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

整合进化和分子微生物学来表征与新型 IV 型休息相关的宿主特异性质粒成本和噬菌体防御

• 批准号: 2438578
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2438578
• 关键词: Integrating; evolutionary; molecular; microbiology; characterise

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.

水平基因转移（HGT）是细菌进化和生态的重要组成部分，促进了包括抗菌素耐药性（AMR）在内的重要性状的传播。然而，进入的基因会带来基因组冲突。这种冲突的基础可能在于特定的基因-基因相互作用，但其机制基础以及这种冲突是否具有宿主特异性尚不清楚。为了抵御外来DNA的入侵，细菌编码了一系列不同的防御系统。为了成功地对抗AMR的全球性和复杂性问题，我们必须更好地理解细菌编码的这种防御与主要负责AMR传播的移动遗传元件（MGE）之间的冲突。几种天然存在的抗汞“pQBR”质粒对荧光假单胞菌SBW25造成了重大影响，先前的研究表明，其主要来源是假想的含有DUF262结构域的染色体蛋白PFLU4242(4242)，这是IV型限制性修饰系统中假定的GmrSD家族成员。因此，4242- 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也不清楚。我们的工作旨在揭示4242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，发现它是附属基因组的一部分，并通过HGT分布。鉴于4242样蛋白与已知的“防御岛”元素明显的基因组共定位，我们假设4242样蛋白是一种基因组防御机制。为了了解4242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冲突是宿主特有的。对于防御系统来说，4242是非常不寻常的，迄今为止，它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尚待确定，但我们推测这可能与4242的预测结构有关，与其他gmrsd样蛋白相比，DUF262中含有关键差异。最后，我们计划研究4242的保守核苷酸水解和HNH核酸酶基序的靶向突变是否足以消除4242介导的pQBR质粒防御。我们的数据提供了更好的理解遗传背景在MGE/宿主动力学中的重要性，并探索了对HGT开放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，对未来AMR和噬菌体治疗的工作具有重要意义。