Characterization of the Molecular Mechanisms of Cell Division In Escherichia coli

描述(申请人提供)：细胞质分裂或细胞分裂是所有生命的生物学基础。从细菌到人类，细胞骨架在细胞分裂中扮演着重要的角色。在细菌中，大肠杆菌FtsZ是真核细胞骨架蛋白TuBulin的同源物，是第一个已知的定位于中细胞的蛋白质，这是未来分裂的地点。至少有19种必需蛋白和辅助蛋白参与这一过程，所有这些蛋白都在中间细胞组装成一个大的环状多蛋白复合体。大肠杆菌的基本细胞分裂机制已经有了很好的特征，然而，关于辅助因子在分裂复合体的维持和活动中的身份和作用(S)，仍有许多有待发现。建议的应用重点是通过在大肠杆菌中使用全基因组亚细胞蛋白定位文库来鉴定3种可能的附属分裂蛋白的分子相互作用，并有3个特定的目标。第一，通过结构域交换、缺失分析和随机突变分析，鉴定和鉴定以前未鉴定的分裂蛋白中的关键结构域和氨基酸残基，使其能够定位到中间细胞。非局部化的衍生物将使用一种新的基因筛查来识别。第二，使用耗竭和过早靶向分析来确定这些假定的辅助蛋白在中细胞的募集模式。辅助蛋白对细胞分裂的协同作用将通过使不同的基因组合失活来检验。第三，通过检查已知的抑制子，并随后扩展遗传筛选来确定其他因素，以确定与以前未鉴定的分裂蛋白相互作用的蛋白质。相互作用将通过双杂交和免疫共沉淀试验得到确认。本研究中确定的在大肠杆菌中推测的细胞分裂辅助因子在许多革兰氏阴性菌中高度保守，包括病原体，如志贺氏菌、沙门氏菌、耶尔森氏菌和弧菌。各种因素如何影响FtsZ环的稳定性和中细胞活性的分子表征可以被用来识别新一代抗菌药物靶点。此外，我们的研究将阐明辅助因素如何在胞质分裂过程中介导FtsZ环的时空调节，这也对全面理解细菌蛋白质的定位具有重要意义。使用大肠杆菌作为模型系统，这项研究解决了细菌细胞如何在中间细胞分裂的一个基本科学问题。辅助蛋白在调节中细胞分裂复合体的稳定性和活性方面的重要作用将被检验。鉴定和表征保守的辅助蛋白如何控制分裂复合体完整性的分子机制可以作为开发新的抗菌药物的靶点。

DESCRIPTION (provided by applicant): Cytokinesis or cell division is the biological basis of all life. From bacteria to humans, the cytoskeleton plays an important role in cell division. In the bacterium, Escherichia coli, FtsZ - a homolog of the eukaryotic cytoskeletal protein tubulin, is the first known protein to localize to midcell, the site of future division. At least 19 essential and accessory proteins are involved in this process, all of which assemble into a large ring like multi- protein complex at midcell. The E. coli essential cell division machinery is well characterized, however, much remains to be discovered about the identities and role(s) of accessory factors in the maintenance and activity of the division complex. The proposed application focuses on characterizing the molecular interactions of 3 putative accessory division proteins identified by using a genome-wide sub-cellular protein localization library in E. coli and has 3 specific goals. One, to identify and characterize key domains and amino acid residues within the previously uncharacterized division proteins that allow localization to midcell using domain swaps, deletion assays, and random mutagenesis analyses. Non-localizing derivatives will be identified using a novel genetic screen. Two, to determine the recruitment pattern of these putative accessory proteins at midcell using depletion and premature targeting assays. Synergistic effects of accessory proteins on cell division will be examined by inactivating different combinations of genes. Three, to identify proteins that interact with the previously uncharacterized division proteins by examining known suppressors, and later extending genetic screens to identify additional factors. Interactions will be confirmed by two- hybrid and co-immunoprecipitation assays. The putative cell division accessory factors identified in this study in E. coli are highly conserved in a number of Gram-negatives, including pathogens, such as Shigella, Salmonella, Yersinia, and Vibrio. Molecular characterization of how the various factors influence FtsZ ring stability and activity at midcell could be exploited to identify a new generation of antimicrobial drug targets. Furthermore, our study will illuminate how accessory factors mediate spatiotemporal regulation of the FtsZ ring during cytokinesis, as also have important implications towards the general understanding of how localization of bacterial proteins is achieved. Using Escherichia coli as a model system, this study addresses a fundamental scientific problem of how bacterial cells divide at midcell. The important role of accessory proteins in regulating the stability and activity of the midcell division complex will be examined. Identification and characterization of the molecular mechanisms of how conserved accessory proteins govern division complex integrity could be exploited as targets in the development of new antibacterials.