D-mannosaminuronic acid constituent of the K-7 antigen of Escherichia coli

Abstract

The identification of d-mannosaminuronic acid (2-amino-2-deoxy-d-mannuronic acid) as one of the main constituents of an acidic polysaccharide material isolated from two Escherichia coli serotypes is described. This material carrying the specificity of the E. coli K7 antigen is isolated from serotypes O14:K7:H− and O7:K7:H4 by extraction of the bacterial dry mass–obtained by growth on solid medium–using the hot phenol-water procedure. Fractionation of the aqueous phase material, after removal of lipopolysaccharide by repeated ultracentrifugation, was achieved by a cetavlon precipitation technique. RNA, representing the bulk of the aqueous phase material, precipitates in the presence of cetavlon already at a NaCl molarity of 0.3 M whereas the K7 active material is only precipitable at a much lower NaCl molarity of 0.06. Examination of HCl-hydrolysates of this substance by high voltage electrophoresis in a non-complexing buffer of pH 5.3 revealed the presence of a positively charged component which was shown to be the lactone of an aminohexuronic acid. The amino sugars present in the K7 specific material were separated from uncharged hydrolysis products by passing the hydrolysate over a Dowex 50 column previously equilibrated with 0.1 N HCl. The amino sugar fraction obtained by elution with 0.5 N HCl consisted of three major components: (a) a hexosaminuronic acid, (b) the lactone of the hexosaminuronic acid and (c) d-glucosamine. (a) and (b) are interconvertible and give rise to the same product after N-acetylation. (a) Shows the characteristic ninhydrin staining of hexosaminuronic acids and does react in the Elson-Morgan reaction with a maximal absorption at 530–533 mμ, thus indicating a free 1-aldo-2-aminogroup and, in addition, an unsubstituted C-3 position. Chromatographic and electrophoretic data differed from those of two hexosaminuronic acid standards available (glucosaminuronic and galactosaminuronic acids) but were in agreement with data reported by Perkins [2] for mannosaminuronic acid isolated from a polysaccharide material of Micrococcus lysodeikticus. Since no reference material was available we tried to elucidate the structure of the hexosaminuronic acid by converting the previously esterified hexosaminuronic acid units of the polymer by NaBH4-reduction to the related hexos-amine units. Esterification was carried out with an aqueous solution of ethylene oxide at room temperature, and NaBH4-reduction in the presence of an excess of glycerol in order to maintain a pH of approximately 8. Examination of the reduced material revealed the presence of a newly formed hexosamine which subsequently was identified as d-mannosamine. The identification is based on the following results: a) identity of Rf- and MGlcn-values in several systems of the free and the N-acetylated amino sugar with authentic samples of d-mannosamine and N-acetyl-d-mannosamine; b) formation of a pentose with the Rf-value of arabinose after ninhydrin degradation; c) reactivity of the amino sugar with hexokinase and formation of a hexosamine-6-phosphate. This reaction in addition proves the d-configuration of the amino sugar. Quantitative analysis shows that d-mannosaminuronic acid forms at least 15–20% of the K7 specific material extracted from E. coli O14:K7, whereas d-glucosamine represents only a minor constituent (2–3%). Hitherto 2-amino-2-deoxy-mannuronic acid was only reported once [2] as a constituent of a cell wall polysaccharide from Micrococcus lysodeikticus. To our knowledge its occurence in the K7 specific material of E. coli is the first report from gramnegative bacteria.