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

水平基因转移（HGT）是细菌进化和生态学的重要组成部分，促进了包括抗菌素耐药性（AMR）在内的重要性状的传播。然而，引入的基因可能会引入基因组冲突。这种冲突的基础可能在于特定的基因-基因相互作用，但机制基础，以及这种冲突是否是宿主特异性的，还不清楚。为了抵御外来DNA的入侵，细菌编码了各种各样的防御系统。为了成功应对全球性的复杂AMR问题，我们必须更好地了解细菌编码的这种防御与移动的遗传元件（MGE）之间的冲突，这是AMR传播的主要原因。几种天然存在的汞抗性“pQBR”质粒对荧光假单胞菌SBW 25造成了显著的成本，并且先前的工作表明主要来源为假设的含DUF 262结构域的染色体蛋白PFLU 4242（4242），其为IV型限制性修饰系统的GmrSD家族的推定成员。4242-因此，pQBR相互作用是泛基因组冲突的典型模型，但4242的功能尚不清楚，4242-pQBR冲突是否具有宿主特异性。我们分析了4242个同源物在不同物种中的分布，表明它是辅助基因组的一部分，并通过HGT分布。鉴于4242样蛋白与已知的“防御岛”元件的明显基因组共定位，我们假设4242样蛋白是基因组防御机制。为了了解4242的生理活性和生态功能，我们随后在其他假单胞菌属物种中表达了4242和一个自然产生的失活突变体。我们的研究结果表明，4242-pQBR冲突是宿主特异性的。对于防御系统来说，4242是非常不寻常的，到目前为止，它只表现出质粒而不是噬菌体的防御能力。结论噬菌体防御调查悬而未决，但我们推测，这可能与4242的预测结构包含DUF 262的关键差异相比，其他GmrSD样蛋白。最后，我们计划调查是否有针对性的突变4242的保守的核苷酸水解和HNH核酸酶基序是足以消除4242介导的pQBR质粒防御在其天然host.Our的数据中看到的MGE/宿主dynamics遗传背景的重要性提供了更好的理解，并探讨了鲜为人知的开放HGT和基因组防御之间的权衡：决定基因组内容，适应能力和泛基因组结构的关键机制，对AMR和噬菌体治疗的未来工作具有重要意义。

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.