TARGETING AND ASSEMBLY OF E COLI CELL DIVISION PROTEINS

细胞分裂对细菌的增殖至关重要，并且似乎依赖于位于分裂部位的分子机器。尽管对这一基本细胞过程中涉及的主要蛋白质有广泛的了解，但对于这些蛋白质如何被靶向，它们如何组装成一个复合体，以及它们如何从根本上改变正常细胞壁生长以合成分裂隔膜，人们知之甚少。该项目的长期目标是利用新的、强大的细菌细胞学方法，以大肠杆菌为模型系统，阐明细胞分裂蛋白靶向和组装背后的分子机制。一个关键的通用细胞分裂蛋白FtsZ在分裂位点的内膜表面自组装成一个聚合环（z环），并招募其他蛋白质，包括FtsA， ZipA和一组完整的膜蛋白，所有这些蛋白质都有助于完成隔膜。本提案的第一个目的是获得FtsZ对FtsA和ZipA招募的分子理解。解决这一问题的一种方法是对FtsZ- ftsa和FtsZ- zipa相互作用中涉及的FtsZ区域进行诱变，以获得相互作用中有缺陷的蛋白质。一旦这些突变体被分离出来，对基因内和基因外抑制子的选择可能会揭示残基特异性接触。新型荧光原位蛋白相互作用测定将用于筛选蛋白-蛋白相互作用，然后进行直接相互作用的生化试验。第二个目的是研究FtsA在细胞分裂中的作用。纯化的FtsA的酶和结构性质将被表征。此外，FtsA与膜中FtsZ、ZipA和下游细胞分裂蛋白相互作用的能力将被测试。该项目的最终目标是了解z环是如何定位到其确切的蜂窝地址的。类核似乎对z环装配产生了负面影响，可能是类核偏析释放了一个负面信号，从而允许精确的z环定位。将对类核结构缺陷和染色体复制缺陷突变体的z环组装进行研究，以确定类核和z环之间的关系。最后，由MinC、D和E组成的Min系统是Z形环装配和定位的重要调节器。Min系统与自由贸易区之间的关系将从生物化学和遗传学两方面进行详细讨论。本研究的结果有望为细胞分裂的分子机制提供更完整的认识，并有助于分离新的和更好的抗菌药物靶点。

Cell division is essential for proliferation of bacteria, and appears to rely on a molecular machine positioned at the site of division. Despite extensive knowledge of the major proteins involved in this fundamental cellular process, very little is understood about how these proteins are targeted, how they assemble into a complex, and how they radically alter normal cell wall growth in order to synthesize the division septum. The long term goals of this project are to elucidate the molecular mechanisms behind the targeting and assembly of cell division proteins, using Escherichia coli as a model system and taking advantage of new, powerful cytological methods for bacteria. A key universal cell division protein, FtsZ, self-assembles into a polymeric ring (the Z-ring) at the surface of the inner membrane at the division site and recruits other proteins, including FtsA, ZipA, and a group of integral membrane proteins, all of which help to complete the septum. The first aim of this proposal is to gain a molecular understanding of the recruitment of FtsA and ZipA by FtsZ. One way that this will be addressed is by mutagenesis of a region of FtsZ that is involved in FtsZ-FtsA and FtsZ-ZipA interactions in order to obtain proteins defective in interaction. Once such mutants are isolated, selection for intragenic and extragenic suppressors may reveal residue-specific contacts. Novel fluorescent in situ protein interaction assays will be used to screen for protein-protein interactions, followed by biochemical tests for direct interaction. The second aim will investigate the role of FtsA in cell division. Enzymatic and structural properties of purified FtsA will be characterized. In addition, the ability of FtsA to interact with FtsZ, ZipA, and downstream cell division proteins in the membrane will be tested. The final aims of the project are to understand how the Z-ring gets targeted to its exact cellular address. The nucleoid appears to negatively influence Z-ring assembly, and it is possible that nucleoid segregation releases a negative signal to allow precise Z-ring positioning. Studies of Z-ring assembly in mutants defective in nucleoid structure and chromosome replication will be carried out to determine the relationship between the nucleoid and the Z-ring. Finally, the Min system, which consists of MinC, D, and E, is an important regulator of Z- ring assembly and positioning. The relationship between the Min system and FtsZ will be addressed in detail both biochemically and genetically. The results of this proposed research are expected to provide a more complete understanding of the molecular mechanisms of cell division, and should facilitate the isolation of new and better antimicrobial drug targets.