The in vitro biosynthesis of the exopolysaccharide produced by Rhizobium leguminosarum bv. trifolii, strain NA 30.

Rhizobium leguminosarum bv. trifolii, strain NA 30 nodulates both red (Trifolium pratense) and white (T. repens) clover and produces an acidic exopolysaccharide (EPS) containing glucose, galactose, glucuronic acid, acetate and ketalpyruvate residues in a 5:1:2:1:2 molar ratio. The in vitro synthesis of this EPS as well as the characterization of five structurally related lipid linked oligosaccharides is described employing EDTA treated cells as enzyme preparation and 14C-labelled UDP-Glc, UDP-GlcA, Acetyl CoA and phosphoenol pyruvate (PEP) and doubly labelled 32P UDP-14C-Glc as precursors. The lipidic derivatives, extracted with chloroform, methanol, water (1:2:0,3) had the properties expected for prenyl-diphospho-sugars, as judged by the pattern of labelling, DEAE cellulose column chromatography, catalytic reduction and acid lability, etc. The sugar moieties of these phosphoprenyl derivatives were identified as the acetylated octasaccharide repeating unit, its mono- and di-ketalpyruvate derivatives and two trisaccharides, one of them acetylated, on the basis of specific labelling, gel filtration, paper electrophoresis and chromatography, TLC, permethylation, etc. In vitro polymer synthesis was greatly increased when electroporated cells were substituted for EDTA treated cells as enzyme system.