Production and function of E coli vesicles

性状（由申请方提供）：大肠埃希菌及其迄今为止研究的所有革兰氏阴性亲缘菌都经历了外膜的囊泡形成或夹断过程。囊泡已被证明能够与细菌和真核细胞膜融合，在此过程中递送可溶性和膜组分。虽然它们的存在已经被认识了几十年，但细菌外膜囊泡尚未在基本的遗传和生物化学水平上进行研究。在前期工作中，已经发展了从大肠杆菌分离和纯化囊泡的方法。杆菌第一个目标是阐明产生囊泡的细胞机制的遗传方法。随机生成的E.将筛选大肠杆菌突变体的囊泡产生缺陷。第二个目标是囊泡如何在正常生理条件下和应激生长条件下有益于“母细胞”。囊泡在细胞之间交流的能力可能有助于竞争性生长环境和传播遗传信息。利用生化分析，我们将研究囊泡介导的蛋白质，脂质和核酸的传输。人工脂质体已被证明在体外与细菌膜融合，这些实验为开始研究可能对融合重要的囊泡膜特征提供了基础。在应激生长条件下，囊泡途径可以被增选以允许外膜的快速重塑。将分析小泡对冷休克后脂多糖组成戏剧性转变的贡献。此外，囊泡形成突变体将用于研究细菌对不同环境胁迫的反应过程中囊泡的功能。该提案的目标是明确的，并不相互依赖，但彼此建立在解决项目假设。这种遗传学和生物化学的强大结合将为细菌生理学这一基本领域的未来发现奠定基础。预计这些研究将揭示膜动力学的一般原理，同时也突出了细菌膜特有的新概念。

DESCRIPTION (provided by applicant): Escherichia coli, as well as all of its gram-negative relatives studied to date, undergo a process of vesiculation, or pinching-off, of the outer membrane. Vesicles have been shown to be capable of fusion with both bacterial and eukaryotic membranes, delivering soluble and membrane components during this process. Although their presence has been recognized for decades, bacterial outer membrane vesicles have yet to be investigated at a basic genetic and biochemical level. In preliminary work, methods have been developed to isolate and purify vesicles from E. coli. The first objective is a genetic approach to elucidate the cellular machinery that produces vesicles. Randomly generated E. coli mutants will be screened for defects in vesicle production. The second objective is to how vesicles benefit the "mother cell" under normal physiological conditions and under stressful growth conditions. The ability of vesicles to communicate between cells may help in a competitive growth environment and to disseminate genetic information. Using biochemical assays, we will investigate vesicle-mediated transmission of proteins, lipids and nucleic acids. Artificial liposomes have been shown to fuse with bacterial membranes in vitro and these experiments provide a basis to begin studying vesicle membrane characteristics that may be important for fusion. Under stressful growth conditions, the vesicle pathway may be co-opted to allow quick remodeling of the outer membrane. The contribution of vesicles to the dramatic switch in lipopolysaccharide composition after cold shock will be analyzed. Further, vesiculation mutants will be used to investigate the function of vesicles during a bacterial response to diverse environmental stresses. The aims of the proposal are distinct and do not depend on one another, yet build on each other to address the project hypotheses. This powerful combination of genetics and biochemistry will set a foundation for future discoveries in this basic area of bacterial physiology. It is anticipated that these studies will reveal general principles of membrane dynamics, while also highlighting new concepts unique to bacterial membranes.