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Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii


Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nif, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nijZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both niJZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.

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  1. Beynon J, Ally A, Cannon M, Cannon F, Jacobson M, Cash V, Dean D (1987) Comparative organization of nitrogen fixationspecific genes from Azotobacter vinelandii and Klebsiella pneumoniae: DNA sequence of the nifUSV genes. J Bacteriol 169:4024–4029

  2. Brigle KE, Weiss MC, Newton WE, Dean DR (1987) Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes nifD and nifK. J Bacteriol 169:1547–1553

  3. Buck M, Miller S, Drummond M, Dixon R (1986) Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature 320:370–378

  4. Earl CD, Ronson C, Ausubel FM (1987) Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes. J Bacteriol 169:1127–1136

  5. Filler WA, Kemp RM, Ng JC, Hawkes TR, Dixon RA, Smith BE (1986) The nifH gene product is required for the synthesis or stability of the iron-molybdenum cofactor of nitrogenase from Klebsiella pneumoniae. Eur J Biochem 160: 371–377

  6. Gubler M, Hennecke H (1986) FixA, B and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett 200:186–192

  7. Hawkes TR, McLean PA, Smith BE (1984) Nitrogenase from nifV mutants of Klebsiella pneumoniae contains an altered form of the iron-molybdenum cofactor. Biochem J 217: 317–321

  8. Hoover TR, Shah VK, Roberts GP, Ludden PW (1986) nifV-de-pendent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase. J Bacteriol 167:999–1003

  9. Hoover TR, Robertson AD, Cerny RC, Hayes RN, Imperial J, Shah VK, Ludden PW (1987) Identification of the V factor needed for synthesis of the iron-molybdenum cofactor of nitrogenase as homocitrate. Nature 329:855–857

  10. Hoover TR, Imperial J, Liang J, Ludden PW, Shah VK (1988a) Dinitrogenase with altered substrate specificity results from the use of homocitrate analogues for in vitro synthesis of the ironmolybdenum cofactor. Biochemistry 27:3647–3652

  11. Hoover TR, Imperial J, Ludden PW, Shah VK (1988b) Homocitrate cures the NifV phenotype in Klebsiella pneumoniae. J Bacteriol 170:1978–1979

  12. Hoover TR, Imperial J, Ludden PW, Shah VK (1989) Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase. Biochemistry 28:2768–2771

  13. Hooward KS, McLean PA, Hansen FB, Lemley PV, Koblan KS, Orme-Johnson WH (1986) Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. J Biol Chem 261:772–778

  14. Howard JB, Davis R, Moldenhauser B, Cash VL, Dean DR (1989) Fe: S cluster ligands are the only cysteines required for nitrogenase Fe-protein activities. J Biol Chem 264:11270–11274

  15. Jacobson MR, Premakumar R, Bishop PE (1986) Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J Bacteriol 167:480–486

  16. Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR (1989) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol 171:1017–1027

  17. Joerger RD, Bishop PE (1988) Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J Bacteriol 170:1475–1487

  18. Joerger RD, Premakumar R, Bishop PE (1986) Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions. J Bacteriol 168:673–682

  19. Kennedy C, Gamal R, Humphrey R, Ramos J, Brigle K, Dean D (1986) The nifH and nifM genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLAFR1 gene banks. Mol Gen Genet 205:318–325

  20. McLean PA, Dixon RA (1981) Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature 292:655–656

  21. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78

  22. O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021

  23. Pagani S, Eldridge M, Eady RR (1987) Nitrogenase of Klebsiella pneumoniae: rhodenase-catalyzed restoration of activity of the inactive 2Fe species of the Fe protein. Biochem J 244:485–488

  24. Page WJ, von Tigerstrom M (1979) Optimal conditions for transformation of Azotobacter vinelandii. J Bacteriol 139:1058–1061

  25. Paul W, Merrick M (1987) The nucleotide sequence of the nifM gene of Klebsiella pneumoniae and identification of a new nif gene: nifZ. Eur J Biochem 170:259–265

  26. Paul W, Merrick M (1989) The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis. Eur J Biochem 178:675–682

  27. Roberts GP, MacNeil T, MacNeil D, Brill WJ (1978) Regulation and Characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol 135:267–279

  28. Robinson AC, Burgess BK, Dean DR (1986) Activity, reconstitution and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J Bacteriol 166:180–186

  29. Robinson AC, Dean DR, Burgess BK (1987) Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein nitrogenase. J Biol Chem 262:14327–14332

  30. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

  31. Sibold L (1982) The polar effect on nifM of mutations in the nifU, -S, -V genes of Klebsiella pneumoniae depends on their plasmid or chromosomal location. Mol Gen Genet 186:569–571

  32. Smith BE, Buck M, Eady RR, Lowe DJ, Thorneley RNF, Ashby G, Deistung J, Eldridge M, Fisher K, Gormal C, Ioannidis I, Kent H, Arber J, Flood A, Garner CD, Hasnain S, Miller R (1988) Recent studies on the structure and function of molybdenum nitrogenase. In: Bothe H, de Bruijn FJ, Newton WE (eds) Nitrogen fixation: hundred years after. Gustav Fischer Verlag, Stuttgart, Federal Republic of Germany, pp 91–100

  33. Strandberg GW, Wilson PW (1968) Formation of the nitrogenfixing enzyme system in Azotobacter vinelandii. Can J Microbiol 14:25–31

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Correspondence to Dennis R. Dean.

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Communicated by J. Schell

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Jacobson, M.R., Cash, V.L., Weiss, M.C. et al. Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii . Molec. Gen. Genet. 219, 49–57 (1989).

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Key words

  • nifU
  • nifS
  • nifV
  • nifW
  • nifZ
  • nifM
  • Azotobacter vinelandii