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.

The in vitro biosynthesis of functional nodulation factors (Nod Rm) produced by Rhizobium meliloti 1021.

Rhizobium meliloti associates symbiotically with alfalfa by forming root nodules in which the bacteria reduce atmospheric N2 into products useful to both organisms. Nod factors are signal molecules, lipooligosaccharides, produced by the bacteria that trigger nodule formation in the plant host. Nod Rm-1 consists of a beta-1,4-N-acetyl glucosamine tetrasaccharide from which the N-acetyl group at the non reducing end is replaced by a fatty acid and the N-acetyl glucosamine at the reducing end is sulfated at position 6. By in vitro incubation of electroporated cells in the presence of [35S]PAPS or UDP-[14C]GlcNAc a labelled compound has been obtained with the properties of the in vivo produced Nod Rm-1 factor, as judged by HPLC, TLC and HPTLC techniques. The [14C]GlcNAc labelled compound has also hair root deformation activity on alfalfa plantlets indicating that a functional Nod Rm-1 factor has been synthesized in vitro.

In vitro biosynthesis of acetan using electroporated Acetobacter xylinum cells as enzyme preparations.

Acetobacter xylinum strain NRRL B42 and its derivative RCGr1 produce a complex exopolysaccharide, acetan, containing glucose, mannose, glucuronic acid and rhamnose in a 4:1:1:1 molar ratio. The in vitro synthesis of acetan, employing electroporated cells as the enzyme system and the respective 14C-labelled sugar nucleotide precursors, is described. The synthesis of the prenyl-linked heptasaccharide repeat unit, already observed in EDTA-treated cells, was confirmed, as well as the formation of other saccharides not related to acetan biosynthesis, including a high molecular mass glucan. The acetan formed was characterized by gel filtration, specific radioactive labelling with each precursor and permethylation analysis. It was also shown that acetan contains acetyl residues and that using [14C]acetyl CoA as donor, radioactivity was detected both at the polysaccharide and at the prenyl-linked oligosaccharide stage.