Phylogeny of Nitrogenase Structural and Assembly Components Reveals New Insights into the Origin and Distribution of Nitrogen Fixation across Bacteria and Archaea

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.

Phylogenetically and catabolically diverse diazotrophs reside in deep-sea cold seep sediments

“…BuS5 in NCBI taxonomy), Desulfatiglandaceae and Syntrophales known to degrade alkanes or aromatic hydrocarbons coupled with sulfate reduction 7 , 57 . The increased diversity of bacterial and archaeal diazotrophic lineages substantially broadens the genomic database of microbial diazotrophs in deep-sea cold seep sediments 48 , which previously only included ANME-2b and SEEP-SRB1g 34 . Indeed, to our knowledge, this represents the first genomic evidence of nitrogen fixation potential in five different phyla, namely Altarchaeia, Omnitrophota, Caldatribacteriota along with two bacterial candidate phyla FCPU426 and UBA6262 48 , 58 .…”

“…2 ) suggested that nifH homologues were classified into distinct bona fide nitrogenase sequences (canonical groups I to III) as well as nitrogenase-like groups (groups IV to VI) 44 – 48 . These include (1) typical Mo-Fe nitrogenases from aerobic and facultative anaerobic bacteria (group I; n = 1); (2) Mo-Fe nitrogenases from anaerobic bacteria and archaea (group II; n = 32); (3) alternative nitrogenases (Mo-independent Anf and Vnf) and some Mo-Fe nitrogenases from Euryarchaeota 48 (group III; n = 11); (4) poorly characterized nif homologues (group IV; n = 123); (5) bacteriochlorophyll and chlorophyll biosynthesis genes 49 (group V; n = 1); and (6) putative tetrapyrrole cofactor biosynthesis genes 44 (group VI; n = 5). Group IV genes include its subclusters B ( n = 19), C ( n = 19) and E ( n = 16) with unknown functions, as well as subcluster D ( n = 69) involved in archaeal methionine biosynthesis 44 – 48 .…”

“…These include (1) typical Mo-Fe nitrogenases from aerobic and facultative anaerobic bacteria (group I; n = 1); (2) Mo-Fe nitrogenases from anaerobic bacteria and archaea (group II; n = 32); (3) alternative nitrogenases (Mo-independent Anf and Vnf) and some Mo-Fe nitrogenases from Euryarchaeota 48 (group III; n = 11); (4) poorly characterized nif homologues (group IV; n = 123); (5) bacteriochlorophyll and chlorophyll biosynthesis genes 49 (group V; n = 1); and (6) putative tetrapyrrole cofactor biosynthesis genes 44 (group VI; n = 5). Group IV genes include its subclusters B (n = 19), C (n = 19) and E (n = 16) with unknown functions, as well as subcluster D (n = 69) involved in archaeal methionine biosynthesis [44][45][46][47][48] . Subcluster A within group IV also includes functional nitrogenases found in Endomicrobium proavitum that can fix nitrogen 50 , but none of the identified nifH homologues from the cold seep assemblies are affiliated with this subcluster.…”

“…The increased diversity of bacterial and archaeal diazotrophic lineages substantially broadens the genomic database of microbial diazotrophs in deep-sea cold seep sediments 48 , which previously only included ANME-2b and SEEP-SRB1g 34 . Indeed, to our knowledge, this represents the first genomic evidence of nitrogen fixation potential in five different phyla, namely Altarchaeia, Omnitrophota, Caldatribacteriota along with two bacterial candidate phyla FCPU426 and UBA6262 48,58 . Among archaea, only lineages related to anaerobic methanogens and closely related anerobic methanotrophs are known or predicted to possess nitrogenases 58,59 .…”

The Microbial Nitrogen Cycling, Bacterial Community Composition, and Functional Potential in a Natural Grassland Are Stable from Breaking Dormancy to Being Dormant Again

“…Earlier reports verify the difficulty in attaining complete coverage of the nifH gene population, with over 50 primer sets described in the literature [ 58 ]. There is simply no sufficiently conserved region in the prokaryotic nifH gene pool [ 59 ]. The degenerate PCR primers that must be used can cause template-specific bias and require challenging annealing temperatures [ 60 ].…”