Biosynthesis of the 09 Antigen of Escherichia coli. Growth of the Polysaccharide Chain

The 09-specific mannan of Escherichia coli was synthesized in vitro from GDP-[14C]mannose by membranes which were obtained from a phosphomannose isomerase-less mutant of E. coli 09:K29−:H−. Subsequent treatment of the membranes with dilute acid liberated a neutral product. whereas with aqueous phenol a charged product was obtained. Chromatography on DEAE-cellulose. incubation with alkaline phosphatase and microdetermination showed that the charged mannan was substituted with one phosphate per chain. The neutral 14C-labelled product of the incubation in vitro was reduced with sodium boro[3H]hydride. After total acid hydrolysis, the radioactive material was chromatographed on paper in the presence of borate. It was found that [3H]glucitol. but no [3H, 14C]mannitol was present. When the neutral product, which was obtained after incubation of 14C-prelabelled membranes with nonradioactive GDP-mannose, was hydrolyzed with and without prior reduction with non-radioactive sodium borohydride, in subsequent paper chromatography, [14C]glucitol or [14C]glucose was found. The glucose was also converted enzymatically to gluconic acid, which was identified by paper electrophoresis. These results show that in the neutral 09-specific mannan glucose is at the reducing end and they indicate that the mannan chain grows at the non-reducing end. This is discussed with respect to the overall mechanism of the biosynthesis of the 09 antigen.