Open AccessBiosynthesis of the 09 Antigen of Escherichia coli
Author(s) -
FLEMMING HansCurt,
JANN Klaus
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12066.x
Subject(s) - mannan , mannose , chemistry , biochemistry , chromatography , sodium borohydride , biosynthesis , hydrolysis , polysaccharide , incubation , acid hydrolysis , enzyme , catalysis
The 09‐specific mannan of Escherichia coli was synthesized in vitro from GDP‐[ 14 C]mannose by membranes which were obtained from a phosphomannose isomerase‐less mutant of E. coli 09:K29 − :H − . Subsequent treatment of the membranes with dilute acid liberated a neutral product. whereas with aqueous phenol a charged product was obtained. Chromatography on DEAE‐cellulose. incubation with alkaline phosphatase and microdetermination showed that the charged mannan was substituted with one phosphate per chain. The neutral 14 C‐labelled product of the incubation in vitro was reduced with sodium boro[ 3 H]hydride. After total acid hydrolysis, the radioactive material was chromatographed on paper in the presence of borate. It was found that [ 3 H]glucitol. but no [ 3 H, 14 C]mannitol was present. When the neutral product, which was obtained after incubation of 14 C‐prelabelled membranes with nonradioactive GDP‐mannose, was hydrolyzed with and without prior reduction with non‐radioactive sodium borohydride, in subsequent paper chromatography, [ 14 C]glucitol or [ 14 C]glucose was found. The glucose was also converted enzymatically to gluconic acid, which was identified by paper electrophoresis. These results show that in the neutral 09‐specific mannan glucose is at the reducing end and they indicate that the mannan chain grows at the non‐reducing end. This is discussed with respect to the overall mechanism of the biosynthesis of the 09 antigen.