The two evolutionary unrelated nitric oxide-producing nitrite reductases NirK and NirS are best known for their redundant role in denitrification. They are also often found in organisms that do not perform denitrification. To assess the functional roles of the two enzymes and to address sequence and structural variation within each, we reconstructed robust phylogenies of both proteins with sequences recovered from 6973 isolate and metagenome-assembled genomes and identified 32 well-supported clades of structurally distinct protein lineages. We then inferred the potential niche of each clade by considering other functional genes of the organisms carrying them, as well as the relative abundances of each nir gene in 4082 environmental metagenomes across diverse aquatic, terrestrial, host-associated, and engineered biomes. We demonstrate that Nir phylogenies recapitulate ecology distinctly from the corresponding organismal phylogeny. While some clades of the nitrite reductase were equally prevalent across biomes, others had more restricted ranges. Nitrifiers make up a sizeable proportion of the nitrite reducing community, especially for NirK in marine surface waters and dry soils. Furthermore, the two reductases showed distinct associations with genes involved in oxidising and reducing other compounds, indicating that NirS and NirK activity may be linked to different elemental cycles. Accordingly, the relative abundance and diversity of NirS vs. NirK varies between biomes. Our results show the divergent ecological roles NirK and NirS-encoding organisms may play in the environment and provide a phylogenetic framework to distinguish traits associated with organisms encoding the different lineages of nitrite reductases.
