Metagenomic selections reveal diverse antiphage defenses in human and environmental microbiomes

To prevent phage infection, bacteria have developed an arsenal of antiphage defenses. Evidence suggests that many examples in nature have not been described. Using plasmid libraries expressing small DNA inserts and functional selections for antiphage defense in Escherichia coli, we identified over 200 putative defenses from 14 bacterial phyla in 9 human and soil microbiomes. Many defenses were unrecognizable based on sequence or predicted structure and thus could only be identified via functional assays. In mechanistic studies, we show that some defenses encode nucleases that distinguish phage DNA via diverse chemical modifications. We also identify outer membrane proteins that prevent phage adsorption and a set of unknown defenses with diverse antiphage profiles and modalities. Most defenses acted against at least two phages, indicating that broadly acting systems are widely distributed. Collectively, these findings highlight the diversity and interoperability of antiphage defense systems.