Chromosome Dynamics in Bacillus subtilis

描述（由申请人提供）：本提案的重点是了解细菌染色体动力学的基本特性：在细胞周期和分化过程中，细菌染色体是如何重塑和分离的？染色体的组织和分离与DNA复制有何联系？在这些过程中，顺式作用因子和反式作用因子的作用是什么？错误分离的DNA如何在细胞质分裂后转位到合适的子细胞中？这些最基本的生物学问题被证明是非常棘手的，部分原因是由于功能冗余和缺乏定量分析。我们拟研究革兰氏阳性细菌枯草芽孢杆菌染色体的组织和分离。这种生物体易于进行遗传、生化和细胞学分析，使其成为研究染色体动力学的理想系统。我们将利用下一代分子和细胞生物学工具以及我们开发的用于研究孢子形成和营养细胞周期期间染色体的定量分析来解决这些问题。作为一个概念和实验框架，细菌染色体分离的过程可以分为三个步骤。i)新复制的起始点被重新定位到细胞极点。ii)姐妹染色体被重塑和分离，然后是细胞分裂。iii)细胞质分裂时存在于分裂平面上的未分离DNA通过隔膜转移到适当的子细胞中。这种动态过程在每个细胞周期中以极高的保真度执行。在这个建议中，我们将研究这三个步骤的分子基础。我们建议：1)确定与其同源parS位点结合的染色体分裂蛋白ParB如何将SMC缩合复合体招募到原点，以及起源定位的SMC如何压缩和组织染色体。2)确定SpoIIIE易位酶如何在分裂到隔膜运输未分离的DNA。3)研究在进入孢子形成时，复制起始和起源分离是如何联系在一起的。公共卫生相关性：染色体忠实分离到子细胞是一个必要的过程。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用于抗菌治疗的新靶点。本文研究了枯草芽孢杆菌生长和产孢过程中的染色体动力学。因此，见解将特别相关的革兰氏阳性病原体和孢子形成梭状芽孢杆菌和炭疽杆菌。

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.