Three Nitrogen Fixation Systems in Azotobacter vinelandii

  • P. E. Bishop
  • R. D. Joerger
  • R. Premakumar
Part of the 41. Colloquium der Gesellschaft für Biologische Chemie 5.–7. April 1990 in Mosbach/Baden book series (MOSBACH, volume 41)

Abstract

In the early 1980s we presented evidence indicating that the N2-fixing bacterium, Azotobacter vinelandii, contained at least two nitrogenase systems: the conventional Mo-containing nitrogenase (nitrogenase-1) system; and an alternative nitrogenase system expressed in the absence of Mo. This evidence primarily centered on the observation that Nif- (unable to fix N2) mutant strains underwent phenotypic reversal (i.e., Nif- to Nif+) under conditions of Mo deprivation. These reports (Bishop et al. 1980; Bishop et al. 1982; Page and Collinson 1982; Premakumar et al. 1984) were received with skepticism because they challenged the long-held belief that Mo was absolutely required for N2 fixation and that nitrogenases were essentially the same regardless of their source. The latter notion was further supported by the results of Southern blot experiments by Ruvkun and Ausubel (1980) which indicated that some of the structural genes encoding nitrogenases from diverse diazotrophic organisms were highly conserved at the nucleotide sequence level.

Keywords

Sulfide Catalysis Cysteine Electrophoresis Vanadium 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bishop, P.E., Jarlenski, D.M.L. & Hetherington, D.R. (1980) Evidence for an alternative nitogen fixation system in Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA 77:7342–7346PubMedCrossRefGoogle Scholar
  2. Bishop, P.E., Jarlenski, D.M.L. & Hetherington, D.R. (1982) Expression of an alternative nitrogen fixation system in Azotobacter vinelandii. J. Bacteriol. 150:1244–1251PubMedGoogle Scholar
  3. Bishop, P.E., Hawkins, M.E. & Eady, R.R. (1986a) Nitrogen fixation in Mo-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem. J. 238:437–442PubMedGoogle Scholar
  4. Bishop, P.E., Premakumar, R., Dean, D.R., Jacobson, M.R., Chisnell, J.R., Rizzo, T.M. & Kopczynski, J. (1986b) Nitrogen fixation by A zotobacter vinelandii strains having deletions in structural genes for nitrogenase. Science 232:92–94PubMedCrossRefGoogle Scholar
  5. Brigle, K.E., Setterquist, R.A., Dean, D.R., Cantwell, J.S., Weiss, M.C. & Newton, W.E. (1987) Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA 84:7066–7069PubMedCrossRefGoogle Scholar
  6. Chisnell, J.R., Premakumar, R. & Bishop, P.E. (1988) Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J. Bacteriol. 170:27–33PubMedGoogle Scholar
  7. Eady, R.R. (1986) Enzymology in free-living diazotrophs. In: Broughton, W.J., Piihler, A. (eds.) Nitrogen fixation. Oxford, Clarendon Press 4:1–49Google Scholar
  8. Eady, R.R., Robson, R.L., Richardson, T.H., Miller, R.W. & Hawkins, M. (1987) The vanadium nitrogenase of Azotobacter chroococcum: purification and properties of the VFe protein. Biochem. J. 244:197–207PubMedGoogle Scholar
  9. Eady, R.R., Richardson, T.H., Miller, R.W., Hawkins, M. & Lowe, D.J. (1988) The vanadium nitrogenase of Azotobacter chroococcum: purification and properties of the Fe protein. Biochem. J. 256:189–196PubMedGoogle Scholar
  10. Fischer, M.-H., Bruderer, T. & Hennecke, H. (1988) Essential and nonessential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues involved in redox reactivity and/or metal binding. Nucleic Acids Res. 16:2207–2224PubMedCrossRefGoogle Scholar
  11. Gillum, W.O., Mortenson, L.E., Chen, J.S. & Holm, R.H. (1977) Quantitative extrusion of the Fe4S4 cores of active sites of ferredoxins and the hydrogenase of Clostridium pasteurianum. J. Am. Chem. Soc. 99:584–595PubMedCrossRefGoogle Scholar
  12. Hales, B.J., Case, E.E., Momingstar, J.E., Dzeda, M.F. & Mauterer, L.A., (1986a) Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25:7251–7255PubMedCrossRefGoogle Scholar
  13. Hales, B.J., Langosch, D.J. & Case, E.E. (1986b) Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J. Biol. Chem. 261:15301–15306PubMedGoogle Scholar
  14. Hausinger, R.P. & Howard, J.B. (1983) Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii. J. Biol. Chem. 258:13486–13492PubMedGoogle Scholar
  15. Jacobson, M.R., Brigle, K.E., Bennett, L.T., Setterquist, R.A., Wilson, M.S., Cash, V.L., Beynon, J., Newton, W.E. & Dean, D.R. (1989) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J. Bacteriol. 171:1017–1027PubMedGoogle Scholar
  16. Joerger, R.D. & Bishop, P.E. (1988) Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J. Bacteriol. 170:1475–1487PubMedGoogle Scholar
