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) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. ThespsK gene ofSphingomonas strain S88 and thepssDE genes ofRhizobium leguminosarum were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in thespsK gene and thepssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form 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 requirements: we propose thatspsL codes for a glucosyl-(β1→4)-glucuronosyl transferase inSphingomonas and thatpssC codes for a glucuronosyl-(β1→4)-glucuronosyl transferase inR. leguminosarum . Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55 -isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.