encoding MoaA, which catalyses the first step in molybdenum cofactor biosynthesis, also prevented nitrate reduction, consistent with the Nars being molybdoenzymes. In contrast to the triple mutant, the mutant was also unable to use nitrate as sole nitrogen source, which indicates that the assimilatory nitrate reductases in are also molybdenum-dependent. Analysis of growth on solid medium demonstrated that Nar activity is present in both spores and mycelium (hypha). Development of a survival assay with the nitrate analogue chlorate revealed that wild-type spores and mycelium were sensitive to chlorate after anaerobic incubation, independent of the presence of nitrate, while both the and triple mutants were chlorate-resistant. Complementation of the triple mutant with the individual operons delivered on cosmids revealed that each operon encoded an enzyme that was synthesized and active in nitrate or chlorate reduction. The data obtained from these studies allow a tentative assignment of Nar1 activity to spores, Nar2 to spores and mycelium, and Nar3 exclusively to mycelium.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.042572-0
2010-10-01
2019-10-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/10/3166.html?itemId=/content/journal/micro/10.1099/mic.0.042572-0&mimeType=html&fmt=ahah

References

  1. Albrecht, A., Ottow, J. C. G., Benckiser, G., Sich, I. & Russow, R. ( 1997; ). Incomplete denitrification (NO and N2O) from nitrate by Streptomyces violaceoruber and S. nitrosporeus revealed by acetylene inhibition and 15N gas chromatography-quadrupole mass spectrometry analyses. Naturwissenschaften 84, 145–147.[CrossRef]
    [Google Scholar]
  2. Ballantine, S. P. & Boxer, D. H. ( 1985; ). Nickel-containing hydrogenase isoenzymes from anaerobically grown Escherichia coli K-12. J Bacteriol 163, 454–459.
    [Google Scholar]
  3. Bancroft, K., Grant, I. F. & Alexander, M. ( 1979; ). Toxicity of NO2: effect of nitrite on microbial activity in an acid soil. Appl Environ Microbiol 38, 940–944.
    [Google Scholar]
  4. Bell, L. C., Richardson, D. J. & Ferguson, S. J. ( 1990; ). Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha: the periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS Lett 265, 85–87.[CrossRef]
    [Google Scholar]
  5. Bell, L. C., Page, M. D., Berks, B. C., Richardson, D. J. & Ferguson, S. J. ( 1993; ). Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity. J Gen Microbiol 139, 3205–3214.[CrossRef]
    [Google Scholar]
  6. Bentley, S. D., Chater, K. F., Cerdano-Tarrago, A.-M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser, H. & other authors ( 2002; ). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef]
    [Google Scholar]
  7. Bertero, M. G., Rothery, R. A., Palak, M., Hou, C., Lim, D., Blasco, F., Weiner, J. H. & Strynadka, N. C. J. ( 2003; ). Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat Struct Biol 10, 681–687.[CrossRef]
    [Google Scholar]
  8. Blasco, F., Guigliarelli, B., Magalon, A., Asso, M., Giordano, G. & Rothery, R. A. ( 2001; ). The coordination and function of the redox centres of the membrane-bound nitrate reductases. Cell Mol Life Sci 58, 179–193.[CrossRef]
    [Google Scholar]
  9. Chèneby, D., Philippot, L., Hartmann, A., Hénault, C. & Germon, J.-C. ( 2000; ). 16S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol 34, 121–128.[CrossRef]
    [Google Scholar]
  10. Cherepanov, P. P. & Wackernagel, W. ( 1995; ). Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14.[CrossRef]
    [Google Scholar]
  11. Datsenko, K. A. & Wanner, B. L. ( 2000; ). One-step inactivation of chromosomal genes in Escherichia coli using PCR products. Proc Natl Acad Sci U S A 97, 6640–6645.[CrossRef]
    [Google Scholar]
  12. Ellington, M. J. K., Bhakoo, K. K., Sawers, G., Richardson, D. J. & Ferguson, S. J. ( 2002; ). Hierarchy of carbon source selection in Paracoccus pantotrophus: strict correlation between reduction state of the carbon substrate and aerobic expression of the nap operon. J Bacteriol 184, 4767–4774.[CrossRef]
