Horizontal gene transfer (HGT), often facilitated by mobile genetic elements (MGE), is an essential component of bacterial evolution and ecology. HGT facilitates the spread of significant traits including antimicrobial resistance (AMR). However, incoming genes can introduce genomic conflict. The basis of such conflict can lie in specific gene-gene interactions, but the mechanistic basis, and whether such conflicts are host specific, is not clear.

There is an intrinsic, universal evolutionary pressure for bacteria to defend themselves against such invasive, foreign DNA. This has led to bacteria developing ways to protect themselves against MGEs such as phage and plasmids by encoding a diverse range of defence systems. The anti-phage defence field is a rapidly emerging and growing discipline of study with the discovery of dozens of novel systems reported upon within the last few years. Crucially, the systems we are currently aware of are thought to be the very tip of the iceberg and it is anticipated that the full antiphage weaponry of bacteria will likely include hundreds, if not thousands, of diverse, mobile defence systems. Furthermore, recent work is beginning to shed light on the ability of phage and plasmids to counter-defend against these systems through the carriage of what has been termed ‘anti-defence’- systems.

In order to successfully combat the global and complex issue of AMR, it is imperative that we better understand the conflict between phage and plasmid defences encoded by bacteria and their mobile genetic elements.

Several naturally-occurring mercury resistance ‘pQBR’ plasmids impose significant costs to Pseudomonas fluorescens SBW25, and previous work indicates the principal source of conflict is hypothetical DUF262 domain-containing chromosomal protein PFLU4242 (4242). DUF262 is predicted to act as an NTPase, and has been identified within several recently discovered novel anti-phage systems including BrxU, Dazbog and PD-T4-2. 4242 has been identified as a putative Type IV restriction-modification (RM) system that may play a role in anti-MGE defence; 4242-pQBR interactions are therefore an exemplary model of MGE conflict. 4242’s mechanism of action is unknown, as is whether 4242-pQBR conflict is genetic background specific.

We analysed 4242 homologue distribution across diverse species, and found that it often resides within Defence Islands and is widely distributed via HGT. Given this genomic co-localisation of 4242-like proteins with other Defence Island elements, we hypothesise that 4242-like proteins are a genome defence mechanism. To explore this further, we cloned 4242 and a naturally-arising inactive mutant into Escherichia coli DH5-alpha and challenged these isogenic strains with a panel of coliphage. 4242 failed to show activity against glc-5hmC, 5mC, or 5hmC modifications despite being a homologue of the well characterised GmrSD and BrxU anti-phage defence systems which are known to provide robust protection against T-even phage.

To understand 4242’s physiological activity and ecological function, we next expressed 4242 and the naturally-arising inactive mutant in other Pseudomonas species. Our results help answer whether 4242-pQBR conflict is specific to P. fluorescens SBW25, or might operate across the Pseudomonas pangenome. Our data provides better understanding of MGE/host dynamics and explores the poorly understood trade-off between openness to HGT and genome defence: a key mechanism determining genome content, adaptive capacity, and pangenome structure.

Our current work focuses on a 4242-homologue found in Salmonella enterica serotype Newport, in order to better understand the anti-phage defence capabilities of such Type IV RM systems in a clinically relevant strain that is rapidly emerging as a pathogen in both animals and humans. We also seek to investigate conserved Defence Island hotspots in this serovar.