Integrating evolutionary and molecular microbiology to characterise host-specific plasmid costs and phage defence associated with a novel Type IV Rest

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Horizontal gene transfer (HGT) is an essential component of bacterial evolution and ecology, facilitating 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. To defend against invasive, foreign DNA, bacteria encode a diverse range of defence systems. In order to successfully combat the global and complex issue of AMR, it is imperative that we better understand the conflict between such defences encoded by bacteria and mobile genetic elements (MGE), largely responsible for the spread of AMR. Several naturally-occurring mercury resistance 'pQBR' plasmids impose significant costs to Pseudomonas fluorescens SBW25, and previous work indicated the principal source as hypothetical DUF262 domain-containing chromosomal protein PFLU4242 (4242), a putative member of the GmrSD family of Type IV Restriction Modification systems. 4242-pQBR interactions therefore presented an exemplary model of pangenome conflict, but 4242's function was unknown, as was whether 4242-pQBR conflict was host specific.Our work has sought to uncover 4242's evolutionary background, ecological function and mechanism of action. We analysed 4242 homologue distribution across diverse species, and show it is part of the accessory genome and distributed via HGT. Given apparent genomic co-localisation of 4242-like proteins with known 'defence island' elements, we hypothesised that 4242-like proteins are a genome defence mechanism. To understand 4242's physiological activity and ecological function, we then expressed 4242 and a naturally-arising inactive mutant in other Pseudomonas species. Our results show that 4242-pQBR conflict is host specific.Highly unusual for a defence system, 4242 has thus far only demonstrated plasmid and not bacteriophage defence capabilities. Concluding phage defence investigations are pending, but we speculate this may be related to 4242's predicted structure containing key differences in DUF262 when compared to other GmrSD-like proteins. Finally, we plan to investigate whether targeted mutations to 4242's conserved nucleotide hydrolysis and HNH nuclease motifs are sufficient to eliminate the 4242 mediated pQBR plasmid defence seen in its native host.Our data provides better understanding of the importance of genetic background in 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, with important implications for future work in AMR and Phage Therapy.