Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in Sphingomonas Strain S88 and Rhizobium leguminosarum

Abstract

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. The spsK gene ofSphingomonas strain S88 and the pssDE genes ofRhizobium leguminosarum were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE 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 that spsL codes for a glucosyl-(β1→4)-glucuronosyl transferase inSphingomonas and that pssC codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in R. 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.

Biochemical characterization of the β-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis A.T.C.C. 31461

“…In Table 2 we show the similarity scores of GelK with other bacterial GTs belonging to GT family 1. The biochemical functions of these proteins have been established based on biochemical assays or genetic complementation studies [8,[31][32][33]. All these GTs have in common the use of the same nucleotide portion (UDP) and of a lipid-linked PP i as acceptor.…”

“…These clusters include genes essential for the assembly and secretion of exopolysaccharide and a four-gene operon needed for the synthesis of dTDP--rhamnose, but not the pgmG gene [6,7]. SpsK is possibly a β-1,4-glucuronosyltransferase, catalysing the addition of GlcA into the glucosyl-α-pyrophosphorylpolyprenol (PPL) intermediate ( Figure 1) [8].…”

The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases

“…trifolii strain 5599 [7]. The PssA protein was characterized as ¢rst glucosyl-IP-transferase in the assembly of the repeat unit of EPS of R. leguminosarum [11].…”

“…The pssDE genes encode glucuronosyl-(L1C4)-glucosyltransferase and catalyse the second step in the biosynthesis of repeat unit [7,11]. The pssC gene was identi¢ed as encoding a glucuronosyl-(L1C4)-glucuronosyl transferase in R. leguminosarum involved in the third step of repeat unit assembly [7,11]. The order of attachment of the main sugars to the C SS -IP carrier was proposed as -GlcA-GlcA-Glc-isoprenylpyrophosphate [11].…”

“…The pssC gene was identi¢ed as encoding a glucuronosyl-(L1C4)-glucuronosyl transferase in R. leguminosarum involved in the third step of repeat unit assembly [7,11]. The order of attachment of the main sugars to the C SS -IP carrier was proposed as -GlcA-GlcA-Glc-isoprenylpyrophosphate [11]. The next steps in biosynthesis of acidic EPS in R. leguminosarum remain to be established.…”