Characterization of the Bacterial BREX Phage Restriction System - Administrative Supplement

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SUMMARY (Originally submitted for grant number 1R15GM140375-01A1) The arms race between bacteria and phage has driven bacteria to evolve a wide range of defense systems, many of which remain to be characterized. A largely uncharacterized phage restriction system called ‘BREX’ (‘Bacteriophage Exclusion’), initially described 35 years ago, is widely distributed in bacterial and archaeal genomes. The mechanism of BREX-mediated phage restriction is likely complex given the large number of genes (4-8 genes, depending on the subfamily) and range of enzymatic activities encoded by BREX systems. Studies of type 1 and type 2 BREX systems (there are 6 total subfamilies) have demonstrated that the PglX DNA methyltransferase establishes self / non-self discrimination by modifying the bacterial genome. However, BREX-mediated restriction does not appear to function by degrading the phage genome, which is a surprise given the well-characterized role for similar methyltransferase enzymes in restriction-modification (R-M) systems. Type 1 BREX systems inhibit phage after adsorption and prior to DNA replication, but further details about the mechanism of phage restriction are not known. The long-term goal is to understand the mechanism by which BREX restricts phage, including elucidating the functional role of each BREX component. Our understanding of BREX-mediated phage restriction is limited because the function of most individual BREX proteins is not understood, nor is it understood how they are regulated. To address these questions, we are characterizing the Acinetobacter type 1 BREX system using biochemical and structural studies combined with biological activity assays using an E. coli-based phage infection system. The first aim will address the hypothesis that Brx0, an Acinetobacter-specific factor (i.e. not present in other BREX Type 1 systems), transcriptionally regulates other BREX ORFs. The second aim will address the hypothesis that BrxL is a AAA+ chambered protease that specifically targets substrate proteins for degradation. Uncovering the roles of both proteins will provide key insight into the mechanism of BREX-mediated phage restriction, not only in Acinetobacter but as a general strategy in other bacterial clades. Gaining a fundamental understanding of bacterial defense systems could impact human health in numerous ways, including contributing to strategies to treat antibiotic-resistant bacteria and understanding how the microbiome relates to health and disease.