A membrane-bound nuclease directly cleaves phage DNA during genome injection

From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defense strategy against viral infection, but it requires mechanisms for distinguishing self from non-self1,2. In bacteria, CRISPR-Cas and restriction modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications. Alternative mechanisms likely remain to be discovered. Here, we characterize SNIPE, a novel anti-phage defense system that constitutively localizes to the bacterial cell membrane in E. coli to block phage {lambda} infection. Using radiolabeled phage DNA and time-lapse microscopy to track phage genomes we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labeling, we find that SNIPE associates with host proteins essential for {lambda} genome entry and with the {lambda} tape measure protein, which facilitates {lambda} genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, likely through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defense system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a novel strategy for distinguishing self from non-self in prokaryotic immune systems.