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

Małgorzata Marczak,Kamila Bobrek,Agnieszka Łupicka-Słowik,Marcin Sieńczyk,Kamil Żebracki,Kamila Talarek,Andrzej Mazur,Piotr Koper,Anna Turska-Szewczuk,Magdalena Wójcik,Dominika Nowak,Leszek Wawiórka,Marceli Romańczuk

doi:10.3390/ijms21207764

Abstract

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.

Highlights

Exopolysaccharides (EPSs) are extracellular carbohydrate polymers produced, secreted, and accumulated outside the cells of various microorganisms
The basic octasaccharide subunit of acidic EPS produced by the clover symbiont Rhizobium leguminosarum bv. trifolii is composed of D-glucose, D-glucuronic acid, and D-galactose residues in a molar ratio 5:2:1 and modified with O-acetyl and pyruvyl groups [9,10,11,12]
Exopolysaccharide synthesis in rhizobia is governed by proteins classified as components of the Wzx/Wzy-dependent system

Summary

Introduction

Exopolysaccharides (EPSs) are extracellular carbohydrate polymers produced, secreted, and accumulated outside the cells of various microorganisms. GTs represent a diverse class of enzymes that catalyze the synthesis of glycosidic linkages by the transfer of a sugar residue from a donor substrate to an acceptor. They play essential roles in the biosynthesis of oligo- and polysaccharides, protein glycosylation, and formation of valuable natural products [16]. According to the Carbohydrate-Active enZYmes database (CAZy) [17], the currently recognized GTs are classified into 111 families. Despite the low identity of nucleotide and amino acid sequences, even if they synthesize the same glycan linkages, similarities between GTs can be identified at a structural level [19,21]. Inverting and retaining enzymes are present in both the GT-A and GT-B families, indicating that the different domain organization does not correlate with the catalytic mechanism [16]

Methods

Results

Conclusion