Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes

Abstract

The structure of the O-antigen from the international reference strain Escherichia coli O93:−:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons and (iii) unassigned 2D 1H,13C-HSQC and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1 → 3)-β-d-Manp2Ac6Ac-(1 → 4)-β-d-GlcpA-(1 → 3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Glycan Shape, Motions, and Interactions Explored by NMR Spectroscopy

“…The ability to predict NMR chemical shifts of molecules by machine learning, − understand the biology of branched N -glycans, or unveil the origin of glycan-mediated host-microbe interactions by deep learning methods opens new avenues for exploring the importance of glycan structure in biology using NMR spectroscopy as the experimental technique. With the introduction of AlphaFold to predict three-dimensional structure of proteins, analysis of carbohydrate binding proteins, structures of glycosyl transferases, and possible functions of proteins as well as modules thereof has been completely changed. One can now with confidence first investigate whether a sequence of amino acids of a protein or a subset of these have a defined three-dimensional structure, and if this is the case, a construct can be made to check the prediction by NMR spectroscopy (Figure ), prior to trying to elucidate the function of the module or of the whole protein.…”