Characterization of the Molecular Mechanisms of Cell Division In Escherichia coli

描述（由申请人提供）：细胞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起着重要作用。在大肠杆菌中，FtsZ -真核细胞骨架蛋白微管蛋白的同源物，是已知的第一个定位于中间细胞的蛋白，即未来分裂的位点。至少有19种必需蛋白和辅助蛋白参与了这一过程，它们在细胞中间组装成一个大的环状多蛋白复合物。大肠杆菌的基本细胞分裂机制已经被很好地描述，然而，关于辅助因子在分裂复合体的维持和活性中的身份和作用，还有很多有待发现。该申请主要是利用大肠杆菌全基因组亚细胞蛋白定位文库鉴定3种推定的辅助分裂蛋白的分子相互作用，并有3个特定目标。首先，利用结构域交换、缺失分析和随机突变分析，鉴定和表征先前未表征的分裂蛋白中的关键结构域和氨基酸残基，这些结构域和氨基酸残基允许定位到中细胞。非定位衍生物将使用一种新的基因筛选来识别。第二，利用耗尽和过早靶向试验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灭活不同的基因组合来检验。第三，通过检查已知的抑制因子来确定与先前未表征的分裂蛋白相互作用的蛋白质，然后扩展遗传筛选以确定其他因素。相互作用将通过双杂交和共免疫沉淀试验来证实。本研究在大肠杆菌中发现的假定的细胞分裂辅助因子在许多革兰氏阴性菌中高度保守，包括志贺氏菌、沙门氏菌、耶尔森氏菌和弧菌等病原体。各种因素如何影响FtsZ环在中细胞的稳定性和活性的分子表征可以用于鉴定新一代抗菌药物靶点。此外，我们的研究将阐明辅助因子如何在细胞分裂过程中介导FtsZ环的时空调节，也对如何实现细菌蛋白定位的一般理解具有重要意义。使用大肠杆菌作为模型系统，本研究解决了细菌细胞如何在中细胞分裂的基本科学问题。辅助蛋白在调节中细胞分裂复合体的稳定性和活性中的重要作用将被检查。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.