Turner, A. A. B.; Stahn, M.; Millard, A.; Sauvageau, D.; Stein, L. Y.

Agriculture is a major source of anthropogenic greenhouse-gas emissions, being the largest source of nitrous oxide (N2O), an extremely potent greenhouse gas and ozone-depleting agent. Soil N2O emissions are largely driven by microbial nitrification, in which ammonia-oxidizing microorganisms catalyze the rate-limiting oxidation of ammonia to nitrite. Nitrification not only mediates N2O fluxes but also reduces fertilization efficiency and contributes to eutrophication through nitrate leaching. Bacteriophage (phage)-based control of microbial communities is rapidly garnering interest in a number of fields; however, phages infecting ammonia-oxidizers are largely uncharacterized, with only one lytic phage having been described, limiting the potential for phage-mediated nitrification inhibition. Here, we show the largest set of phages infecting ammonia-oxidizing bacteria (AOB) to date: 45 dsDNA phages identified from urban wastewater, infecting four AOB species, with 16 demonstrating cross-genus host ranges and capable of eliminating nitrification activity in liquid cultures. Phylogenetic and taxonomic analyses revealed six proposed families of Caudoviricetes and numerous monophyletic clades, likely representing higher-level lineages. Structure-guided genome annotation revealed these phages to carry diverse and seldom-seen auxiliary metabolic genes, ranging from a complete ABC transporter cassette to a large antimicrobial resistance gene cluster. These results unveil the previously unrecognized diversity of AOB phages and their potential to alter host physiology. Our data demonstrates a broad taxonomic and functional repertoire of cultured AOB phages, greatly expanding the panel of known AOB phages, suggesting that viruses play a more significant and complex role in nitrification than previously understood. Moreover, we outline an effective methodological framework for isolating AOB phages from environmental samples. These results will help reframe our understanding of environmental nitrification and enable intensified selection and use of phages for its control.