Cloning, DNA Sequencing, and Characterization of a NifD-homologous Gene from the Archaeon Methanosarcina Barkeri 227 Which Resembles NifD1 from the Eubacterium Clostridium Pasteurianum

L. Sibold, M. Henriquet, O. Possot, and J.-P. Aubert (Res. Microbiol. 142:5-12, 1991) cloned and sequenced two nifH-homologous open reading frames (ORFs) from Methanosarcina barkeri 227. Phylogenetic analysis of the deduced amino acid sequences of the nifH ORFs from M. barkeri showed that nifH1 clusters with nifH genes from alternative nitrogenases, while nifH2 clusters with nifH1 from the gram-positive eubacterium Clostridium pasteurianum. The N-terminal sequence of the purified nitrogenase component 2 (the nifH gene product) from M. barkeri was identical with that predicted for nifH2, and dot blot analysis of RNA transcripts indicated that nifH2 (and nifDK2) was expressed in M. barkeri when grown diazotrophically in Mo-containing medium. To obtain nifD2 from M. barkeri, a 4.7-kbp BamHI fragment of M. barkeri DNA was cloned which contained at least five ORFs, including nifH2, ORF105, and ORF125 (previously described by Sibold et al.), as well as nifD2 and part of nifK2. ORFnifD2 is 1,596 bp long and encodes 532 amino acid residues, while the nifK2 fragment is 135 bp long. The deduced amino acid sequences for nifD2 and the nifK2 fragment from M. barkeri cluster most closely with the corresponding nifDK1 gene products from C. pasteurianum. The predicted M. barkeri nifD2 product contains a 50-amino acid insert near the C terminus which has previously been found only in the clostridial nifD1 product. Previous biochemical and sequencing evidence indicates that the C. pasteurianum nitrogenase is the most divergent of known eubacterial Mo-nitrogenases, most likely representing a distinct nif gene family, which now also contains M. barkeri as a member. The similarity between the methanogen and clostridial nif sequences is especially intriguing in light of the recent findings of sequence similarities between gene products from archaea and from low-G+C gram-positive eubacteria for glutamate dehydrogenase, glutamine synthetase I, and heat shock protein 70. It is not clear whether this similarity is due to horizontal gene transfer or to the resemblance of the M. barkeri and C. pasteurianum nitrogenase sequences to an ancestral nitrogenase.