Mechanism of Assembly of the Outer Membrane of Salmonella typhimurium: SITE OF SYNTHESIS OF LIPOPOLYSACCHARIDE

Abstract

The site of synthesis of lipopolysaccharide has been investigated in cytoplasmic and outer membrane fractions isolated by isopycnic sucrose gradient centrifugation of the total membrane fraction from lysozyme-EDTA spheroplasts. Evidence that synthesis of O-antigen occurs exclusively in cytoplasmic membrane was obtained by pulse-chase experiments in vivo and by assay of biosynthetic enzymes in isolated membrane fractions. O-Antigen chains pulse-labeled in vivo with [14C]mannose appeared initially in cytoplasmic membrane, but were rapidly transferred to outer membrane during a subsequent chase with nonradioactive mannose. In accord, the specific activities of enzymes of O-antigen synthesis in isolated cytoplasmic membrane fractions were 15- to 30-fold greater than in outer membrane. The cytoplasmic membrane fractions were also enriched for glycosyltransferase activities involved in biosynthesis of the core region of lipopolysaccharide. However, unequivocal localization of core transferase enzymes in the isolated membranes was not possible since these activities were also found with soluble fraction, and secondary binding of soluble enzyme to both cytoplasmic and outer membrane was shown to occur during the isolation procedure. Evidence that the cytoplasmic membrane is the site of synthesis of the core region was derived from pulse-chase experiments in a mutant in which [14C]galactose is incorporated exclusively into the core portion of the polysaccharide. Over 90% of the [14C]galactose incorporated into lipopolysaccharide during a 1-min pulse was recovered in the cytoplasmic membrane fraction. The mechanism of translocation of lipopolysaccharide to the outer membrane is unknown. Evidence suggesting that the process is not readily reversible was obtained in experiments in mutants with conditional defects in lipopolysaccharide synthesis. Incomplete lipopolysaccharides incorporated into outer membrane during growth under nonpermissive conditions could not be completed following subsequent shift to permissive growth conditions.