Nucleotide Sequence and Functional Analysis of the Two nifH Copies of Rhizobium ORS571

Summary: In Rhizobium ORS571, which is able to form both root and stem nodules on the tropical plant Sesbania rostrata and to grow in the free-living state at the expense of N2, two copies of the nifH gene, which codes for the nitrogenase Fe-protein, were characterized. One copy, nifH1, was localized in the nifHDK operon; the other, nifH2, was localized elsewhere. nifH1 and nifH2 differed in only six nucleotides and both encoded a polypeptide of 296 amino acids with a single change at position 282, a serine in nifH1 and a threonine in nifH2 At position 102, arginine in the sequence Gly-Arg-Gly-Val-Ile-Thr might, as in Rhodospirillum rubrum, be the site of ‘switch-off’ inactivation. Compared with other nifH genes, the highest SAB was found with Bradyrhizobium japonicum and Rhizobium sp. (Parasponia). A sequence identical (nifH1) or very similar (nifH2) to the nif consensus sequence was found upstream from the initiation codon. Homology with sequences upstream from Klebsiella pneumoniae nif promoters, which affect nifA-mediated activation, was also found in both cases. From mutants carrying either a single nifH1 or nifH2 deletion, or a double nifH1-nifH2 deletion, it appeared that both genes were functional ex planta and in Planta and that no other nifH copy seemed to be present in ORS571. The optimal expression of nifH1 and of nifH2 might depend on different physiological conditions.