Demonstration by membrane reconstitution of a butanol-soluble intermediate in the biosynthesis of the O9 antigen of Escherichia coli.

The activity in vitro of the mannan-synthesizing system of Escherichia coli O9 depends on the presence of glucose in the growth medium of the bacteria. Inactive membranes of E. coli strain F988 grown without gain mannan-synthesizing activity by reconstitution with a butanol extract obtained from the same bacteria grown with glucose. Inactive membranes could also be restored to biosynthetic activity by incubation with UDP-glucose in the presence of magnesium chloride. In this magnesium-ion-dependent reaction, a glucolipid was formed which was extractable with butanol. It could be used for the reconstitution of inactive membranes. The products of incubations with GDP-mannose of reconstituted and active membranes were analysed for electrophoretic mobility in sodium dodecylsulfate/polyacrylamide gel electrophoresis, molecular weight and composition. In all cases they proved to be the mannan attached to a hydrophobic mannose carrier, presumably a glucolipid. These results suggest that a glucolipid is the intermediary mannose acceptor in the biosynthesis of the O9 antigen.