Phylogenetic diversity of NO reductases,new tools for nor monitoring,and insights into N_(2)O production in natural and engineered environments

Nitric oxide reductases(NORs)have a central role in denitrification,detoxification of nitric oxide(NO)in host-pathogen interactions,and NO-mediated cell-cell signaling.In this study,we focus on the phylogeny and detection of qNOR and cNOR genes because of their nucleotide sequence similarity and evolutionary relatedness to cytochrome oxidases,their key role in denitrification,and their abundance in natural,agricultural,and wastewater ecosystems.We also include nitric oxide dismutase(NOD)due to its similarity to qNOR.Using 548 nor sequences from publicly accessible databases and sequenced isolates from N2O-producng bioreactors,we constructed phylogenetic trees for 289 qnor/nod genes and 259 cnorB genes.These trees contain evidence of horizontal gene transfer and gene duplication,with 13.4%of the sequenced strains containing two or more nor genes.By aligning amino acid sequences for qnor+cnor,qnor,and cnor,we identified four highly conserved regions for NOR and NOD,including two highly conserved histidine residues at the active site for qNOR and cNOR.Extending this approach,we identified conserved sequences for:1)all nor(nor-universal);2)all qnor(qnor-universal)and all cnor(cnor-universal);3)qnor of Comamonadaceae;4)Clade-specific sequences;and 5)nod of Candidates Methylomirabilis oxyfera.Examples of primer performance were confirmed experimentally.