GENETIC ANALYSIS OF THE RFA LOCUS IN ESCHERICHIA COLI

大肠杆菌的RFA基因座的结构将是 调查过了。该基因座是由10个或更多基因组成的簇 实现内毒素核心区的生物合成 (LP)。编码核心早期步骤的一些基因的突变 生物合成有一种表型，称为“深粗糙”，在这种表型中，细胞 外膜蛋白减少，并成为 对洗涤剂和疏水抗生素过敏。我们 希望对这些基因及其产物的分析可能表明 稳定细胞的结构或生物合成途径 肠道细菌的外膜。我们已经隔离了 映射了一组定义操纵子的Tn10插入突变 它对扩展核心所必需的函数进行编码 七糖区域。我们还证明了一个片段 从RFA基因座克隆的基因部分地补充了其中的一些 插入突变。通过一种联合分离的方法 额外的突变和互补测试 我们希望克隆的片段能够定义操纵子和功能 它包括。我们还将在这个范围内找到一个反终结点 操纵子及其启动子(S)的调控定位研究 这个操纵台。将使用化学分析来确定之前的 未知的不稳定的内毒素组分，其中一个似乎缺失 或更多突变体，这些突变体似乎会影响侧向内毒素 互动。在长期研究中，我们将使用类似的方法 为了扩大这项研究，编码生物合成的基因 核心的七糖区域。因为有证据表明基因敲除 这些基因的突变是致命的，我们将分离出 在符合以下条件的菌株中发生条件突变或插入突变 RFA区域的二倍体。这些技术将被用于 确定参与内毒素核心早期步骤的必要基因 生物合成。

The structure of the rfa locus of Escherichia coli will be investigated. This locus is a cluster of 10 or more genes which carry out the biosynthesis of the core region of lipopolysaccharide (LPS). Mutation of some of the genes encoding early steps in core biosynthesis have a phenotype called "deep rough" in which cells have a decreased amount of outer membrane protein and become hypersensitive to detergents and hydrophobic antibiotics. We hope that analysis of these genes and their products may indicate structures or biosynthetic pathways involved in stabilizing the outer membrane of enteric bacteria. We have isolated and mapped a set of Tn10 insertion mutations which define an operon which encodes functions necessay to extend the core beyond the heptose region. We have also demonstrated that a fragment cloned from the rfa locus partially complements some of these insertion mutations. By a combined approach of isolating additional mutations and complementation tests with various cloned fragments we hope to define the operon and the functions it includes. We will also locate an antitermination site within this operon and locate and study the regulation of the promoter(s) of this operon. Chemical analysis will be used to identify previously unknown labile LPS components which appear to be missing in one or more mutants and which appear to affect lateral LPS interactions. In long-range studies we will use a similar approach to extend this study the genes encoding biosynthesis of the heptose region of the core. Since there is evidence that knockout mutations in these genes are lethal, we will isolate either conditional mutants or insertion mutations in strains which are diploid for the rfa region. These techniques will be used to identify essential genes involved in the early steps of LPS core biosynthesis.