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Nucleic Acids Res. Mar 25, 1988; 16(5 Pt B): 2207–2224.
PMCID: PMC338210

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.

Abstract

The amino acid sequence of the Bradyrhizobium japonicum nitrogen fixation regulatory protein NifA, as derived from the nucleotide sequence of the nifA gene, was aligned to the corresponding protein sequences from Klebsiella pneumoniae, Rhizobium meliloti and Rhizobium leguminosarum biovar viciae. High conservation was found in the central domain and in the COOH-terminal, putative DNA binding domain, whereas very little homology was present within the first 250 amino acids from the NH2-terminus. Upon deletion of the first 218 amino acids (37% of the protein) and expression of the remainder as a Cat'-'NifA hybrid protein, a fully active, nif-specific transcriptional activator protein was obtained which also retained oxygen sensitivity, a characteristic property of the wild-type B. japonicum NifA protein. In contrast, an unaltered COOH-terminal domain was required for an active NifA protein. Between the central and the DNA binding domains, a so-called interdomain linker region was identified which was conserved in all rhizobial species but missing in the K.pneumoniae NifA protein. Two conserved cysteine residues in this region were changed to serine residues, by oligonucleotide-directed mutagenesis. This resulted in absolutely inactive NifA mutant proteins. Similar null phenotypes were obtained by altering two closely adjacent cysteine residues in the central domain to serine residues. 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. On the basis of these observations and previous data on oxygen sensitivity we raise the hypothesis that at least some, if not all, of the four essential cysteine residues may be involved in oxygen reactivity or metal binding or both.

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Selected References

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