Production and function of E coli vesicles

大肠杆菌囊泡的产生和功能

• 批准号: 6532066
• 负责人: META J KUEHN
• 金额: $ 11.55万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6532066
• 关键词: Escherichia coli; bacterial DNA; bacterial genetics; biological transport; cold temperature; environmental adaptation; gene mutation; lipid transport; membrane fusion; microorganism culture; protein transport; vesicle /vacuole

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.

描述（由申请人提供）：大肠杆菌以及迄今为止研究的所有革兰氏阴性近缘细菌都会经历外膜形成囊泡或夹断的过程。囊泡已被证明能够与细菌和真核细胞膜融合，在此过程中传递可溶性成分和膜成分。尽管细菌外膜囊泡的存在几十年来已被认识，但尚未在基本的遗传和生化水平上进行研究。在前期工作中，已经开发出从大肠杆菌中分离和纯化囊泡的方法。第一个目标是通过遗传方法来阐明产生囊泡的细胞机制。随机产生的大肠杆菌突变体将被筛选出囊泡产生的缺陷。第二个目标是在正常生理条件和应激生长条件下囊泡如何使“母细胞”受益。囊泡在细胞之间通讯的能力可能有助于竞争性生长环境并传播遗传信息。使用生化测定，我们将研究囊泡介导的蛋白质、脂质和核酸的传递。人工脂质体已被证明可以在体外与细菌膜融合，这些实验为开始研究对融合可能很重要的囊泡膜特性提供了基础。在应激生长条件下，可以选择囊泡途径以允许外膜快速重塑。将分析囊泡对冷休克后脂多糖组成急剧变化的贡献。此外，囊泡突变体将用于研究细菌对不同环境应激做出反应期间囊泡的功能。该提案的目标是明确的，互不依赖，但又相互基础以解决项目假设。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，同时也强调细菌膜独特的新概念。