GENETIC ANALYSIS OF THE RFA LOCUS IN ESCHERICHIA COLI

大肠杆菌的rfa基因座的结构将是 研究了 该基因座是10个或更多个基因的簇， 进行脂多糖核心区的生物合成， （LPS）。 编码核心蛋白早期步骤的一些基因突变 生物合成具有称为“深粗糙”的表型，其中细胞 外膜蛋白的数量减少， 对洗涤剂和疏水性抗生素过敏。 我们 希望对这些基因及其产物分析可以表明， 结构或生物合成途径参与稳定 肠细菌的外膜。 我们已经分离出 绘制了一组Tn10插入突变， 其编码将核心扩展到 庚糖区。 我们还证明了一个碎片 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 通过隔离和隔离 其他突变和互补试验， 克隆片段，我们希望定义操纵子和功能 它包括。 我们还将在这个细胞内找到一个抗终止位点 操纵子，并定位和研究启动子的调控， 这个操纵子 化学分析将用于识别以前 未知的不稳定LPS成分似乎在一个 或更多的突变体，似乎影响侧向LPS 交互. 在长期研究中，我们将使用类似的方法 为了扩展这项研究，编码生物合成的基因 核心的庚糖区。 既然有证据表明 这些基因的突变是致命的，我们将分离出 条件突变体或插入突变的菌株， RFA区为二倍体。 这些技术将用于 确定参与LPS核心的早期步骤的必需基因 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.