    [Google Scholar]
  13. Goddard, A. D., Moir, J. W. B., Richardson, D. J. & Ferguson, S. J. ( 2008; ). Interdependence of two NarK domains in a fused nitrate/nitrite transporter. Mol Microbiol 70, 667–681.[CrossRef]
    [Google Scholar]
  14. Gregory, M. A., Till, R. & Smith, M. C. M. ( 2003; ). Integration site for Streptomyces phage φBT1 and development of site-specific integrating vectors. J Bacteriol 185, 5320–5323.[CrossRef]
    [Google Scholar]
  15. Gust, B., Challis, G. L., Fowler, K., Kieser, K. & Chater, K. F. ( 2003; ). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100, 1541–1546.[CrossRef]
    [Google Scholar]
  16. 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 Bacteriol 177, 4121–4130.
    [Google Scholar]
  17. Hartley, A. M. & Asai, R. I. ( 1963; ). Spectrophotometric determination of nitrate with 2,6-xylenol reagent. Anal Chem 35, 1207–1213.[CrossRef]
    [Google Scholar]
  18. Hoffmann, T., Troup, B., Szabo, A., Hungerer, C. & Jahn, D. ( 1995; ). The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system. FEMS Microbiol Lett 131, 219–225.[CrossRef]
    [Google Scholar]
  19. Hoffmann, T., Frankenberg, N., Marino, M. & Jahn, D. ( 1998; ). Ammonification in Bacillus subtilis utilizing dissimilatory nitrate reductase is dependent on resDE. J Bacteriol 180, 186–189.
    [Google Scholar]
  20. Hopwood, D. A. ( 2006; ). Soil to genomics: the Streptomyces chromosome. Annu Rev Genet 40, 1–23.[CrossRef]
    [Google Scholar]
  21. Hormann, K. & Andreesen, J. R. ( 1994; ). Purification and characterization of a pyrrole-2-carboxylate oxygenase from Arthrobacter strain Py1. Biol Chem Hoppe Seyler 375, 211–218.
    [Google Scholar]
  22. Jones, R. W. & Garland, P. B. ( 1977; ). Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem J 164, 199–211.
    [Google Scholar]
  23. Jormakka, M., Richardson, D., Byrne, B. & Iwata, S. ( 2004; ). Architecture of NarGH reveals a structural classification of Mo-bisMGD enzymes. Structure 12, 95–104.[CrossRef]
    [Google Scholar]
  24. Kieser, Y., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. ( 2000; ). Practical Streptomyces Genetics. Norwich: The John Innes Foundation.
    [Google Scholar]
  25. Kumon, Y., Sasaki, Y., Kato, I., Takaya, N., Shoun, H. & Beppu, T. ( 2002; ). Codenitrification and denitrification are dual metabolic pathways through which dinitrogen evolves from nitrate in Streptomyces antibioticus. J Bacteriol 184, 2963–2968.[CrossRef]
    [Google Scholar]
  26. Lanciano, P., Vergnes, A., Grimaldi, S., Guigliarelli, B. & Magalon, A. ( 2007; ). Biogenesis of a respiratory complex is orchestrated by a single accessory protein. J Biol Chem 282, 17468–17474.[CrossRef]
    [Google Scholar]
  27. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. ( 1951; ). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275.
    [Google Scholar]
  28. Lund, K. & DeMoss, J. A. ( 1976; ). Association-dissociation behaviour and subunit structure of heat-released nitrate reductase from Escherichia coli. J Biol Chem 251, 2207–2216.
    [Google Scholar]
  29. MacGregor, C. H. ( 1975; ). Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli. J Bacteriol 121, 1117–1121.
    [Google Scholar]
  30. MacNeil, D. J., Gewain, K. M., Ruby, C. L., Dezeny, G., Gibbons, P. H. & MacNeil, T. ( 1992; ). Analysis of Streptomyces avermitilis genes required for avermectin synthesis utilizing a novel integration vector. Gene 111, 61–68.[CrossRef]
    [Google Scholar]
  31. Malm, S., Tiffert, Y., Micklinghoff, J., Schultze, S., Joost, I., Weber, I., Horst, S., Ackermann, B., Schmidt, M. & other authors ( 2009; ). The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology 155, 1332–1339.[CrossRef]
    [Google Scholar]
  32. Noji, S. & Taniguchi, S. ( 1987; ). Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells. J Biol Chem 262, 9441–9443.
    [Google Scholar]
  33. Packter, N. M. & Collins, J. S. ( 1974; ). Effect of inhibitors of protein synthesis on the formation of phenols derived from acetate and shikimic acid in Aspergillus fumigatus. Eur J Biochem 42, 291–302.[CrossRef]
