In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen.

The heteropolysaccharide chains of enterobacterial common antigen (ECA) are made up of linear trisaccharide repeat units with the structure----3)-alpha-D-Fuc4NAc-(1----4)- beta-D-ManNAcA-(1----4)-alpha-D-GlcNAc-(1----, where Fuc4NAc is 4-acetamido-4,6-dideoxy-D-galactose, ManNAcA is N-acetyl-D-mannosaminuronic acid, and GlcNAc is N-acetyl-D-glucosamine. The assembly of these chains involves lipid-linked intermediates, and both GlcNAc-pyrophosphorylundecaprenol (lipid I) and ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) are intermediates in ECA biosynthesis. In this study we demonstrated that lipid II serves as the acceptor of Fuc4NAc residues in the assembly of the trisaccharide repeat unit of ECA chains. Incubation of Escherichia coli membranes with UDP-GlcNAc, UDP-[14C]ManNAcA, and TDP-[3H]Fuc4NAc resulted in the synthesis of a radioactive glycolipid (lipid III) that contained both [14C]ManNAcA and [3H]Fuc4NAc. The oligosaccharide moiety of lipid III was identified as a trisaccharide by gel-permeation chromatography, and the in vitro synthesis of lipid III was dependent on prior synthesis of lipids I and II. Accordingly, the incorporation of [3H]Fuc4NAc into lipid III from the donor TDP-[3H]Fuc4NAc was dependent on the presence of both UDP-GlcNAc and UDP-ManNAcA in the reaction mixtures. In addition, the in vitro synthesis of lipid III was abolished by tunicamycin. Direct conversion of lipid II to lipid III was demonstrated in two-stage reactions in which membranes were initially incubated with UDP-GlcNAc and UDP-[14C]ManNAcA to allow the synthesis of radioactive lipid II. Subsequent addition of TDP-Fuc4Nac to the washed membranes resulted in almost complete conversion of radioactive lipid II to lipid III. The in vitro synthesis of lipid III was also accompanied by the apparent utilization of this lipid intermediate for the assembly of ECA heteropolysaccharide chains. Incubation of membranes with UDP-[3H]GlcNAc, UDP-ManNAcA, and TDP-Fuc4NAc resulted in the apparent incorporation of isotope into ECA polymers, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In addition, the in vitro incorporation of [3H]Fuc4NAc into ECA heteropolysaccharide chains was demonstrated with ether-treated cells that were prepared from delta rfbA mutants of Salmonella typhimurium. These mutants are defective in the synthesis of TDP-Fuc4NAc; as a consequence, they are also defective in the synthesis of lipid III and they accumulate lipid II. Accordingly, incubation of ether-permeabilized cells of delta rfbA mutants with TDP-[3h]Fuc4NAc resulted in the incorporation of isotope into both lipid III and ECA heteropolysaccharide chains.