Functional and Structural Insights into Novel Bacteriophage
Defence Islands

Abstract

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.