Antonie van Leeuwenhoek

, Volume 85, Issue 3, pp 229–235 | Cite as

Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum

  • Leonardo Velasco
  • Socorro Mesa
  • Chang-ai Xu
  • María J. Delgado
  • Eulogio J. Bedmar


The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a PnosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK2.

Bradyrhizobium japonicum Denitrification Microerobiosis Respiratory nitrous oxide reductase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anthamatten D. and Hennecke H. 1991. The regulatory status of the fixL-and fixJ-like genes in Bradyrhizobium japonicum may be different from that in Rhizobium meliloti. Mol. Gen. Genet. 225: 38-48.Google Scholar
  2. Arai H., Kodama T. and Higarashi 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.Google Scholar
  3. Berks B.C., Sargent F. and Palmer T. 2000. The Tat protein export pathway. Mol. Microbiol. 35: 260-74.Google Scholar
  4. Blight M.A. and Holland I.B. 1990. Structure and function of haemolysin B,P-glycoprotein and other members of a novel family of membrane translocators. Mol. Microbiol. 4: 873-880.Google Scholar
  5. Brown K., Tegoni M., Prudencio M., Pereira A.S., Besson S., Moura J.J., Moura I. and Cambillau C. 2000a. A novel type of catalytic copper cluster in nitrous oxide reductase. Nat. Struct. Biol. 7: 191-195.Google Scholar
  6. Brown K., Djinovic-Carugo K., Haltia T., Cabrito I., Saraste M., Moura J.J., Moura I., Tegoni M. and Cambillau C. 2000b. Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur. J. Biol. Chem. 275: 41133-41136.Google Scholar
  7. Chan Y.K., McCormick W.A. and Watson R.J. 1997. A new nos gene downstream from nosDFY is essential for dissimilatory reduction of nitrous oxide by Rhizobium (Sinorhizobium) meliloti. Microbiology 143: 2817-2824.Google Scholar
  8. Cuypers H., Viebrock-Sambale A. and Zumft W.G. 1992. NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri. J. Bacteriol. 174: 5332-5339.Google Scholar
  9. Fischer H.M. 1994. Genetic regulation of nitrogen fixation in rhizobia. Microbiol. Rev. 58: 352-386.Google Scholar
  10. Fischer H.M., Velasco L., Delgado M.J., Bedmar E.J., Schären S., Zingg D., Göttfert M. and Hennecke H. 2001. One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis. J. Bacteriol. 183: 1300-1311.Google Scholar
  11. Fischer H.M., Sciotti M.A. and Hennecke H. 2002. The network controlling sysmbiotic nitrogen fixation genes in Bradyrhizobium japonicum. In: Finan T.M., O'Brian M.R., Layzell D.B., Vesser J.K. and Newton W. (eds), Nitrogen fixation: Global Perspectives. Cabi Publishing, UK, pp. 213-217.Google Scholar
  12. Hoeren F.U., Berks B.C., Ferguson S.J. and McCarthy J.E. 1993. Sequence and expression of the gene encoding the respiratory nitrous-oxide reductase from Paracoccus denitrificans. New and conserved structural and regulatory motifs. Eur. J. Biochem. 218: 49-57.Google Scholar
  13. Holloway P., McCormick W., Watson R.J. and Chan Y.K. 1996. Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti. J. Bacteriol. 178: 1505-1514.Google Scholar
  14. Holm L., Saraste M. and Wikström M. 1987. Structural models of the redox centres in cytochrome oxidase. EMBO J. 6: 2819-2823.Google Scholar
  15. Honisch U. and Zumft W.G. 2003. Operon structure and regulation of the nos gene region of Pseudomonas stutzeri, encoding an ABC-Type ATPase for maturation of nitrous oxide reductase. J. Bacteriol. 185: 1895-1902.Google Scholar
  16. Kwiatkowski A. and Shapleigh J.P. 1996. Requirement of nitric oxide for induction of genes whose products are involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J. Biol. Chem. 271: 24382-24388.Google Scholar
  17. Matsubara T. and Zumft W.G. 1982. Identification of a copper protein as part of nitrous oxide-reducing system in nitrite respiring (denitrifying) pseudomonads. Arch. Microbiol. 132: 322-328.Google Scholar
  18. McGuirl M.A., Nelson L.K., Bollinger J.A., Chan Y.K. and Dooley D.M. 1998. The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: cloning, sequence analysis, and expression. J. Inorg. Biochem. 70: 155-69.Google Scholar
  19. McGuirl M.A., Bollinger J.A., Cosper N., Scott R.A. and Dooley D.M. 2001. Expression, purification, and characterization of NosL, a novel Cu(I) protein of the nitrous oxide reductase (nos) gene cluster. J. Biol. Inorg. Chem. 6: 189-195.Google Scholar
  20. Mesa S., Velasco L., Manzanera M.E., Delgado M.J. and Bedmar E.J. 2002. Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum. Microbiology 148: 3553-3560.Google Scholar