    [Google Scholar]
  34. Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. A. ( 1996; ). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, 77–96.[CrossRef]
    [Google Scholar]
  35. Richardson, D. J., Berks, B. C., Russell, D. A., Spiro, S. & Taylor, C. J. ( 2001; ). Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58, 165–178.[CrossRef]
    [Google Scholar]
  36. Rider, B. F. & Mellon, M. G. ( 1946; ). Colorimetric determination of nitrites. Ind Eng Chem Anal Ed 18, 96–99.[CrossRef]
    [Google Scholar]
  37. Robertson, L. A. & Kuenen, J. G. ( 1984; ). Aerobic denitrification – old wine in new bottles? Antonie van Leeuwenhoek 50, 525–544.[CrossRef]
    [Google Scholar]
  38. Rothery, R. A., Blasco, F., Magalon, A. & Weiner, J. H. ( 2001; ). The diheme cytochrome b subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI): structure, function and interaction with quinols. J Mol Microbiol Biotechnol 3, 273–283.
    [Google Scholar]
  39. 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]
  40. Schwarz, G., Mendel, R. R. & Ribbe, M. W. ( 2009; ). Molybdenum cofactors, enzymes and pathways. Nature 460, 839–847.[CrossRef]
    [Google Scholar]
  41. Shoun, H., Kano, M., Baba, I., Takaya, N. & Matsuo, M. ( 1998; ). Denitrification by actinomycetes and purification of dissimilatory nitrite reductase and azurin from Streptomyces thioluteus. J Bacteriol 180, 4413–4415.
    [Google Scholar]
  42. Showe, M. K. & DeMoss, J. A. ( 1968; ). Localization and regulation of synthesis of nitrate reductase in Escherichia coli. J Bacteriol 95, 1305–1313.
    [Google Scholar]
  43. Sohaskey, C. D. ( 2005; ). Regulation of nitrate reductase activity in Mycobacterium tuberculosis by oxygen and nitric oxide. Microbiology 151, 3803–3810.[CrossRef]
    [Google Scholar]
  44. Sohaskey, C. D. & Modesti, L. ( 2009; ). Differences in nitrate reduction between Mycobacterium tuberculosis and Mycobacterium bovis are due to differential expression of both narGHJI and narK2. FEMS Microbiol Lett 290, 129–134.
    [Google Scholar]
  45. Sohaskey, C. D. & Wayne, L. G. ( 2003; ). Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis. J Bacteriol 185, 7247–7256.[CrossRef]
    [Google Scholar]
  46. Tiffert, Y., Supra, P., Wurm, R., Wohlleben, W., Wagner, R. & Reuther, J. ( 2008; ). The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomyces. Mol Microbiol 67, 861–880.[CrossRef]
    [Google Scholar]
  47. van Keulen, G., Jonkers, H. M., Claessen, D., Dijkhuizen, L. & Wösten, H. A. B. ( 2003; ). Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor. J Bacteriol 185, 1455–1458.[CrossRef]
    [Google Scholar]
  48. van Keulen, G., Alderson, J., White, J. & Sawers, R. G. ( 2005; ). Nitrate respiration in the actinomycete Streptomyces coelicolor. Biochem Soc Trans 33, 210–212.[CrossRef]
    [Google Scholar]
  49. van Keulen, G., Alderson, J., White, J. & Sawers, R. G. ( 2007; ). The obligate aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods of anaerobic stress. Environ Microbiol 9, 3143–3149.[CrossRef]
    [Google Scholar]
  50. Wang, J. & Zhao, G.-P. ( 2009; ). GlnR positively regulates nasA transcription in Streptomyces coelicolor. Biochem Biophys Res Commun 386, 77–81.[CrossRef]
    [Google Scholar]
  51. Wayne, L. G. & Hayes, L. G. ( 1998; ). Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis. Tuber Lung Dis 79, 127–132.[CrossRef]
    [Google Scholar]
  52. Wood, N. J., Allzadeh, T., Richardson, D. J., Ferguson, S. J. & Moir, J. W. B. ( 2002; ). Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus denitrificans. Mol Microbiol 44, 157–170.[CrossRef]
    [Google Scholar]
  53. 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.0.042572-0
Loading
/content/journal/micro/10.1099/mic.0.042572-0
Loading

Data & Media loading...

Supplements

vol. , part 10, pp. 3166 - 3179

Oligonucleotides used in this study [PDF](40 KB)



PDF
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