The Biosynthesis of Cell Wall Lipopolysaccharide in Escherichia coli: VII. CHARACTERIZATION OF HETEROGENEOUS “CORE” OLIGOSACCHARIDE STRUCTURES

Abstract

The core portion of the cell wall lipopolysaccharide of Escherichia coli J-5, an UDP-Gal epimerase-less mutant of E. coli 0111:B4, was subjected to detailed structural analysis. Lipopolysaccharide was isolated from cell envelope preparations by extraction with hot aqueous phenol followed by ultracentrifugation of the aqueous phase. Mild acid (pH 3.5) hydrolysis of lipopolysaccharide yielded a mixture of phosphorylated core oligosaccharides which were dephosphorylated by treatment with hydrofluoric acid at 0°. The mixture of dephosphorylated core oligosaccharides was resolved by paper chromatography, paper electrophoresis, and ion exchange chromatography into three components, designated A, B and C. Each of the oligosaccharides was shown to possess a single titratable carboxyl group and a single terminal reducing 3-keto-3-deoxyoctulosonate (KDO) residue. Analyses indicated the following molar ratios of constituents: A, glucose, heptose, glucosamine, and KDO (2:2:1:1); B, glucose, heptose, and KDO (1:3:1); and C, glucose, heptose, and KDO (1:2:1). The presence of unsubstituted glucosamine in A was confirmed by formation and subsequent isolation of dinitrophenylated glucosamine after treatment of the oligosaccharide with dinitrofluorobenzene and by demonstration of a titratable, free amino group in the polymer. The hexosamine isolated from A by hydrolysis was shown to be glucosamine by degradation to arabinose with ninhydrin. Linkage of the glucosamine moiety to heptose was established by isolation and characterization of glucosaminyl-heptose by controlled hydrolysis of A. Oligosaccharides A and C were characterized structurally by Smith degradation and specific enzymatic and chemical techniques as follows: [see PDF for equation] Because of uncertainty regarding the homogeneity of oligosaccharide B, its detailed structure was not determined unequivocally although it is tentatively proposed to be identical to oligosaccharide C except for a third heptose moiety substituting the heptose linked to KDO at position 6 or 7. Using cell envelope particulate fractions as a source of galactosyl transferase, it was demonstrated that [14C]galactose is incorporated into core oligosaccharides A and C with identical efficiency whereas no detectable quantity of galactose was incorporated into the B structure. The galactosyltransferase system exclusively transfers galactose residues from UDP-galactose to terminal nonreducing glucose residues of oligosaccharides A and C.