Functional and Structural Insights into Novel Bacteriophage Defence Islands

Bacteriophages are the most abundant organisms on the planet and are a major driving force in bacterial evolution. As obligate intracellular parasites, phages are reliant on their bacterial host for propagation, but bacteria have evolved means to prevent phage infections. Bacteriophage exclusion (BREX) is a novel phage-resistance system that confers resistance to a wide array of phages, functioning independently of restriction-modification, CRISPR-Cas and abortive infection mechanisms. BREX loci are present in ~10% of bacterial and archaeal genomes, including pathogenic strains such as non-typhoidal invasive Salmonella enterica and multidrug resistant Escherichia fergusonii. Whilst investigating the mechanism of BREX in E. fergusonii, a putative endonuclease was discovered, clustered within the BREX locus. This enzyme, BrxU, was biochemically and structurally characterised, and shown to be a standalone phage defence system that targets modified phage genomes. It became clear that the BREX and BrxU phage defence systems were organised into a phage defence island, constituting a bacterial immune system capable of resisting multiple phage types. Both systems detailed in this thesis represent novel antiphage mechanisms with potential for biotechnological application. The BrxU endonuclease structure has been solved to 2.12 Å and reveals insight into key protein domains implicated in type IV restriction enzymes. BrxU has been observed to utilise a range of nucleotide and metal cofactors and confers extensive protection to its bacterial host against phage infection.