Systematic discovery of bacterial anti-phage systems through a protein domain-centric strategy.

Bacterial exposure to constant phage attack drives rapid diversification of anti-phage defense systems, often through the exchange of modular defensive domains. Here, we leverage this modularity signature to identify new defense systems by systematically searching for operons encoding known defensive domains in non-canonical configurations. Using this approach, we identified 39 848 candidate defense operons in Escherichia coli genomes. Annotation of the operons based on their shared defensive domains with known systems reveals that the operons represent variants of 82 defense families. Experimental testing of nine candidates validated six with anti-phage activity. These include DarTG and ietAS system variants that have acquired helicase modules, and a Gabija system in which a MazF-like protein replaces GajA, implying novel anti-phage mechanisms. We also identified a new clade of Pycsar that synergizes with type IV Thoeris to broaden phage protection. Our findings demonstrate that mining modular defensive domains provides a powerful strategy to predict and characterize new anti-phage systems, expanding the known repertoire of bacterial immunity.