Nuclease-NTPase systems use shared molecular features to control bacterial anti-phage defense

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein–protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.