Identification and Characterization of the Two Glycosyltransferases Required for the Polymerization of the HS:1 Serotype Capsular Polysaccharide of Campylobacter jejuni G1.

Abstract

Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in poultry and is the leading cause of gastrointestinal infections in the United States. Similar to other Gram-negative bacteria, C. jejuni possesses an extracellular carbohydrate-based capsular polysaccharide (CPS) composed of repeating units of monosaccharides bound via glycosidic linkages. The gene cluster for serotype 1 (HS:1) of C. jejuni contains 13 different genes required for the production and presentation of the CPS. Each repeating unit within the HS:1 CPS structure contains a backbone of glycerol phosphate and d-galactose. Here, the enzyme HS1.11 was shown to catalyze the formation of CDP-(2R)-glycerol from MgCTP and l-glycerol-3-phosphate. HS1.09 was found to be a multidomain protein that catalyzes the polymerization of l-glycerol-3-phosphate and d-galactose using UDP-d-galactose and CDP-(2R)-glycerol as substrates. The domain of HS1.09 that extends from residues 286 to 703 was shown to catalyze the transfer of l-glycerol-P from CDP-glycerol to the hydroxyl group at C4 of the d-galactose moiety at the nonreducing end of the growing oligosaccharide. The transfer of d-galactose to the C2 hydroxyl group of the glycerol-phosphate moiety was shown to be catalyzed with retention of configuration by the domain of HS1.09 that extends from residues 704 to 1095. Primers as short as a single d-galactoside were accepted as initial substrates. Oligosaccharide products were isolated by ion exchange chromatography and identified by high-resolution ESI-mass spectrometry and NMR spectroscopy.