Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn 5 mutant

A sequence homologous to the conventional nifH gene has been cloned from a different region of the Azotobacter vinelandii genome. Tn 5 insertions were obtained in this clone and the mutagenized plasmid was used for marker exchange with A. vinelandii strain CA12 (ΔnifHDK) to obtain Tn 5 mutants. These mutants exhibited a Nif^- phenotype in the presence of vanadium, unlike CA12 which was Nif⁺ on vanadium-containing medium. The gene in the cloned nifH-like region is therefore apparently involved in the vanadium dependent alternative pathway of nitrogen fixation. This gene, nifH2, has been sequenced and encodes a protein of 289 amino acids that is similar to nifH in nucleotide sequence, deduced amino acid sequence, predicted secondary structure and hydrophobicity profile. A second open reading frame downstream of nifH2 codes for a protein of 64 amino acids, similar to the ferredoxin (Fd)-like protein encoded downstream of nifH^* in A. chroococcum. Sequence analysis suggests that the nifH2 and Fd-like genes are in a single operon.