Biosynthesis of the 09 Antigen of Escherichia coli

Abstract

The O9 antigen of Escherichia coli O9 consists of a mannan, bound to the core oligosaccharide and lipid A. The mannan contains α‐1,2‐ and α‐1,3‐linked mannose in an unknown sequence. The serological specificity of the O9‐antigen is not expressed through the mannan but through an immuno‐dominant group in which isopropylidene ribose seems to play a role.From a non‐capsulated (K−) form of E. coli O9:K29(A):H− a mutant lacking phosphomannose isomerase pmi−) was selected. Membrane preparations were obtained from this mutant using plasmolysis, spheroplasting and sonic rupture of the spheroplasts. Cytoplasmic and outer membranes were separated by sucrose gradient centrifugation.Incorporation in vivo of [14C]mannose into late log phase bacteria resulted in the formation of complete lipopolysaccharide (O9 antigen) which accumulated to a large extent on the cytoplasmic membrane before being exported to the outer membrane. The product was extracted with 45% aqueous phenol and characterized by chemical analysis, dodecylsulfate polyacrylamide gel electro‐ phoresis, gel permeation chromatography and antigen binding using anti‐O9 antiserum.Incorporation in vitro from GDP‐[14C]mannose into membrane mixtures or isolated cytoplasmic membrane resulted in the formation of the mannan part of the lipopolysaccharide which was not substituted by the determinant group and was not transferred to the core‐lipid A part. The product, isolated with 45% aqueous phenol, was characterized as was the product in vivo. Furthermore, it was subjected to methylation analysis with the same results as obtained with authentic O9 (lipo)poly‐saccharide. By monitoring the formation of radioactive polymer the over‐all reaction was found to have an apparent Km= 5 × 10−5 moles per 1, based on GDP‐mannose. It was found that the preferred activated form of mannose in the 09 antigen synthesis is GDP‐mannose, other purine or pyrimidine bases being much less effective; it was also found that the reaction is not dependent on bivalent cations such as Mg2+ or Mn2+, these ions even inhibiting the reaction partially at concentrations above 5 mM; it was further found that bacitracin as well as EDTA are not inhibitory. All attempts to extract an intermediate into organic solvents were unsuccessful. Oligosaccharides could not be isolated at any stage of the synthesis, starting 5 s after the addition of GDP‐[14C]mannose.In pulse‐chase experiments in vitro it was found that an intermediate was formed which can be detected by dodecylsulfate polyacrylamide gel electrophoresis. With increasing incubation time this material shows decreasing charge to mass ratios. Treatment of the incubation mixture with mild acid or mild alkali prior to dodecylsulfate polyacrylamide gel electrophoresis showed that the radio‐ active material (mannan) was bound to a carrier through an alkali‐stable but acid‐labile linkage. The nature of the carrier is discussed.From the results it is concluded that the O9 mannan is synthesized in a single chain mechanism which does not utilize lipid‐bound oligosaccharides.