Phage homing endonuclease amplifies anti-defense genes to evade bacterial immunity [SITE-seq]

Bacteriophages must overcome diverse bacterial immune systems, yet the molecular principles enabling such escape remain poorly understood. Here, we show that the phage homing endonuclease SegB facilitates immune evasion by promoting the segmental amplification of anti-defense loci. The antiphage defense Septu inhibits phage T6 replication by cleaving the variable loop of tRNATyr. We show that SegB enables immune evasion by amplifying a genomic segment that contains the full-length tRNATyr gene. This repeat expansion increases tRNATyr expression, allowing the phage to overcome Septu immunity. Remarkably, SegB also mediates in trans amplification of distinct anti-defense genes that counteract OLD and toxin-antitoxin ToxIN defense systems. Collectively, our findings demonstrate that SegB-mediated segmental amplification represents a versatile mechanism by which phages rapidly adapt to and circumvent diverse bacterial antiphage defenses. Overall design: Identification of genomic positions cleaved by SegB homing endonuclease in high-molecular-weight T6 phage genomic DNA using the selective enrichment and identification of tagged genomic DNA ends by sequencing (SegB treatment vs. Mock treatment)