A Type IV restriction system targets glucosylated 5-hydroxyl methyl cytosine to protect against phage infection.

A major challenge faced by is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, has evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized more were encoded in given the relative paucity of phage that have been isolated which infect this species. Using a genomic library, we identified a Type IV restriction system consisting of two genes within a 16kB region of the pathogenicity island-2 that we name TgvA and TgvB (ype I-embedded mrSD-like system of PI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modification are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We show that additional phage defense genes are encoded in VPI-2 that protect against other phage like T3, secΦ18, secΦ27 and λ. Our study uncovers a novel Type IV restriction system in , increasing our understanding of the evolution and ecology of while highlighting the evolutionary interplay between restriction systems and phage genome modification.