REGULATION OF THE MAJOR OUTER MEMBRANE PORIN PROTEINS

肠道细菌产生一组蛋白质，称为孔蛋白， 被动扩散孔，以允许小的亲水性分子穿过 外膜通透性屏障。 大肠杆菌K12含有两个 主要的非特异性孔蛋白，OmpF和OmpC。 总的来说， 这两个孔蛋白存在于细胞中是恒定的，然而， 浓度随各种环境条件而波动。 一 环境因素似乎是特别重要的是媒体 渗透压 如果摩尔渗透压浓度高，则OmpC占优势。 反之，在 通过对渗透压的敏感，推测E. 大肠杆菌可以区分其共同的栖息地，即宿主内部或外部 动物 我们希望了解这种调控的分子基础 系统 我们实验室和其他人以前的工作已经确定了两个 调控基因ompR和envZ，其包含操纵子ompB。 这两 基因指定激活孔蛋白基因所需的蛋白质 表情 几种ompR-lacZ融合体，其指定了具有以下的杂合蛋白： 已分离出部分OmpR活性。 选择利用这些 双功能融合已经设计，并将用于详细的 OmpR结构/功能分析 类似的策略将用于 EnvZ 研究蛋白质-蛋白质相互作用并分析顺式作用 调节蛋白的靶位点，第二位点抑制突变 将通过体外试验和DNA测序进行分离和表征 分析. 最后，将研究非线性调节的一般性 通过分离和表征大量具有代表性的lacZ 融合到受调控的基因。

Enteric bacteria produce a set of proteins, termed porins, that act as passive diffusion pores to allow small hydrophilic molecules to cross the outer membrane permeability barrier. Escherichia coli K12 contains two major, non-specific porins, OmpF and OmpC. In general, the total amount of these two porins present in the cell is constant, however, the relative levels fluctuate in response to a variety of environmental conditions. One environmental factor that appears to be particularly important is media osmolarity. If osmolarity is high, then OmpC predominates. Conversely, in dilute media, OmpF is found. It is thought that by sensing osmolarity, E. coli can distinguish its common habitats, i.e. inside or out of a host animal. We wish to understand the molecular basis for this regulatory system. Previous work from our lab and others have identified two regulatory genes, ompR and envZ, which comprise an operon, ompB. These two genes specify proteins that are required to activate porin gene expression. Several ompR-lacZ fusions that specify hybrid proteins with partial OmpR activities have been isolated. Selections that exploit these bifunctional fusions have been designed and will be employed for a detailed structure/function analysis of OmpR. A similar strategy will be used with EnvZ. To study protein-protein interactions and to analyze the cis-acting target sites of the regulatory proteins, second-site suppressor mutations will be isolated and characterized by in vitro assay and DNA sequence analysis. Finally, the generality of osmoregulation will be investigated by isolating and characterizing a large and representative series of lacZ fusions to osmoregulated genes.