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 δ - Proteobacteria as the primary donor reservoir. Extreme GC3 differentiation between δ - and ε - Proteobacteria and uniformly negative ΔENC values indicated strong lineage-specific translational selection. We identified inter-phylum HGT to Planctomycetes and Bacteroidetes , one inter-domain transfer ( Archaea → δ - 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.