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)在内的重要特征的传播。然而，传入的基因可能会导致基因组冲突。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有的，尚不清楚。为了防御入侵的外来DNA，细菌编码了一系列不同的防御系统。为了成功地抗击AMR这一全球性而复杂的问题，我们必须更好地了解细菌编码的这种防御与移动遗传元件(MGE)之间的冲突，MGE是AMR传播的主要原因。几个自然产生的汞抗性‘pQBR’质粒给荧光假单胞菌SBW25带来了巨大的成本，以前的工作表明，主要来源是假设的DUF262结构域含有染色体蛋白PFLU4242(4242)，它是GmrSD家族IV型限制性内切酶修饰系统的成员。因此，4242-pQBR互作提供了一个典型的群体冲突模型，但4242‘S的功能未知，也不知道4242-pQBR冲突是否是宿主特有的。我们的工作试图揭示4242’S的进化背景、生态功能和作用机制。我们分析了4242个同源基因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类蛋白与已知的“防御岛”元件明显的基因组共定位，我们假设4242类蛋白是一种基因组防御机制。为了了解4242‘S的生理活性和生态功能，我们在其他假单胞菌中表达了4242和一个自然产生的失活突变体。我们的结果表明，4242-pQBR的冲突是宿主特有的。对于防御系统来说，非常不寻常的是，4242到目前为止只展示了质粒而不是噬菌体的防御能力。结论噬菌体防御研究仍在进行中，但我们推测这可能与4242‘S预测的结构有关，该结构包含DUF262与其他GmrSD样蛋白的关键差异。最后，我们计划调查4242‘S保守的核苷酸水解和HNH核酸酶基序的靶向突变是否足以消除在其天然宿主中看到的4242介导的pQBR质粒防御。我们的数据为更好地理解遗传背景在MGE/宿主动态中的重要性提供了更好的理解，并探索了对HGT的开放和基因组防御之间鲜为人知的权衡：这是决定基因组含量、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.