Diversity of Novel Bacteriophages Infecting Ammonia-Oxidizing Bacteria

Agriculture is a major source of anthropogenic greenhouse-gas emissions and the largest source of nitrous oxide (N2O), an extremely potent greenhouse gas and ozone-depleting agent. Soil N2O emissions are largely driven by nitrification, in which ammonia-oxidizing microorganisms catalyze the 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 gathering interest; however, phages infecting ammonia-oxidizers are mostly uncharacterized, with only one lytic phage having been described, limiting the potential for developing phage-mediated nitrification control. Here, we characterized the largest set of cultivated phages infecting ammonia-oxidizing bacteria (AOB) to date: 45 dsDNA phages collected 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 several monophyletic clades, likely representing higher-level lineages. Structure-guided genome annotation revealed a diverse collection of auxiliary metabolic genes encoded by phage, ranging from a complete ABC transporter cassette to a large antimicrobial resistance gene cluster. These results unveil previously unrecognized diversity of cultivated AOB phages and their potential to alter host physiology. Our data describes a broad taxonomic and functional repertoire of cultured AOB phages, suggesting that viruses play a significant and complex role in nitrification. Moreover, we outline an effective methodological framework for isolating AOB phages from environmental samples. These results reframe our understanding of environmental nitrification and enable intensified cultivation, characterization and use of phages for its control.