  17. Joerger, R.D., Premakumar, R. & Bishop, P.E. (1986) Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions. J. Bacteriol. 168:673–682PubMedGoogle Scholar
  18. Joerger, R.D., Jacobson, M.R. & Bishop, P.E. (1989a) Two nifA like genes required for expression of alternative nitrogenases by Azotobacter vinelandii. J. Bacteriol. 171:3258–3267PubMedGoogle Scholar
  19. Joerger, R.D., Jacobson, M.R., Premakumar, R., Wolfinger, E.D. & Bishop, P.E. (1989b) Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J. Bacteriol. 171:1075–1086PubMedGoogle Scholar
  20. Joerger, R.D., Loveless, T.M., Pau, R.N., Mitchenall, L.A., Simon, B.H. & Bishop, P.E. (1990) Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J. Bacteriol. 172:3400–3408PubMedGoogle Scholar
  21. Kennedy, C., Gamal, R., Humphrey, R., Ramos, J., Brigle, K. & Dean, D. (1986) The nifH, and nifN genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLAFRl gene banks. Mol. Gen. Genet. 205:318–325CrossRefGoogle Scholar
  22. McLean, P.A., Papaefthymiou, V., Münck, E. & Orme-Johnson, W.H. (1988) Use of isotopic hybrids of the MoFe protein to study the mechanism of nitrogenase catalysis. In: Bothe, H., deBruijn, F.J., Newton, W.E. (eds.) Nitrogen fixation: hundred years after. Fischer, Stuttgart, pp. 101–106Google Scholar
  23. Page, WJ. & Collinson, S.K. (1982) Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif mutant. Can. J. Microbiol. 28:1173–1180CrossRefGoogle Scholar
  24. Pau, R.N., Mitchenall, L.A. & Robson, R.L. (1989) Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J. Bacteriol. 171:124–129PubMedGoogle Scholar
  25. Premakumar, R., Lemos, E.M. & Bishop, P.E. (1984) Evidence for two dinitrogenase reductases under regulatory control by molybdenum in Azotobacter vinelandii. Biochim. Biophys. Acta 797:64–70CrossRefGoogle Scholar
  26. Raina, R., Reddy, M.A., Ghosal, D. & Das, H.K. (1988) Characterization of the gene for the Fe-protein otihe vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tα5 mutant. Mol. Gen. Genet. 214:121–127PubMedCrossRefGoogle Scholar
  27. Robson, R.L. (1986) Nitrogen fixation in strains of Azotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase. Arch. Microbiol. 146:74–79CrossRefGoogle Scholar
  28. Robson, R.L., Eady, R.R., Richardson, T.H., Miller, R.W., Hawkins, M. & Postgate, J.R. (1986a) The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme. Nature (Lond) 322:388–390CrossRefGoogle Scholar
  29. Robson, R.L., Woodley, P.R. & Jones, R. (1986b) Second gene (nifH*) coding for a nitrogenase iron-protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein. EMBO J. 5:1159–1163PubMedGoogle Scholar
  30. Robson, R.L., Woodley, P.R., Pau, R.N. & Eady, R.R. (1989) Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J. 8:1217–1224PubMedGoogle Scholar
  31. Ruvkun, G.B. & Ausubel, F.M. (1980) Interspecies homology of nitrogenase genes. Proc. Natl. Acad. Sci. USA 77:191–195PubMedCrossRefGoogle Scholar
  32. Smith, B.E., Bishop, P.E., Dixon, R.A., Eady, R.R., Filler, W.A., Lowe, D.J., Richards, A.J.M., Thomson, A.J., Thornley, R.N.F. & Postgate, J.R. (1985) The iron-molybdenum cofactor of nitrogenase. In: Evans, H.J., Bottomley, P.J., Newton, W.E. (eds.) Nitrogen fixation research progress. Nijhoff, Dordrecht Boston, pp. 597–603CrossRefGoogle Scholar
  33. Smith, B.E., Eady, R.R., Lowe, D.J. & Gormal, C. (1988) The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor. Biochem. J. 250:299–302PubMedGoogle Scholar
  34. Souillard, N. & Sibold, L. (1989) Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotro-phicus. Mol. Microbiol. 3:441–552CrossRefGoogle Scholar
  35. Thöny, B., Kaluza, K. & Hennecke, H. (1985) Structural and functional homology between the α and β subunits of the nitrogenase MoFe protein as revealed by sequencing the Rhizobium japonicum nifK gene. Mol. Gen. Genet. 198:441–448CrossRefGoogle Scholar
  36. Wang, S.-Z., Chen, J.-S. & Johnson, J.L. (1988) The presence of five nifH-like sequences in Clostridium pasteurianum: sequence divergence and transcriptional properties. Nucl. Acids Res. 16:439–454PubMedCrossRefGoogle Scholar
  37. Wolfinger, E.D., Pau, R.N. & Bishop, P.E. (1989) Multiple nifE-and nifN-like genes in Azotobacter vinelandii. Ann. Mtg. Am. Soc. Microbiol. (Abstr H-97) 185Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1990

Authors and Affiliations

  • P. E. Bishop
  • R. D. Joerger
  • R. Premakumar
    • 1
  1. 1.North Carolina State UniversityDepartment of Microbiology and United States Department of Agriculture, Agricultural Research ServiceRaleighUSA

Personalised recommendations