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.
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