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

 描述（由申请人提供）：本申请的长期目标是了解超分子机制（称为分裂体）在进行细菌细胞分裂中的工作机制。分裂体由30多种蛋白质组成，这些蛋白质在正确的时间和空间进行组装和功能，以确保成功的胞质分裂。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，对于提供潜在的新的抗菌靶标以治疗 细菌感染在过去，该领域的一个主要焦点是在分裂体的基本组分的作用上，分裂体是由微管蛋白同源蛋白FtsZ形成的环状结构（Z环），在产生机械力以主动收缩膜中。分裂体的其他两个组成部分，隔细胞壁肽聚糖（PG）合成机制和染色体分离机制的作用，被认为是遵循Z-环的主动收缩。最近积累的证据表明，Z-环可能不是主要的力发生器，但一个关键的调节器/调解器，细胞壁合成和染色体分离机制有更大的作用，在驱动隔膜关闭比Z-环收缩。 这里描述的项目将使用单分子成像，遗传，生物化学和结构方法的组合来检查这一假设。第一个目的是确定的Z-环在隔PG蛋白的时空组织的作用。然后将野生型FtsZ细胞中的动力学和组织与具有改变的结构、动力学、GTdR活性和蛋白质-蛋白质相互作用的Z环的突变细胞中的动力学和组织进行比较。第二个目的是确定隔PG合成在驱动隔关闭中的作用。将使用靶向突变和药物治疗系统性干扰间隔PG合成，并使用各种单分子成像和生化方法测量相应的时间依赖性间隔闭合率、间隔PG掺入率、间隔形态和组成。第三个目的是确定染色体分离和隔膜关闭之间协调的结构基础。将使用遗传、生物化学、计算和结构方法来确定一套蛋白质之间的分子界面，这些蛋白质彼此相互作用并将Z环锚定到染色体上。 本项目的预期成果是：（1）高分辨率的全E.杆菌 分裂体，（2）重新定义的作用和相对贡献的三个主要分裂体组分在胞质分裂，（3）分子洞察分裂体的工作机制作为一个整体，和（4）一套创新的成像为基础的工具和分析，使细菌细胞生物学家。

 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.