Chromosome Dynamics in Bacillus subtilis

描述(申请人提供)：这项建议侧重于了解细菌染色体动力学的基本特性：细菌染色体在细胞周期和分化过程中是如何重塑和分离的？染色体组织和分离是如何与DNA复制联系在一起的？在这些过程中涉及的顺式作用元件和反式作用因子的作用是什么？在细胞质分裂后，错误分离的DNA是如何转移到合适的女儿体内的？事实证明，这些最基本的生物学问题非常棘手，部分原因是功能冗余和缺乏定量分析。我们建议研究模式革兰氏阳性细菌枯草芽孢杆菌的染色体的组织和分离。在这种生物体中进行遗传、生化和细胞学分析非常容易，这使它成为研究染色体动力学的理想系统。我们将使用下一代分子和细胞生物学工具以及我们为研究孢子形成和营养细胞周期中的染色体而开发的定量分析方法来解决这些问题。作为一个概念和实验框架，细菌的染色体分离过程可以分为三个步骤。I)将新复制的起始点朝向细胞极点重新定位。Ii)姐妹染色体重塑和分离，然后细胞分裂。3)胞质分裂时存在于分裂平面的未分离的DNA通过隔膜转移到适当的子细胞中。这一动态过程在每个细胞周期中都以极高的保真度执行。在这项提案中，我们将研究所有三个步骤的分子基础。我们建议：1)确定与其同源部分结合的染色体分割蛋白PARB如何将SMC凝聚复合体招募到起源，以及起源定位的SMC如何紧凑和组织染色体。2)确定SpoIIIE转位酶如何在分裂到隔膜运输未分离的DNA。3)研究复制起始和起始分离在进入产孢期后是如何联系的。与公共卫生相关：将染色体忠实地分离到子代细胞是一个基本的过程。了解在细菌中控制这一过程的分子机制可能会导致发现适合于抗菌治疗的新靶点。在这项建议中，我们研究了枯草芽孢杆菌生长和产孢期的染色体动力学。因此，对于革兰氏阳性病原体和梭状芽胞杆菌和炭疽杆菌的孢子形成者的见解将特别相关。

DESCRIPTION (provided by applicant): This proposal is focused on understanding fundamental properties of bacterial chromosome dynamics: How is the bacterial chromosome remodeled and segregated during the cell cycle and differentiation? How is chromosome organization and segregation linked to DNA replication? What are the roles of the cis-acting elements and trans-acting factors implicated in these processes? How is mis-segregated DNA translocated into the appropriate daughter after cytokinesis? These most basic biological questions have proven remarkably intractable, in part, due to functional redundancy and the lack of quantitative assays. We propose to study the organization and segregation of the chromosome in the model gram-positive bacterium Bacillus subtilis. The ease with which genetic, biochemical and cytological analysis can be carried out in this organism makes it an ideal system to study chromosome dynamics. We will address these questions using the next generation of molecular and cell biological tools and quantitative assays that we have developed to study the chromosome during spore-formation and the vegetative cell cycle. As a conceptual and experimental framework, the process of chromosome segregation in bacteria can be divided into three steps. i) The newly replicated origins are re-positioned towards the cell poles. ii) The sister chromosomes are remodeled and segregated followed by cell division. iii) Unsegregated DNA present at the division plane at the time of cytokinesis is translocated across the septum into the appropriate daughter cell. This dynamic process is executed in every cell cycle with extremely high fidelity. In this proposal we will investigate the molecular underpinnings of all three steps. We propose to: 1) Determine how the chromosomal partitioning protein ParB bound to its cognate parS sites recruits the SMC condensation complex to the origin and how origin-localized SMC compacts and organizes the chromosome. 2) Determine how the SpoIIIE translocase functions at the division to septum to transport unsegregated DNA. 3) Investigate how replication initiation and origin segregation are linked upon entry into sporulation. PUBLIC HEALTH RELEVANCE: The faithful segregation of chromosomes to daughter cells is an essential process. Understanding the molecular mechanisms that govern this process in bacteria could lead to the discovery of new targets appropriate for antimicrobial therapeutics. In this proposal we investigate chromosome dynamics during growth and sporulation in Bacillus subtilis. Accordingly, insights will be particularly relevant to gram-positive pathogens and spore-formers of the Clostridiales and B. anthracis.