Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium Leguminosarum bv. Trifolii.

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a ΔpssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.