Isolation of mutants conditionally blocked in the biosynthesis of the 3-deoxy-D-manno-octulosonic-acid--lipid-A part of lipopolysaccharides derived from Salmonella typhimurium

Abstract

A procedure is described for the selection of conditional 3-deoxy-D-manno-octulosonic-acid--Lipid A mutants which depends on temperature sensitivity for both synthesis of complete lipopolysaccharide and for growth. Using this procedure new types of mutants were isolated which cease growth and accumulate lipid A precursors following a shift to nonpermissive temperatures. All precursor molecules differ in their charge as judged by DEAE-cellulose chromatography. While they all contain glucosamine, phosphate and 3-hydroxymyristic acid, they lack detectable 3-deoxy-D-manno-octulosonic acid (dOclA) as well as the nonhydroxylated fatty acids of the complete lipid A structure. Three mutants proved to be conditionally defective in dOclA metabolism, whereas one seems to be blocked at a relatively early step in lipid A synthesis. The phenotypes of all these mutants appear to be due to single mutations by reversion analysis and by characterization of the temperature-resistant revertants. Studies of these mutants may shed light on the essential role of the complete dOclA--lipid A part of lipopolysaccharides in membrane function.