Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis

Abstract

We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 M, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases.Nitrogen fixation, the conversion of atmospheric N 2 to ammonia, is carried out by a variety of Bacteria and Archaea (23). Among the Bacteria, it occurs in at least four groups, the Proteobacteria, gram-positive bacteria, cyanobacteria, and green sulfur bacteria. Among Archaea, nitrogen fixation is found in all three orders of methanogens (10). Despite its wide phylogenetic distribution, the mechanism of nitrogen fixation is conserved. A typical dinitrogenase is an ␣ 2 ␤ 2 tetramer encoded by nifD and nifK and containing an iron-molybdenum cofactor, FeMoCo (22). Mo-independent dinitrogenases (2) have cofactors that coordinate vanadium in place of molybdenum (Vdinitrogenases) or that have neither molybdenum nor vanadium (Fe-dinitrogenases); these are encoded by the nifD and nifK homologs vnfD and vnfK and anfD and anfK, respectively. An additional small subunit (␦) found with the Mo-independent dinitrogenases is encoded by vnfG or anfG. Included in all nitrogenase complexes is dinitrogenase reductase, typically a homodimer in Bacteria, encoded by nifH, vnfH, or anfH.The discovery of nitrogen fixation in methanogens in 1984 redefined the phylogenetic extremity of nitrogen fixation and opened the door to a series of new questions regarding biological nitrogen fixation (1, 12). Many of the properties common to nitrogen fixation in the Bacteria hold true in the Archaea as well. Lobo and Zinder found the nitrogenase complex from Methanosarcina barkeri to resemble those from Bacteria in subunit composition (except that dinitrogenase reductase appeared to be a homotetramer), substrate range, and immunological cross-reactivity of dinitrogenase reductase (9). nif genes from a variety of methanogens proved to be homologous and similar in organization to those from Bacteria (10). Phylogenetic analysis of nifH, nifD, and nifK showed the methanogen genes to be related to certain groups of bacterial genes (discussed below).One question that has not been resolved is which metals are required for nitrogen fixation in methanogens and might therefore be incorporated into the dinitrogenase cofactor. Lobo and Zinder found that molybdenum but not vanadium stimulated nitrogen fixation in M. barkeri 227 (8). In contrast, Scherer reported stimulation by molybdenum and to a lesser extent by vanadium in two strains of M. barkeri, 227 and Fusaro (14). The issue is further complicated by the fact that M. barkeri 227 has two potentially active nitrogenases, as evidenced by the existence of two sets o...

Bacteroidales ectosymbionts of gut flagellates shape the nitrogen-fixing community in dry-wood termites

“…It has been shown that bacteria possessing both nif and anf genes preferentially express their anf paralogs in the absence of molybdenum cofactor (Masepohl and Klipp, 1996;Kessler et al, 1997;Braun et al, 1999). Based on their detailed study of nitrogenase activity, nifH gene expression and nif operon structure in N. koshunensis, Noda et al (1999) had suggested that preferential expression of anfH in Kalotermitidae is related to the lack of molybdenum in sound wood, which may have favored the acquisition of a molybdenum-independent nitrogenase (that is, anfH) by their gut symbionts.…”

Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1

“…Downstream of nifK, the nifE and nifN genes, which are essential for FeMo-cofactor assembly (Dean et al 1993), are found in a separate operon. In Archaea, genes homologous to the bacterial nif genes have been identified, and nitrogen fixation has been observed in several methanogenic species (Lobo and Zinder 1992, Young 1992, Bult et al 1996, Chien and Zinder 1996, Haselkorn and Buikema 1996, Kessler et al 1997, Smith et al 1997. The discovery of genes homologous to nifH, nifD and nifK suggests that the basic mechanism of nitrogen fixation is similar in Bacteria and Archaea and predicts that most methanogenic nitrogenases contain a molybdenum-cofactor Zinder 1996, Kessler et al 1997).…”

Nitrogen Fixation In Methanogens: The Archaeal Perspective

“…The presence of two genes in M. barkeri that are phylogenetically related to functional nifH genes (19) allows for the possibility that both molybdenum and vanadium nitrogenases are present in this organism, and recently a vanadium nitrogenase has been confirmed (25a). In M. maripaludis, molybdenum was clearly required for diazotrophic growth (26). Vanadium failed to stimulate, and tungsten inhibited, so this species may contain a single nitrogenase of the molybdenum type.…”

“…Vanadium failed to stimulate, and tungsten inhibited, so this species may contain a single nitrogenase of the molybdenum type. The presence of molybdenum nitrogenases in other methanogens is corroborated by nif gene phylogeny (26), and by the apparent absence from the nif gene clusters of a gene corresponding to vnfG or anfG, which encode the δ subunits of alternative nitrogenases in Bacteria (27). Thus, molybdenum nitrogenases seem to predominate in methanogens, but vanadium nitrogenases may be present as well.…”