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. Posset, 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 o f the deduced amino acid sequences of the nifH ORFs from M. barkeri sh owed that nifH1 clusters with nifH genes from alternative nitrogenases , while nifH2 clusters with nifH1 from the gram-positive eubacterium C lostridium pasteurianum. The N-terminal sequence of the purified nitro genase component 2 (the nifH gene product) from M. barkeri was identic al with that predicted for nifH2, and dot blot analysis of RNA transcr ipts indicated that nifH2 (and nifDK2) was expressed in M. barkeri whe n grown diazotrophically in Mo-containing medium. To obtain nifD2 from M. barkeri, a 4.7-kbp BamHI fragment of M. barkeri DNA was cloned whi ch contained at least five ORFs, including nifH2, ORF105, and ORF125 ( previously described by Sibold et al.), as well as nifD2 and part of n ifK2. ORFnifD2 is 1,596 bp long and encodes 532 amino acid residues, w hile the nifK2 fragment is 135 bp long. The deduced amino acid sequenc es for nifD2 and the nifK2 fragment from M. barkeri cluster most close ly with the corresponding nifDK1 gene products from C, pasteurianum. T he predicted M. barkeri nifD2 product contains a 50-amino acid insert near the C terminus which has previously been found only in the clostr idial nifD1 product. Previous biochemical and sequencing evidence indi cates that the C. pasteurianum nitrogenase is the most divergent of kn own eubacterial Mo-nitrogenases, most likely representing a distinct n if gene family, which now also contains M. barkeri as a member. The si milarity between the methanogen and clostridial nif sequences is espec ially intriguing in light of the recent findings of sequence similarit ies between gene products from archaea and from low-G+C gram-positive eubacteria for glutamate dehydrogenase, glutamine synthetase I, and he at 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. pasterianum nitrogenase sequences to an ancestral nitrogenase.