Biogenesis and maintenance of the outer membrane of Gram-negative bacteria

项目总结 革兰氏阴性菌的细胞膜由内(IM)和外(OM)两层膜和一个 房水隔室称为位于它们之间的周质。我的实验室的长期目标一直是 以大肠埃希菌为模型系统，了解包膜生物发生的机制。这 该建议涉及OM的生物发生和维持细胞膜生理的应激反应。所有的 有机质、磷脂、脂多糖、脂蛋白和β桶蛋白的组成 (OMP)，在IM的细胞质或内部小叶中合成。我们已经确定了必需的蛋白质 需要在OM中组装内毒素(LptABCDEFG)和OMP(BamABCDE)，我们已经提供了证据 在IM和OM之间的磷脂运输的扩散机制。在本供资期间， 我们已经证明，Bamb BAME双突变体的条件致死表型可以被简单地抑制 通过删除表面暴露的脂蛋白，我们揭示了替代脂蛋白运输的存在 途径，我们揭示了肠道细菌共同抗原的环状形式在维持OM中的作用 屏障，我们发现了激活或启动σE应激反应的突变，这些突变抑制了各种omp 和巴姆缺陷。在翻译研究中，我们利用我们对OM生物发生的知识发现了一类新的 在细胞表面抑制BAMA的抗生素。 我们建议使用我们的大量突变来改变BAM成分或各种OMP底物 再加上我们收集的抑制器，作为探测OMP组装过程的工具。特别是，我们将 探索非必需的BamBCE脂蛋白的功能，并检验我们的假设，即BAMD不 发挥真正必要的机械作用，而不是作为一个调节器来控制巴马的活动。我们 将测试伴侣蛋白Skp作为周质蛋白酶DegP的特异性接头的作用。我们还假设 主要OMP的三聚体性质通过提供以下功能作为OM架构的全球组织者 多个相互作用的面，以允许形成蛋白质岛所需的蛋白质-蛋白质相互作用。 我们对内毒素组装的研究将利用突变的O-抗原连接酶和酶分类酶来粘附肽 或蛋白质到脂多糖，以挑战LptDE转位蛋白的能力。我们还将测试我们的模型 必需的IM蛋白、YejM、YciM和FtsH组成了一条新的途径，调节内毒素的合成 与OM的脂质状态有关。 MlaA*突变通过增加内毒素水平来破坏OM的稳定。这会导致OM造成膜损失 囊泡和IM请流入OM以弥补损失。我们已经确定了一种使这种脂质减慢的突变 我们相信，对这一基因的持续研究可能会为了解这一鲜为人知的过程提供洞察。 顺行的物流运输。

PROJECT SUMMARY The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and β-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins required to assemble LPS (LptABCDEFG) and OMPs (BamABCDE) in the OM and we have provided evidence of a diffusive mechanism of phospholipid transport between the IM and the OM. In the current funding period, we have shown that the conditional lethal phenotype of bamB bamE double mutants can be suppressed simply by deleting a surface-exposed lipoprotein, we revealed the existence of an alternate lipoprotein trafficking pathway, we uncovered a role for the cyclic form of Enterobacterial Common Antigen in maintaining the OM barrier, and we identified mutations that activate or prime the σE stress response that suppress a variety of OMP and Bam defects. In translational studies, we used our knowledge of OM biogenesis to discover a new class of antibiotics that work to inhibit BamA at the cell surface. We propose to use our large collection of mutations that alter the Bam components or various OMP substrates together with our collection of suppressors as tools to probe the OMP assembly process. In particular, we will probe the function of the non-essential BamBCE lipoproteins and test our hypothesis that BamD does not perform a truly essential mechanistic role, but rather functions as a regulator to control the activity of BamA. We will test the role of the chaperone Skp as a specific adaptor for the periplasmic protease DegP. We also posit that the trimeric nature of the major OMPs functions as a global organizer of OM architecture by providing multiple interacting faces to allow the protein-protein interactions necessary for the formation of protein islands. Our studies on LPS assembly will utilize a mutant O-antigen ligase and the enzyme sortase to attach peptides or proteins to LPS to challenge the capabilities of the LptDE translocon. We will also test our model that three essential IM proteins, YejM, YciM, and FtsH comprise a novel pathway that regulates LPS synthesis in response to the lipid status of the OM. The mlaA* mutation destabilizes the OM by increasing LPS levels. This causes membrane loss by OM vesiculation and IM PLs flow into the OM to replace the loss. We have identified a mutation that slows this lipid flow and we believe that continued study of this gene may provide insights into the poorly understood process of anterograde PL transport.