Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof

The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: -> 3)[beta-l-Rhap-(1 -> 4)]-beta-d-GlcpNAc-(1 -> 4)-alpha-d-GalpA-(1 -> 3)-alpha-d-Manp-(1 -> 3)-beta-d-GlcpNAc-(1 ->. Cross-peaks of low intensity, corresponding to a beta-l-Rhap-(1 -> 4)-beta-d-GlcpNAc-(1 -> structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-d-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of the l-Rhap residue (similar to 0.70 per repeating unit) as well as at O-2 and O-3 of the d-GalpA residue (similar to 0.03 and similar to 0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286-290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.