Bacterial antiviral immunity systems can be toxic to their hosts and must be tightly regulated. Immunity genes are often encoded by AT-rich mobile genetic elements subjected to silencing by nucleoid binding protein H-NS. Using RNA-seq, ChIP-seq, and chromosome 3D reconstruction with Micro-C, we find that H-NS binds and compacts bacterial immunity loci. H-NS deletion reveals anti-phage activity in model Escherichia coli strains generally considered phage-sensitive. Extending this approach to environmental isolates, we show that removal of H-NS silencing enhances defense, allowing bacteria to restrict phages with anti-defense proteins. We also discover Madara, a novel immunity system that complements the co-regulated BREX defense. Our results establish H-NS as a master regulator of bacterial immunity and highlight the importance of expression levels for anti-phage activity in native hosts.

Bacteriophages are ubiquitous biological entities that profoundly influence microbiology research and biotechnology. Among coliphages, T1-like viruses (family Drexlerviridae) are notoriously known for their environmental stability and propensity to contaminate laboratory cultures and equipment. Despite this, the genomic features that may underlie their persistence and recurrent detection as laboratory contaminants remain insufficiently characterized. Here, we describe a novel T1-like bacteriophage, KanT1, identified as a recurrent contaminant emerging from environmental samples. Comparative genomics and phylogenetic analyses position KanT1 within the Tunavirus lineage, confirming its close relationship to canonical T1-like phages. Structure-informed annotation enabled the functional characterization of previously unannotated proteins, highlighting the importance of integrating structural predictions into phage genome analysis. Notably, we provide novel details regarding the distribution of superinfection exclusion cassette cor and identify an SH3 domain-containing protein associated with the lysis cassette. We show that SH3 is widespread, though non-universal, across Drexlerviridae genomes. Given the established role of SH3 domains as determinants of cell-wall binding specificity for endolysins of phages infecting Gram-positive bacteria, we propose that this protein represents an auxiliary component of the T1-like lysis module. Together, these findings expand the current understanding of T1-like phage genome organization and provide new insights into molecular features that may contribute to their broad host range and persistence in laboratory environments.