The Biosynthesis of Capsular Polysaccharide in Aerobacter aerogenes

Abstract

Abstract Aerobacter aerogenes produces a capsular polysaccharide composed of tetrasaccharide repeating units containing galactose, mannose, and glucuronic acid in a molar ratio of 2:1:1. Particulate cell envelope fractions of the organism catalyze the incorporation of galactose, mannose, and glucuronic acid from their respective nucleotide derivatives into capsular polysaccharide and into a lipid fraction. Kinetic studies of galactose-14C transfer from UDP-galactose-14C into the lipid fraction and into capsular polysaccharide suggested that a lipid-bound oligosaccharide intermediate is a precursor of the capsular polysaccharide. The biosynthesis of the intermediate oligosaccharide was shown to be initiated by the incorporation of galactose into an endogenous phospholipid. This reaction was characterized as a phosphogalactosyltransferase on the basis of its reversibility by UMP, but not UDP, and the alkali-catalyzed release of cyclic galactose 1,2-phosphate from the intermediate, indicating that galactose is bound to the lipid by a pyrophosphate linkage. The carrier phospholipid was isolated and purified by column chromatography on silicic acid and DEAE-cellulose, and finally, by preparative thin layer chromatography. The pure phospholipid, identified as undecaprenyl phosphate by mass spectrometry, restored the polysaccharide-synthesizing capacity of enzyme preparations which had been depleted of endogenous lipid by treatment with organic solvents. The particulate fraction catalyzes the sequential transfer of mannose, glucuronic acid, and a second galactose moiety to galactosyl-P-P-undecaprenol to form the di-, tri-, and tetrasaccharide derivatives, each of which was isolated and characterized, establishing the following sequence: UDP-Gal + P-lipid ⇌ Gal-P-P-lipid + UMP Gal-P-P-lipid + GDP-Man → Man-Gal-P-P-lipid + (GDP) Man-Gal-P-P-lipid + UDP-GlcUA → GlcUA-Man-Gal-P-P-lipid + (UDP) GlcUA-Man-Gal-P-P-lipid + UDP-Gal → Gal-(GlcUA)-Man-Gal-P-P-lipid + (UDP) The lipid-linked tetrasaccharide is polymerized to form a macromolecular product which is identical with native capsular polysaccharide, based on its composition and its susceptibility to degradation by a specific, phage-induced depolymerase.