Bacteria and their bacteriophages have antagonistically co-evolved for billions of years, driving the evolution of diverse immune strategies and elaborate countermeasures. Among the most striking viral adaptations is the chemical hypermodification of genomic DNA, which decorates canonical nucleobases with bulky substituents to evade host recognition. These modifications, such as glycosylated hydroxymethylcytosine (ghmC) in T4-like phages, can add hundreds of Daltons to a single base and are thought to provide broad protection against host-mediated restriction and cleavage.
Here we identify Azaca as a widespread three-component defense system that senses and degrades hypermodified phage DNA immediately after cell entry. Using functional assays, live cell microscopy, RNA-seq, single-particle cryo-electron microscopy, mutagenesis, and phylogenetics, we demonstrate that Azaca is triggered by diverse DNA hypermodifications to provide potent and non-abortive protection against hypermodified phages. Azaca assembles into an elongated trimeric helicase–nuclease complex that scans DNA using ATP-driven translocation activity in search of hypermodified DNA. Recognition of hypermodified DNA triggers allosteric activation of the Azaca complex leading to targeted genome degradation and immunity. Building on this mechanism, we exploited Azaca’s unique modification specificity to uncover previously hidden hypermodified genomes in phage collections. Together, these findings reveal hypermodification sensing as a fundamental axis of bacterial immunity and establish Azaca as both a mechanistic paradigm and a tool for hypermodified genome discovery.