Expansion of eNOR diversity
Notably, the majority of genomes with the unique C1-C2-NirS structure do not appear to contain a nitric oxide reductase gene (nor ). Though the absence of the nor gene in genomes with nirS is not unprecedented, previous genomic surveys suggest it is relatively uncommon, and the toxicity of the product of Nir (nitric oxide, NO) makes this absence counterintuitive (Hendriks et al. , 2000; Graf, Jones and Hallin, 2014). However, analysis of the SURF MAG 42 metagenome—the original assembled genome in which the novelnirS ORF was observed—did reveal the presence of an unusualnor homolog. Previous studies have identified the established cNOR and qNOR family enzymes (which contain cytochrome c or quinols as electron donors, respectively) in Chloroflexi, as well as a broad distribution of Proteobacteria (Hendriks et al. , 2000; Zumft, 2005; Hemp and Gennis, 2008). However, the predicted NOR in SURF MAG 42 included an active site glutamine substitution characteristic of eNOR (Hemp and Gennis, 2008; Hemp et al. , 2015) (Table S4). eNOR has been previously described in Archaea (Hemp and Gennis, 2008) and at least one isolated Anaerolineales bacterium (Hemp et al. , 2015).
Phylogenetic analysis indicates the presence of eNOR in an expanded diversity of genomes (Fig. 5). Previous studies have described eNOR in Natronomonas ; these data indicate a cluster ofeNOR genes throughout other Halobacteria as well. Additional putative eNOR genes appear in multiple members of Anaerolineales, as well as other Chloroflexi, and in many members of the Alpha-, Beta-, Gamma-, and Deltaproteobacteria. Many of these putative eNOR hits appear to have been misannotated or mislabeled as cytochrome c oxidase genes, likely because of the structural similarity of the heme-copper oxidase subunits (Hemp and Gennis, CDD).