Assembly of Outer Membrane Proteins in Escherichia coli

描述（由申请人提供）：外膜（OM）是革兰氏阴性菌的基本细胞器，作为选择性渗透屏障，保护细胞免受抗生素等有毒化合物的侵害。OM生物发生的关键步骤之一是跨越OM的整合β桶蛋白（OMP）的组装。最近在大肠杆菌中发现了OMP组装所需的机器。这种多亚基复合物被称为BAM复合物，含有一种必需的OMP BamA和四种相关的OM脂蛋白BamB、BamC、BamD和BamE。BamA和BamD都是生长所必需的;相比之下，bamB、bamC和bamE突变体都是可行的，尽管每个突变体在OMP组装中都有缺陷。现在已知BamCDE形成与BamA相互作用的稳定亚复合物，尽管该亚复合物的功能尚不清楚。本研究的目的是确定BamCDE亚复合物在大肠杆菌OMP组装过程中的作用。杆菌在具体目标1和2中，我提出了几种遗传方法来探测BamCDE亚复合体成员之间的相互作用和BamCDE和BamA之间的相互作用。我将产生一系列BamD突变体，以确定正常OMP组装所需的这种蛋白质的残基。在《特定目标3》中，我将描述我使用抑制基因分析产生的突变体。还将对每种突变体进行生物化学表征，以鉴定新型OMP组装中间体。 公共卫生相关性：蛋白质在外膜中的组装是一个从细菌到人类都保守的基本过程。了解这一过程在细菌中的作用可能会导致识别新的抗生素靶标。这些研究结果也可以阐明高等生物包括植物和人类的蛋白质运输和蛋白质组装途径的机制。

DESCRIPTION (provided by applicant): The outer membrane (OM) is an essential organelle of Gram-negative bacteria which acts as a selective permeability barrier to protect the cell from toxic compounds like antibiotics. One of the key steps in OM biogenesis is assembly of integral beta-barrel proteins (OMPs) that span the OM. The machinery required for OMP assembly has recently been discovered in Escherichia coli. Called the BAM complex, this multi- subunit complex contains one molecule each of an essential OMP BamA, and four associated OM lipoproteins BamB, BamC, BamD, and BamE. BamA and BamD are both essential for growth; in contrast, bamB, bamC, and bamE mutants are all viable, although each mutant has defects in OMP assembly. BamCDE is now known to form a stable subcomplex that interacts with BamA, although the function of this subcomplex is unclear. The goal of this proposal is to determine the role of the BamCDE subcomplex during OMP assembly in E. coli. In Specific Aims 1 and 2, I propose several genetic approaches to probe the interactions among the BamCDE subcomplex members and between BamCDE and BamA. I will generate a collection of BamD mutants to identify residues of this protein that are required for normal OMP assembly. In Specific Aim 3, I will characterize the mutants I generate using suppressor analysis. Each mutant will also be characterized biochemically to identify novel OMP assembly intermediates. PUBLIC HEALTH RELEVANCE: Assembly of proteins in the outer membrane is an essential process that is conserved from bacteria to man. Understanding how this process works in bacteria could lead to identification of novel antibiotic targets. Results from these studies could also illuminate the mechanisms of protein trafficking and protein assembly pathways in higher organisms including plants and humans.