Expression and functional analysis of the Rhizobium meliloti nifA gene

Abstract

The translational initiation point for Rhizobium meliloti nifA, the specific activator for nitrogen fixation (nif) genes, was determined. When expressed in an Escherichia coli linked transcriptional-translational system the DNA coding for the R.meliloti nifA gene produced four polypeptide bands of 71 000, 67 000, 62 000 and 59 000 daltons. There are three in-frame ATG codons at the N-terminus of the gene; by replacing the poor ribosome binding sites of the native DNA with a synthetic consensus ribosome binding site prior to each of ATG codons the polypeptides were produced at enhanced levels and related to each of the initiation codons. The ability of these specifically expressed polypeptides to activate nif promoters fused to lacZ was determined. Only the fulllength polypeptide activated the Klebsiella pneumoniae nifH, R.meliloti nifH and fixA and Bradyrhizobium japonicum nifH and nifD promoters. The R.meliloti fixA promoter, contrary to previous evidence, could be activated in E.coli. Deletion of the putative N-terminal domain of the R.meliloti nifA gene product greatly increased the ability of the protein to activate nif promoters. However, deletions retaining part of this domain were not functional. This shows that the N-terminal domain is not essential for activity and that its presence decreases the full potential function of the protein. Our results are consistent with the suggesting that this domain has a regulatory role. In contrast to K.pneumoniae nifA protein, the function of the full length and domain deleted forms of R.meliloti nifA gene product was sensitive to oxygen in E.coli.