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J Bacteriol. Dec 1991; 173(23): 7565–7572.
PMCID: PMC212524

Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii.


The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnfENX. The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+). However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated. AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions.

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  • Aguilar OM, Taormino J, Thöny B, Ramseier T, Hennecke H, Szalay AA. The nifEN genes participating in FeMo cofactor biosynthesis and genes encoding dinitrogenase are part of the same operon in Bradyrhizobium species. Mol Gen Genet. 1990 Dec;224(3):413–420. [PubMed]
  • Arnold W, Rump A, Klipp W, Priefer UB, Pühler A. Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol. 1988 Oct 5;203(3):715–738. [PubMed]
  • Beynon J, Cannon M, Buchanan-Wollaston V, Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. [PubMed]
  • Bishop PE, Hawkins ME, Eady RR. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J. 1986 Sep 1;238(2):437–442. [PMC free article] [PubMed]
  • Bishop PE, Premakumar R, Dean DR, Jacobson MR, Chisnell JR, Rizzo TM, Kopczynski J. Nitrogen Fixation by Azotobacter vinelandii Strains Having Deletions in Structural Genes for Nitrogenase. Science. 1986 Apr 4;232(4746):92–94. [PubMed]
  • Brigle KE, Newton WE, Dean DR. Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene. 1985;37(1-3):37–44. [PubMed]
  • Brigle KE, Weiss MC, Newton WE, Dean DR. 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. 1987 Apr;169(4):1547–1553. [PMC free article] [PubMed]
  • Bulen WA, LeComte JR. The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci U S A. 1966 Sep;56(3):979–986. [PMC free article] [PubMed]
  • Burgess BK, Jacobs DB, Stiefel EI. Large-scale purification of high activity Azotobacter vinelandII nitrogenase. Biochim Biophys Acta. 1980 Jul 10;614(1):196–209. [PubMed]
  • DE WITT CW, ROWE JA. N,O-Diacetylneuraminic acid and N-acetylneuraminic acid in Escherichia coli. Nature. 1959 Aug 1;184(Suppl 6):381–382. [PubMed]
  • Chisnell JR, Premakumar R, Bishop PE. Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol. 1988 Jan;170(1):27–33. [PMC free article] [PubMed]
  • Dean DR, Brigle KE. Azotobacter vinelandii nifD- and nifE-encoded polypeptides share structural homology. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5720–5723. [PMC free article] [PubMed]
  • Dean DR, Setterquist RA, Brigle KE, Scott DJ, Laird NF, Newton WE. Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein alpha-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not. Mol Microbiol. 1990 Sep;4(9):1505–1512. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Gosink MM, Franklin NM, Roberts GP. The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon. J Bacteriol. 1990 Mar;172(3):1441–1447. [PMC free article] [PubMed]
  • Hales BJ, Langosch DJ, Case EE. Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem. 1986 Nov 15;261(32):15301–15306. [PubMed]
  • Jacobson MR, Brigle KE, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR. Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol. 1989 Feb;171(2):1017–1027. [PMC free article] [PubMed]
  • Joerger RD, Bishop PE. Bacterial alternative nitrogen fixation systems. Crit Rev Microbiol. 1988;16(1):1–14. [PubMed]
  • Joerger RD, Jacobson MR, Bishop PE. Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii. J Bacteriol. 1989 Jun;171(6):3258–3267. [PMC free article] [PubMed]
  • Joerger RD, Jacobson MR, Premakumar R, Wolfinger ED, Bishop PE. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J Bacteriol. 1989 Feb;171(2):1075–1086. [PMC free article] [PubMed]
  • Joerger RD, Loveless TM, Pau RN, Mitchenall LA, Simon BH, Bishop PE. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J Bacteriol. 1990 Jun;172(6):3400–3408. [PMC free article] [PubMed]
  • Johnston RE, Mackenzie JM, Jr, Dougherty WG. Assembly of overlapping DNA sequences by a program written in BASIC for 64K CP/M and MS-DOS IBM-compatible microcomputers. Nucleic Acids Res. 1986 Jan 10;14(1):517–527. [PMC free article] [PubMed]
  • Kent HM, Baines M, Gormal C, Smith BE, Buck M. Analysis of site-directed mutations in the alpha- and beta-subunits of Klebsiella pneumoniae nitrogenase. Mol Microbiol. 1990 Sep;4(9):1497–1504. [PubMed]
  • Messing J, Gronenborn B, Müller-Hill B, Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. [PMC free article] [PubMed]
  • Moreno-Vivian C, Schmehl M, Masepohl B, Arnold W, Klipp W. DNA sequence and genetic analysis of the Rhodobacter capsulatus nifENX gene region: homology between NifX and NifB suggests involvement of NifX in processing of the iron-molybdenum cofactor. Mol Gen Genet. 1989 Apr;216(2-3):353–363. [PubMed]
  • Page WJ, von Tigerstrom M. Optimal conditions for transformation of Azotobacter vinelandii. J Bacteriol. 1979 Sep;139(3):1058–1061. [PMC free article] [PubMed]
  • Paustian TD, Shah VK, Roberts GP. Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6082–6086. [PMC free article] [PubMed]
  • Paustian TD, Shah VK, Roberts GP. Apodinitrogenase: purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase. Biochemistry. 1990 Apr 10;29(14):3515–3522. [PubMed]
  • Robinson AC, Burgess BK, Dean DR. Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J Bacteriol. 1986 Apr;166(1):180–186. [PMC free article] [PubMed]
  • Robson RL, Woodley PR, Pau RN, Eady RR. Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J. 1989 Apr;8(4):1217–1224. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Setterquist R, Brigle KE, Beynon J, Cannon M, Ally A, Cannon F, Dean DR. Nucleotide sequence of the nifE and nifN genes from Klebsiella pneumoniae. Nucleic Acids Res. 1988 Jun 10;16(11):5215–5215. [PMC free article] [PubMed]
  • Shah VK, Brill WJ. Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3249–3253. [PMC free article] [PubMed]
  • Simon R, Quandt J, Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene. 1989 Aug 1;80(1):161–169. [PubMed]
  • Smith BE, Eady RR, Lowe DJ, Gormal C. The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor. Biochem J. 1988 Feb 15;250(1):299–302. [PMC free article] [PubMed]
  • Strandberg GW, Wilson PW. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol. 1968 Jan;14(1):25–31. [PubMed]
  • Ugalde RA, Imperial J, Shah VK, Brill WJ. Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase. J Bacteriol. 1984 Sep;159(3):888–893. [PMC free article] [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

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