ASSIGNMENT OF BIOCHEMICAL FUNCTIONS TO GLYCOSYL TRANSFERASE GENES WHICH ARE ESSENTIAL FOR BIOSYNTHESIS OF EXOPOLYSACCHARIDES IN SPHINGOMONAS STRAIN S88 AND RHIZOBIUM-LEGUMINOSARUM

Glycosyl transferases which recognize identical substrates (nucleotide -sugars and lipid-linked carbohydrates) ran substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes origina te in different genera of bacteria, This substitution can be used to i dentify the substrate specificities of uncharacterized transferase gen es, The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rh izobium leguminosarum were identified as encoding glucuronosyl-(beta 1 -->4)-glucosyl transferases based ore reciprocal genetic complementati on of mutations in the spsK gene and lire pssDE genes by segments of c loned DNA and by the SpsK-dependent incorporation of radioactive gluco se (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells, Bg contrast, glycosyl transferases which Br im alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host, The negative effects also suggested specific substrate requirem ents: we propose that spsL, codes for a glucosyl-(beta 1-->4)-glucuron osyl transferase in Sphingomonas and that pssC codes for a glucuronosy l-(beta 1-->4)-glucuronosyl transferase in X. leguminosarum. Finally, the complementation results indicate the order of attachment of sphing an maim-chain sugars to the C-55-isoprenylphosphate carrier as -Glc-Gl cA-Glc-isoprenylpyrophosphate.