Structural characterisation and re-engineering of the PglX methyltransferase and specificity factor for BREX phage defence.
Bacteria must defend from infection by bacteriophages (phages), as they are outnumbered by viral parasites that outnumber them by aboutn estimated ten-to-one. Bacteria have evolved a broad range of systems that provide phage defence, many of which have been harnessed for valuable biotechnological purposesco-opted as useful biotechnologies. Bacteriophage Exclusion (BREX) systems recognize and methylate 6 bp non-palindromic motifs within the host genome. An unknown mechanism then prevents replication of non-modified phage DNA that encodesing the same motifs, but how BREX recognizes cognate motifs has not been fully understood. We have characterised BREX from pathogenic Salmonella and have generated the first X-ray crystallographic structure of the conserved BREX protein, PglX. The PglX N-terminal domain is a methyltransferase homolog and the C-terminal domain is implicated in DNA-binding. Through rational engineering of PglX, we it was possible to broadened both the range of phages targeted, and the host motif sequences that are methylated. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr. Our analyses propose modes for DNA-binding by PglX and indicate show that larger BREX complexes are required for methyltransferase activity and defence. ONevertheless, our data demonstrate that PglX is the sole specificity factor for BREX activity which, providesing motif recognition for both phage defence and host methylation.