Probing the structure, dynamics and functiion of the E. coli divisome

 描述(申请人提供)：本申请的长期目标是了解超分子机械，称为分裂体，在执行细菌细胞分裂的工作机制。分裂体由30多种蛋白质组成，这些蛋白质经过精心编排，在正确的时间和空间组装并发挥作用，以确保细胞质分裂成功。了解这一过程中的关键限速步骤以及不同蛋白质如何相互协调对于为治疗提供潜在的新抗菌靶点非常重要 细菌感染。过去，该领域的一个主要焦点是分裂体的基本成分，即由微管蛋白同源蛋白FtsZ形成的环状结构(Z环)，在产生主动收缩膜的机械力中所起的作用。分裂体的另外两个组成部分，隔壁细胞壁肽聚糖(PG)合成机制和染色体分离机制，被认为是在Z环的主动收缩之后发挥的作用。最近越来越多的证据表明，Z环可能不是主要的作用力产生者，而是一个关键的调节/调节因子，细胞壁合成和染色体分离机制在驱动隔膜关闭方面比Z环收缩起到更大的作用。这里描述的项目将使用单分子成像、遗传、生化和结构方法的组合来检验这一假说。第一个目的是确定Z环在间隔PG蛋白的时空组织中的作用。然后将野生型FtsZ细胞中的动力学和组织与含有Z环的突变细胞中的动力学和组织进行比较，Z环的结构、动力学、GTPase活性和蛋白质-蛋白质相互作用发生变化。第二个目的是确定间隔PG合成在间隔关闭中的作用。靶向突变和药物治疗将系统地干扰间隔PG的合成，并将使用各种单分子成像和生化方法测量相应的依赖时间的间隔闭合率、间隔PG掺入率、间隔形态和组成。第三个目标是确定负责染色体分离和隔膜关闭之间协调的结构基础。一组相互作用并将Z环固定在染色体上的蛋白质之间的分子界面将使用遗传、生化、计算和结构方法来确定。该项目的预期成果是：(1)建立了完整的大肠杆菌的高分辨率结构和动力学模型 (2)重新定义了三个主要分裂体组分在胞质分裂中的作用和相对贡献；(3)从分子上深入了解了分裂体整体的工作机制；(4)一套创新的基于成像的工具和分析方法，使细菌细胞生物学家能够使用这些工具和方法。

 DESCRIPTION (provided by applicant): The long-term goal of this application is to understand the working mechanism of a supramolecular machinery, termed divisome, in carrying out bacterial cell division. The divisome is composed of more than thirty proteins that are orchestrated to assemble and function at the correct time and space to ensure successful cytokinesis. Understanding key rate-limiting steps in this process and how different proteins coordinate with each other is important to provide potential new antimicrobial targets for treating bacterial infections. In the past a major focus of the field is on the role of the essential component of the divisome, a ring-like structure (Z-ring) formed by the tubulin homolog protein FtsZ, in generating a mechanic force to constrict the membrane actively. The roles of the other two components of the divisome, septal cell wall peptidoglycan (PG) synthesis machinery and chromosome segregation machinery, are thought to follow the active contraction of Z-ring. Recently accumulating evidence suggests that the Z-ring may not be the main force generator but a key regulator/mediator, and that cell wall synthesis and chromosome segregation machineries have larger roles in driving septum closure than Z-ring contraction. The project described here will examine this hypothesis using a combination of single-molecule imaging, genetic, biochemical and structural methods. The first Aim is to determine the role of the Z-ring in the spatiotemporal organization of septal PG proteins. The dynamics and organizations in wild-type FtsZ cells will then be compared to those in mutant cells harboring Z-rings with altered structures, dynamics, GTPase activity, and protein-proteins interactions. The second Aim is to determine the role of septal PG synthesis in driving septum closure. Septal PG synthesis will be systematically perturbed using targeted mutations and drug treatments, and the corresponding time-dependent septum closure rate, septal PG incorporation rate, septa morphology and composition will be measured using a variety of single-molecule imaging and biochemical methods. The third Aim is to determine the structural basis responsible for the coordination between chromosome segregation and septum closing. Molecular interfaces between a suite of proteins that interact with each other and anchor the Z-ring to the chromosome will be determined using genetic, biochemical, computational and structural methods. The expected outcomes of the project are: (1) a high-resolution structural and dynamic model of the full E. coli divisome, (2) a redefined roles and relative contributions of the three major divisome components in cytokinesis, (3) molecular insight into the working mechanism of the divisome as a whole, and (4) a set of innovative imaging- based tools and assays enabling bacterial cell biologists.