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Gene Review

nifA  -  nitrogen fixation protein NifA

Sinorhizobium fredii NGR234

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Disease relevance of nifA

  • In Azotobacter vinelandii (gamma-subgroup) nifA is cotranscribed with a second gene nifL [1].
  • We report the sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE [2].
  • The nifA gene has two possible translation start sites, both of which are used in an Escherichia coli background, resulting in proteins with apparent molecular weights of 58 kD and 57 kD; initiation at the second site is preferred over initiation at the first [2].
  • Five Tn5-induced Nif- mutants of Azotobacter vinelandii were characterized as regulatory mutants because they were restored to Nif+ by the introduction of constitutively expressed nifA from Klebsiella pneumoniae [3].
  • A gene bank of Azospirillum lipoferum Br17 constructed in the vector lambda GEM11 was screened with a Bradyrhizobium japonicum nifA gene probe [4].

High impact information on nifA

  • Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA [5].
  • The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression [5].
  • This activation required direct interaction of NifA with the essential -173 to -88 regulatory region [5].
  • The presence of four conserved cysteine residues in the NifA protein might be an indication that NifA is directly sensitive to oxygen [1].
  • Activation of NifA upon removal of fixed N seems to involve, either directly or indirectly, the signal transduction protein P(II) [1].

Chemical compound and disease context of nifA

  • Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding [6].
  • NifA-dependent expression of glutamate dehydrogenase in Rhizobium etli modifies nitrogen partitioning during symbiosis [7].

Biological context of nifA

  • Construction of a nifA-lacZ fusion and mapping of the RNA transcriptional start site showed that the nifA-like gene was expressed from an unidentified promoter, under conditions of nitrogen fixation and in the presence of oxygen and ammonia [4].
  • The nucleotide sequence of nifA of A. brasilense Sp7 was determined [4].
  • The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel) [8].
  • To study the functions of the DNA 5' to these promoters, plasmids carrying deletions in this region were constructed and analyzed in vivo in a heterologous system consisting of an E. coli (NtrA+) background with a plasmid that constitutively expresses the K. pneumoniae nifA gene [9].
  • The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined [10].

Associations of nifA with chemical compounds

  • The ability of both nifA and nfrX mutants to grow on nitrogen-free medium with vanadium, but not on medium with molybdenum, suggests that neither gene is required for expression of the alternative V-containing nitrogenase of A. vinelandii [3].
  • Nif gene activation in vivo by the B.japonicum NifA protein, but not by the K.pneumoniae NifA protein, was sensitive to treatment with chelating agents, and this inhibition could be overcome by the addition of divalent metal ions [6].
  • In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream [11].
  • In order to generate oxygen-tolerant variants of the NifA protein a plasmid carrying the R. meliloti nifA gene was mutagenized in vitro with hydroxylamine [12].

Other interactions of nifA

  • One of these loci corresponded to nifA and the other to nifB [4].

Analytical, diagnostic and therapeutic context of nifA

  • Ligation of two adjacent EcoRI fragments of A. chroococcum yielded an intact nifA gene that activated expression of nifH-lac fusions and also restored MV3 to Nif+ [3].
  • Sequence analysis revealed four open reading frames (ORFs), two of them with rightward polarity, termed nfe1 and nfe2, are preceded by functional nif consensus sequences and NifA-binding motifs [13].
  • Using the techniques of oligonucleotide-directed mutagenesis, we report here that several of the four intervening amino acids can be replaced by others without loss of NifA function [14].
  • Studies of high-copy-number nifH promoter constructs showed that partial deletion of the consensus UAS does not alter the ability to inhibit nitrogen fixation by titration of NifA suggesting that NifA can also complex with RNA polymerase containing the alternative sigma-factor RpoN [15].


  1. Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. Steenhoudt, O., Vanderleyden, J. FEMS Microbiol. Rev. (2000) [Pubmed]
  2. Nucleotide sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE: transcriptional control sites and expression in Escherichia coli. Roelvink, P.W., Hontelez, J.G., van Kammen, A., van den Bos, R.C. Mol. Microbiol. (1989) [Pubmed]
  3. Identification and characterization of two nitrogen fixation regulatory regions, nifA and nfrX, in Azotobacter vinelandii and Azotobacter chroococcum. Santero, E., Toukdarian, A., Humphrey, R., Kennedy, C. Mol. Microbiol. (1988) [Pubmed]
  4. Identification of a nifA-like regulatory gene of Azospirillum brasilense Sp7 expressed under conditions of nitrogen fixation and in the presence of air and ammonia. Liang, Y.Y., Kaminski, P.A., Elmerich, C. Mol. Microbiol. (1991) [Pubmed]
  5. Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein nifA. Brito, B., Martínez, M., Fernández, D., Rey, L., Cabrera, E., Palacios, J.M., Imperial, J., Ruiz-Argüeso, T. Proc. Natl. Acad. Sci. U.S.A. (1997) [Pubmed]
  6. Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding. Fischer, H.M., Bruderer, T., Hennecke, H. Nucleic Acids Res. (1988) [Pubmed]
  7. NifA-dependent expression of glutamate dehydrogenase in Rhizobium etli modifies nitrogen partitioning during symbiosis. Mendoza, A., Valderrama, B., Leija, A., Mora, J. Mol. Plant Microbe Interact. (1998) [Pubmed]
  8. Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum. Rossen, L., Ma, Q.S., Mudd, E.A., Johnston, A.W., Downie, J.A. Nucleic Acids Res. (1984) [Pubmed]
  9. Activation of the Bradyrhizobium japonicum nifH and nifDK operons is dependent on promoter-upstream DNA sequences. Alvarez-Morales, A., Betancourt-Alvarez, M., Kaluza, K., Hennecke, H. Nucleic Acids Res. (1986) [Pubmed]
  10. The symbiotic nitrogen fixation regulatory operon (fixRnifA) of Bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifA-independent type of activation. Thöny, B., Fischer, H.M., Anthamatten, D., Bruderer, T., Hennecke, H. Nucleic Acids Res. (1987) [Pubmed]
  11. Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes. Valderrama, B., Dávalos, A., Girard, L., Morett, E., Mora, J. J. Bacteriol. (1996) [Pubmed]
  12. A defined amino acid exchange close to the putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein. Krey, R., Pühler, A., Klipp, W. Mol. Gen. Genet. (1992) [Pubmed]
  13. Nucleotide sequence and characterization of Rhizobium meliloti nodulation competitiveness genes nfe. Soto, M.J., Zorzano, A., Mercado-Blanco, J., Lepek, V., Olivares, J., Toro, N. J. Mol. Biol. (1993) [Pubmed]
  14. Critical spacing between two essential cysteine residues in the interdomain linker of the Bradyrhizobium japonicum NifA protein. Fischer, H.M., Fritsche, S., Herzog, B., Hennecke, H. FEBS Lett. (1989) [Pubmed]
  15. The nifH promoter region of Rhizobium leguminosarum: nucleotide sequence and promoter elements controlling activation by NifA protein. Roelvink, P.W., Harmsen, M., van Kammen, A., van den Bos, R.C. Gene (1990) [Pubmed]
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