Elucidation of the O-antigen structure of Escherichia coli O63

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-β-d-Quip3N(d-allo-ThrAc)-(1→2)-β-d-Ribf-(1→4)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element β-d-Galp-(1→3)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.