TARGETING AND ASSEMBLY OF E COLI CELL DIVISION PROTEINS

细胞分裂对于细菌的增殖是必不可少的，并且似乎依赖于位于分裂位点的分子机器。 尽管对参与这一基本细胞过程的主要蛋白质有广泛的了解，但对这些蛋白质如何靶向，它们如何组装成复合物，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的了解甚少。 该项目的长期目标是阐明细胞分裂蛋白的靶向和组装背后的分子机制，使用大肠杆菌作为模型系统，并利用新的，强大的细菌细胞学方法。 一种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聚合物环（Z环），并招募其他蛋白质，包括FtsA，ZipA和一组完整的膜蛋白，所有这些都有助于完成隔膜。 该提案的第一个目的是获得FtsZ招募FtsA和ZipA的分子理解。 解决这一问题的一种方法是诱变参与FtsZ-FtsA和FtsZ-ZipA相互作用的FtsZ区域，以获得相互作用缺陷的蛋白质。 一旦分离出这样的突变体，基因内和基因外抑制子的选择可以揭示残基特异性接触。 新的荧光原位蛋白质相互作用试验将用于筛选蛋白质-蛋白质相互作用，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的酶和结构特性的特点。 此外，将测试FtsA与膜中的FtsZ、ZipA和下游细胞分裂蛋白相互作用的能力。该项目的最终目标是了解Z环如何定位到其确切的蜂窝地址。 类核似乎对Z环组装产生负面影响，并且类核分离可能释放负信号以允许精确的Z环定位。 将进行类核结构和染色体复制缺陷突变体中Z环组装的研究，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系统和FtsZ之间的关系将在生物化学和遗传学上详细讨论。 这项拟议研究的结果预计将提供对细胞分裂分子机制的更完整理解，并应有助于分离新的和更好的抗菌药物靶标。

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.