Högskolan i Skövde

Denitrification is a key microbial process that completes the nitrogen cycle by reducing nitrate to dinitrogen gas. A critical intermediate step in this pathway is the reduction of nitrite to nitric oxide, catalyzed by two non-homologous enzymes: copper nitrite reductase (NirK) and cytochrome cd₁ nitrite reductase (NirS). This process enables microbial respiration under oxygen-limited conditions and plays a vital role in both natural and engineered ecosystems. Given the ecological importance of denitrification, understanding the mobility of nir genes is essential, especially in environments subject to anthropogenic influence or rapid microbial turnover. To explore the potential for horizontal gene transfer, public databases of mobile genetic elements (MGEs)—including plasmids (IMG-PR), viruses (IMG-VR; PhageScope), and integrative/conjugative elements (ICEberg 3.0)—were systematically screened. The analysis focused on identifying instances of nirK and nirS within these MGEs, determining their host taxa, and evaluating their distribution across various biomes. Enrichment analysis was performed to detect overrepresentation in specific ecological contexts, particularly aquatic and engineered environments. Across all MGEs, nirK was consistently more prevalent than nirS, although both genes remained rare overall. Enrichment analysis revealed that nirK is disproportionately found in aquatic and engineered sub-biomes such as bioreactors, built environments, and industrial production systems. In contrast, nirS was largely confined to engineered niches. Example rates included approximately 1–3% nirK among plasmids from wastewater-related contexts, while nirS was detected at ≤0.1% overall, with occasional spikes in industrial settings. Viral nirK was observed sporadically in Caudoviricetes infecting Gammaproteobacteria, whereas nirS was not detected in any viral datasets surveyed. These findings support a model in which plasmids serve as the dominant vectors for nirK dissemination across taxonomically broad hosts and ecologically engineered niches, while nirS shows restricted mobility. This asymmetry in gene transfer potential has important implications for microbial community structure, process stability, and nitrous oxide (N₂O) emissions in managed ecosystems. Understanding the differential mobility of these genes can inform strategies to optimize nitrogen removal and mitigate greenhouse gas emissions in wastewater treatment and other engineered environments.