Biosynthesis of the O9 Antigen of Escherichia coli. Initial Reaction and Overall Mechanism

Abstract

The mannan-synthesizing system of Escherichia coli O9 was studied with membranes of the rfe− mutant 1357, which also lacks phosphomannose isomerase. These membranes did not incorporate mannose from GDP-mannose. Their reconstitution with UDP-glucose/magnesium chloride was studied under different conditions. Simultaneous incubation of rfe−membranes with 10 mM UDP-glucose, 20 mM magnesium chloride and 5 μM GDP-[14C]mannose resulted in the formation of a glycolipid, the carbohydrate moiety of which was characterized as mannosyl-(1→3)-mannosyl-glucose. This glycolipid was not a mannose acceptor when used in membrane reconstitution experiments. Sequential incubation of rfe− membranes first with 10 mM UDP-glucose/20 mM magnesium chloride and then with GDP-[14C]mannose gave rise to the radioactive mannan of E. coli O9. After the incubation of rfe− membranes with 10 mM UDP-glucose/20 mM magnesium chloride in the absence of GDP-mannose, a glucolipid was extracted with butanol and purified by ion-exchange chromatography on DEAE-cellulose. In reconstitution experiments this glucose-containing lipid functions as a mannose acceptor. It is characterized in the accompanying paper [Weisgerber, C. and Jann, K. (1982) Eur. J. Biochem. 127, 165–168] as α-glucosyldiphosphoundecaprenol. The formation of this glucolipid could be reversed with [14C]UMP using rfe−membranes as enzyme source. In the reverse reaction [14C]UDP-glucose was formed in about 20% yield. When rfe− membranes were incubated with 2 μM UDP-[14C]glucose and 20 mM magnesium chloride, a lipid containing [14C]glucose was formed, although no stimulation of mannose incorporation could be observed at this UDP-glucose concentration. The glucose is bound to a lipid moiety (presumably undecaprenol) via a phosphodiester bridge. On the basis of these results the mechanism of the biosynthesis of the E. coli O9 antigen is discussed. We propose that in the initial reversible reaction α-glucosyldiphosphoundecaprenol is formed by the transfer of α-glucosyl 1-phosphate from UDP-glucose to undecaprenol phosphate. The product functions as mannose acceptor in the subsequent sequential transfer of mannose from GDP-mannose directly. The mannan, together with the glucose unit, is finally translocated from undecaprenol diphosphate to core lipid A, with the formation of the complete lipopolysaccharide (O9 antigen).