Free flow electrophoresis as a tool for enrichment of mutants with temperature-dependent lethal mutations in lipid A synthesis.
Free flow electrophoresis was shown to be a useful tool to enrich for mutants 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 dOclA-8-P synthesis was isolated (mutant Ts5). Following a shift to nonpermissive conditions it accumulates a mixture of at least two 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 dOclA and the nonhydroxylated fatty acids lauric, myristic and 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.
D000446:Aldehyde-Lyases / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004586:Electrophoresis / Q000379:methods; D008050:Lipid A / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature
D008050:Lipid A; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases