Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on site-specific epigenetic DNA methylation to discriminate between the host and invading DNA. We demonstrate that in Type I BREX systems, defense and methylation require BREX site DNA binding by the BrxX (PglX) methyltransferase employing S-adenosyl methionine as a cofactor. We determined 2.2-Å cryoEM structure of Escherichia coli BrxX bound to target dsDNA revealing molecular details of BREX DNA recognition. Structure-guided engineering of BrxX expands its DNA specificity and dramatically enhances phage defense. We show that BrxX alone does not methylate DNA, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters BrxX in an inactive dimeric form. We propose that BrxX-mediated foreign DNA sensing is a necessary first step in activation of BREX defense.

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on epigenetic DNA methylation to discriminate between the host and invading DNA, but their mechanism of protection remains enigmatic. We demonstrate that in Type I BREX systems, both defense and methylation are based on site-specific DNA recognition by the BrxX (PglX) methyltransferase and require the S-adenosyl methionine cofactor. We present a 2.2-Å cryoEM structure of Escherichia coli BrxX bound to target dsDNA, which reveals the molecular details of DNA recognition by BREX and paves the way for rational engineering of BREX specificity. We show that BrxX alone does not support methylation, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters an inactive dimeric form of BrxX. Together, these results allow us to propose a model of BREX-mediated DNA sensing and anti-phage defense.

Using Escherichia coli BW25113 as a host, we isolated a novel lytic phage from the commercial poly-specific therapeutic phage cocktail Sextaphage^® (Microgen, Russia). We provide genetic and phenotypic characterization of the phage and describe its host range on the ECOR collection of reference E. coli strains. The phage, hereafter named Sxt1, is a close relative of classical coliphage T3 and belongs to the Teetrevirus genus, yet its internal virion proteins, forming an ejectosome, differ from those of T3. In addition, the Sxt1 lateral tail fiber (LTF) protein clusters with those of the phages from the Berlinvirus genus. A comparison of T7, T3, and Sxt1 LTFs reveals the presence of insertions leading to the elongation of Sxt1 tail fibers, which, together with the difference in the HRDRs (host range-determining regions), might explain the expanded host specificity for the Sxt1.

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.