Glucosyldiphosphoundecaprenol, the mannose acceptor in the synthesis of the O9 antigen of Escherichia coli. Biosynthesis and characterization

We describe the biosynthesis in vitro of the mannose acceptor of the O9 mannan synthesis by Escherichia coli membranes and its analysis with chemical, enzymatic and physical means. Membranes from E. coli 1357 (O9:K29-:H-his,pmi,rfe) were incubated with 10 mM UDP-glucose and 20 mM magnesium chloride in large scale. The incubation mixtures were extracted with butan-1-ol and the extract was fractionated by ion-exchange chromatography on DEAE-cellulose. The presence of the mannose acceptor was detected in the column effluent by using aliquots of the fractions in membrane-reconstitution experiments. The purified mannose acceptor was hydrolyzed for 10 min in 0.1 M hydrochloric acid at 100 degrees C and the hydrolyzate was extracted with light petroleum. Mass spectrometric analysis of the material from the organic phase showed it to be undecaprenol. The aqueous phase contained phosphate and glucose (as determined with glucose oxidase peroxidase) in the ratio of 1.9, alpha-Galactosyldiphosphoundecaprenol and beta-glucosylphosphoundecaprenol were prepared for comparison in these experiments. The results obtained showed that the mannose acceptor in the synthesis of the O9 mannan of E. coli is alpha-glucosyldiphosphoundecaprenol.