  21. Miller J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Habor, New York, USA.Google Scholar
  22. Nellen-Anthamatten D., Rossi P., Kullik P.I., Babst M., Fisher H.M. and Hennecke H. 1998. Bradyrhizobium japonicum FixK2, a crucial distributor in the FixLJ-dependent regulatory cascade for control of genes inducible by low oxygen. J. Bacteriol. 180: 5251-5255.Google Scholar
  23. Okata E. and Ooi T. 1987. Examination of protein sequence homologies IV. Twenty-seven bacterial ferredoxins. J. Mol. Evol. 26: 45-54.Google Scholar
  24. Philippot L., Mirleau P., Mazurier S., Siblot A., Hartmann A., Lemanceau P. and Germon J.C. 2001. Characterization and transcriptional analysis of Pseudomonas fluorescens denitrifying clusters containing the nar, nir, nor, and nos genes. Biochim. Biophys. Acta 1517: 436-440.Google Scholar
  25. Prentki P. and Krisch H.M. 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29: 303-13.Google Scholar
  26. Pugsley A.P. 1993. The complete general secretory pathway in gram-negative bacteria. Microbiol. Rev. 57: 50-108.Google Scholar
  27. Rasmussen T., Berks B.C., Sanders-Loehr J., Dooley D.M., Zumft W.G. and Thomson A.J. 2000. The catalytic center in nitrous oxide reductase, CuZ, is a copper-sulfide cluster. Biochemistry 39: 12753-12756.Google Scholar
  28. Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.Google Scholar
  29. Saraste M., Sibbald P.R. and Wittinghoffe A. 1990. The P-loop, a common motif in ATP-and GTP-binding proteins. Trends Biochem. Sci. 15: 430-434.Google Scholar
  30. Saunders N.F., Hornberg J.J., Reijnders W.N., Westerhoff H.V., de Vries S. and van Spanning R.J. 2000. The NosX and NirX proteins of Paracoccus denitrificans are functional homologues: their role in maturation of nitrous oxide reductase. J. Bacteriol. 182: 5211-5217.Google Scholar
  31. Simon R., Priefer U. and 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
  32. Spaink H.P., Okker H.R.J., Wijffelman C.A., Pees E. and Lugtenberg B.J.J. 1987. Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol. Biol. 9: 27-39.Google Scholar
  33. Spiro S. 1994. The FNR family of transcriptional regulators. Antonie Van Leeuwenhoek 66: 23-36.Google Scholar
  34. Tosques I.E., Shi J. and 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
  35. Vairinhos F., Wallace W. and Nicholas D.J.D. 1989. Simultaneous assimilation and denitrification of nitrate by Bradyrhizobium japonicum. J. Gen. Microbiol. 135: 189-193.Google Scholar
  36. van Spanning R.J.M., de Boer A.P., Reijnders W.N., Spiro S., Westerhoff H.V., Stouthamer A.H. and 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
  37. Velasco L., Mesa S., Delgado M.J. and Bedmar E.J. 2001. Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum. Biochim. Biophys. Acta 1521: 130-134.Google Scholar
  38. Viebrock A. and Zumft W.G. 1988. Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri. J. Bacteriol. 170: 4658-4668.Google Scholar
  39. Vincent J.M. 1974. Root-nodule symbioses with Rhizobium. In: Quispel A. (ed.), The Biology of nitrogen fixation. American Elsevier Publishing Company Inc., New York, NY, USA. pp. 265-341.Google Scholar
  40. Vollack K.U. and Zumft W.G. 2001. Nitric oxide signaling and transcriptional control of denitrification genes in Pseudomonas stutzeri. J. Bacteriol. 183: 2516-2526.Google Scholar
  41. Zumft W.G. 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 4: 533-616.Google Scholar
  42. Zumft W.G., Viebrock-Sambale A. and Braun C. 1990. Nitrous oxide reductase from denitrifying Pseudomonas stutzeri. Genes for copper-processing and properties of the deduced products, including a new member of the family of ATP/GTP-binding proteins. Eur. J. Biochem. 192: 591-599.Google Scholar
  43. Zumft W.G., Dreusch A., Lochelt S., Cuypers H., Friedrich B. and Schneider B. 1992. Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase. Eur. J. Biochem. 208: 31-40.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Leonardo Velasco
    • 1
  • Socorro Mesa
    • 2
  • Chang-ai Xu
    • 3
  • María J. Delgado
    • 4
  • Eulogio J. Bedmar
    • 4
  1. 1.Departamento de Biotecnología. Centro de Investigación y Desarrollo AgroalimentarioMurciaSpain
  2. 2.Institut für MikrobiologieZürichSwitzerland
  3. 3.Agriculture and Agri-Food Canada, Potato Research CentreFredericton, NBCanada
  4. 4.Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSICGranadaSpain

Personalised recommendations