Horizontal gene transfer drives DNRA capacity in wastewater treatment systems

Dissimilatory nitrate reduction to ammonium (DNRA) is a key biological nitrogen retention pathway, yet the evolutionary mechanisms remain poorly understood. Here, we combined phylogenomic analysis, codon usage bias assessment, horizontal gene transfer (HGT) detection, and gene tree--species tree reconciliation to investigate the evolutionary history of nrfA. Analysis of 103 DNRA-capable taxa and 45 curated nrfA sequences revealed that DNRA capability evolved polyphyletically. Reconciliation analysis identified six discrete HGT events, establishing {delta}-Proteobacteria as the primary donor reservoir. Extreme GC3 differentiation between {delta}- and {varepsilon}-Proteobacteria and uniformly negative {Delta}ENC values indicated strong lineage-specific translational selection. We identified inter-phylum HGT to Planctomycetes and Bacteroidetes, one inter-domain transfer (Archaea [->] {delta}-Proteobacteria), and provided direct molecular evidence for IS-element-mediated transfer. To validate the generalizability, we expanded the analysis to 180 nrfA sequences, identifying 112 cross-phylum phylogenetic clusters that further support HGT as a widespread dissemination. These findings established a predictive framework linking molecular evolutionary signatures to DNRA capability, with implications for understanding nitrogen cycling.