Abstract
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.
Highlights
The nitrogen biogeochemical cycle requires reduction of atmospheric nitrogen to ammonia
Most organisms were from Proteobacteria, followed by Firmicutes and Cyanobacteria
Labels in the gray bar represent the number of genomes containing NifHDKENB in that phylum. (b) Distribution of organisms having all six nif genes by habitat and (c) pie diagram showing the lifestyle adopted by various diazotrophs
Summary
The nitrogen biogeochemical cycle requires reduction of atmospheric nitrogen to ammonia. The other half is contributed through a combination of synthetically fixed nitrogen and agricultural promotion of bacterial fixation in legumes. Biological nitrogen fixation or diazotrophy probably evolved 3.6–3.2 Ga ago to support expansion of biota in the nitrogen poor environment of that era [2,3]. Today it is the second most vital process for life on earth after photosynthesis [4]. Diazotrophs are divided into two major types: (1) symbiotic nitrogen-fixing bacteria which form symbiotic relation with legumes like Rhizobium, with actinorhizal plants such as Frankia, and Cyanobacteria associated with cycads, and (2) freeliving nitrogen fixers belonging to genera such as Azotobacter and Clostridium [5]
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