TARGETING AND ASSEMBLY OF E COLI OUTER MEMBRANE PROTEINS

细菌细胞表面与宿主相互作用形成致病性或致病性 共生联盟。这些交互作用直接受 革兰氏阴性菌(如大肠杆菌)的外膜 作为环境和内部之间的接口 手机。越来越清楚的是，外膜 在使细菌对宿主防御因子产生抵抗力方面极其重要 通过提供一种有效的屏障来阻止胆汁的洗涤作用 盐类、消化酶降解和疏水致死 抗生素，如新诺贝菌素和红霉素。小分子水溶性 营养物质和抗生素如青霉素和头孢菌素穿透 细胞通过由一组独特的 蛋白质，称为孔蛋白。OmpF是一种形式 非特定的毛孔，而羔羊形成的毛孔特别有利于 麦芽糊精的扩散(麦芽糊精最高可达7个葡萄糖单位)。这些 孔蛋白还为细菌病毒提供受体位置。波林 蛋白质与其他膜成分紧密结合，如 如脂多糖(LPS)和肽聚糖。 这项研究的长期目标是了解生物发生 外膜与分子间的相互作用 膜组件。这项提案的具体目的是研究如何 蛋白质(OmpF和Lamb)定位于外膜和什么 如果有的话，内毒素在这一过程中发挥作用。这些目标将是 通过利用遗传和生化的组合实现的 接近了。我们设计了一种基因选择方案来分离 温度敏感的折叠突变，有条件地影响 OmpF和Lamb的组装。集合的条件积累 中间体将使我们能够确定它们的结构和细胞 地点。该实验方法是一种新颖的研究方法。 膜蛋白，并将揭示关键残基参与 外膜蛋白的靶向和组装。这些研究将是 扩展到通过抑制器检查其他组件的作用 分析。

Bacterial cell surfaces interact with the host to form a pathogenic or symbiotic association. These interactions are directly influenced by the outer membrane of Gram negative bacteria (such as Escherichia coli) which serves as the interface between the environment and the interior of the cell. It is becoming increasingly clear that the outer membrane is extremely important in making bacteria resistant to host defence factors by providing an effective barrier against the detergent action of bile salts, degradation by digestive enzymes, and killing by hydrophobic antibiotics such as novobiocin and erythromycin. Small water-soluble nutrients and antibiotics such as penicillin and cephalothin penetrate the cell by diffusing through pores or channels formed by a unique set of proteins, called porins. OmpF is an example of a porin that forms nonspecific pores, whereas LamB forms pores that specifically facilitate diffusion of maltodextrins (maltooligomers up to 7 glucose units). These porin proteins also provide receptor sites for bacterial viruses. Porin proteins exist in tight association with other membrane components such as lipopolysaccharide (LPS) and peptidoglycan. The long term goal of this research is to understand the biogenesis of the outer membrane and molecular interactions between the various membrane components. The specific aim of this proposal is to examine how proteins (OmpF and LamB) are targeted to the outer membrane and what role, if any, LPS plays in this process. These objectives will be achieved by utilizing a combination of genetic and biochemical approaches. We have devised a genetic selection scheme to isolate temperature sensitive folding mutations that conditionally affect assembly of OmpF and LamB. The conditional accumulation of assembly intermediates will allow us to determine their structure and cellular location. The experimental approach described is novel for studying membrane proteins and will reveal the key residues involved in the targeting and assembly of outer membrane proteins. These studies will be extended to examine the role of other components through suppressor analysis.