内源性质粒pMF1存在机制及其对粘球菌宿主进化的影响

Myxobacteria are a group of bacteria that possess complex multicellular social behaviors and big genomes. The endogenous plasmid pMF1 is the only plasmid that is able to replicate autonomously in myxobacterial cells. Based on the previous work, we finished the sequencing of the whole genome of the pMF1-harboring strain Myxococcus fulvus 124B02, which is obviously larger than the other sequenced Myxococcus genomes. In order to investigate the existence mechanisms of pMF1 and effects of the plasmid on the host evolution, we cured pMF1 from 124B02 and performed laboratory evolution experiments. The results showed that the plasmids varied stabilities under different culture conditions, and significantly affected the phenotypes of the hosts. In this project, we are planning to re-sequence these laboratory-evolving strains to determine the mutated genes; genetically testify the key factors for host changes; and study functions of the key genes. The studies are not only important for our understanding of the formation of the complex living patterns and big genomes in myxobacteria, but also provide insights into the co-evolution of bacterial host and endogenous low-copy plasmids.

粘细菌是具有复杂群体细胞行为和大基因组特征的细菌类群。pMF1是到目前为止发现的唯一能在粘细菌细胞中稳定存在的内源性质粒。我们在前期工作基础上，完成了发现质粒的菌- - 橙色粘球菌（Myxococcus fulvus）124B02的全基因组测序。该菌基因组显著大于其他已测序的粘球菌。为研究pMF1的存在机制及其对粘细菌宿主进化的影响，我们从124B02中消除了pMF1，并设置不同实验组进行人工进化实验。结果显示，随着培养条件的不同，质粒表现出差异的稳定性，并显著影响宿主细胞的表型变化。本申请拟对人工进化菌株进行基因组重测序，寻找突变基因；通过遗传学实验验证关键基因；并开展关键基因的功能分析。研究结果对认识粘细菌复杂生活方式和大基因组的形成有重要意义，并为研究细菌宿主与内源性低拷贝质粒的共同进化模式提供参考。

pMF1是我们前期发现的粘细菌内源性质粒，是到目前为止发现的唯一能在粘细菌细胞中稳定存在的质粒。在本项目中，（1）我们完成了发现该质粒的菌株-橙色粘球菌124B02的全基因组测序，并进行了基因组横向分析，尝试从组学层面上解析pMF1存在于124B02中的机制；（2）为研究pMF1的存在机制及其对粘细菌宿主进化的影响，我们从124B02中消除了pMF1，并设置不同实验组进行人工进化实验。结果表明，pMF1质粒的存在具有条件依赖性，重测序结果说明124B02/pMF1菌株的基因组变异的整体趋势在正常范围，而124B02/free菌株基因组突变率发生了异常的增高；（3）我们证明pMF1.19和pMF1.20是一对核酸酶毒素-免疫蛋白，以后分离自杀的方式在质粒稳定遗传中发挥作用——结合我们之前对pMF1质粒中复制和分配系统的研究结果，可以确定多种质粒维持机制共同帮助这个低拷贝质粒提高稳定性；（4）以纯化后的ParA进行pulldown和western检测结果发现，ParA能够钓取ParC；结构模建显示，ParA能够形成同源二聚体，并借助ParC 蛋白中介，形成ParA-ParA-ParC-ParA-ParA 排列的五聚体，通过ParA同源二聚体和ParC蛋白的交替结合，可以产生细长的纤维丝结构来介导pMF1质粒的分配。