Free flow electrophoresis as a tool for enrichment of mutants with temperature‐dependent lethal mutations in lipid A synthesis

Abstract

Free flow electrophoresis was shown to be a useful tool to enrich for mutants of Salmonella typhimurium conditionally defective in lipid A synthesis. The method was based on the observation that electrophoretic mobility of bacterial cells is dependent on the structure of lipopolysaccharides and is influenced by lesions in the synthesis of the O-specific chains as well as by lesion in the synthesis of the complete 3-deoxy-D-manno-octulosonic acid (dOclA) lipid A region. Using this procedure a new mutant conditionally, defective in dOcIA-8-P synthesis was isolated (mutant Ts5). Following a shift to nonpermissive conditions it accumulates a mixture of at least 2 equally represented lipid A precursor structures. One is made up of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio 1.0:1.0:2.0 and lacks dOcIA and the nonhydroxylated fatty acids lauric, myristic and palmitic acid (precursor Ia). The other one (precursor Ib) has the same basic composition with additional palmitic acid. The precursor preparation derived from mutant Ts5 thus differs from previously described lipid A intermediates by the relatively high substitution by palmitic acid. The implications of the above findings to the biosynthesis of lipid A are discussed.