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

项目摘要 本应用程序的长期目标是了解 一种超分子机械，称为分裂体，用于进行细菌细胞分裂。这个 分裂组由三十多种蛋白质组成，这些蛋白质被精心编排，以组装和 在正确的时间和空间发挥作用，以确保细胞质分裂成功。理解 这一过程中的关键限速步骤以及不同蛋白质如何与每个步骤协调 其他重要的是为治疗细菌提供潜在的新的抗微生物靶点 感染。过去，该领域的一个主要焦点是基本组成部分的作用 在分裂体中，由微管蛋白同源蛋白FtsZ形成的环状结构(Z环)， 在产生机械力的情况下主动收缩膜。他者的角色 分裂体的两个组成部分，隔膜细胞壁肽聚糖(PG)的合成 机械和染色体分离机械，被认为遵循活跃的 Z-环的收缩。最近积累的证据表明，Z环可能不会 是主要的力量产生者，但也是关键的调节器/介体，而细胞壁合成 而染色体分离机制在驱动间隔关闭方面发挥了更大的作用 而不是Z环收缩。 这里描述的项目将使用以下组合来检验这一假设 单分子成像、遗传学、生化和结构方法。第一个目标是 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结构与 含有Z-环的突变细胞，其结构、动力学、GTP酶活性和 蛋白质之间的相互作用。第二个目的是确定间隔前列腺素的作用。 在驱动间隔关闭中的综合。间隔PG的合成将受到系统性的干扰 使用靶向突变和药物治疗，以及相应的时间依赖 隔膜闭合率、隔膜PG掺入率、隔膜形态和成分 将使用各种单分子成像和生化方法进行测量。 第三个目标是确定负责协调的结构基础 染色体分离和隔膜关闭之间的关系。分子间的界面 一组相互作用并将Z环固定在染色体上的蛋白质 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)高分辨率的结构和 完整的大肠杆菌分裂体的动态模型，(2)重新定义的角色和相对贡献 胞质分裂中的三个主要分裂组分，(3)对 以及(4)一套创新的成像技术-- 使细菌细胞生物学家能够使用的工具和检测方法。

Project Abstract The long-term goal of this application is to understand the working mechanism of a supramolecualr 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 suit 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.