The phylogeny of nitrogenase has only been analyzed using the structural proteins NifHDK. As nifHDKENB has been established as the minimum number of genes necessary for in silico prediction of diazotrophy, we present an updated phylogeny of diazotrophs using both structural (NifHDK) and cofactor assembly proteins (NifENB). Annotated Nif sequences were obtained from InterPro from 963 culture-derived genomes. Nif sequences were aligned individually and concatenated to form one NifHDKENB sequence. Phylogenies obtained using PhyML, FastTree, RapidNJ, and ASTRAL from individuals and concatenated protein sequences were compared and analyzed. All six genes were found across the Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota. The phylogenies of individual Nif proteins were very similar to the overall NifHDKENB phylogeny, indicating the assembly proteins have evolved together. Our higher resolution database upheld the three cluster phylogeny, but revealed undocumented horizontal gene transfers across phyla. Only 48% of the 325 genera containing all six nif genes are currently supported by biochemical evidence of diazotrophy. In addition, this work provides reference for any inter-phyla comparison of Nif sequences and a quality database of Nif proteins that can be used for identifying new Nif sequences.
Keywords: NifHDKENB; diazotroph; horizontal gene transfer; niche; nifH; nitrogenase.