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

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.

A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide

“…The mutants carried mainly a Tn insertion or antibiotic resistance cassettes upstream or inside the pssA coding sequence [ 19 , 27 , 28 , 31 ], and the phenotypes of these mutants were described. However, for the purpose of this study, we constructed a markerless pssA deletion mutant using the pCM351 allelic exchange vector [ 32 ] according to the previously implemented method [ 33 , 34 ]. The constructed deletion comprised the entire coding sequence of pssA ( Supplementary Figure S1 ).…”

“…Given the importance of PssA as a priming phosphoglycosyl transferase, we screened for possible PssA interactions with other glycosyltransferases and components of the polymerization and transport system of EPS using the bacterial two-hybrid system (BTH). Genes encoding glycosyltransferases and PssP, PssT, PssL, and PssP2 proteins were previously cloned into BTH vectors [ 33 , 40 , 41 ]. The co-transformations of the reporter strain E. coli DHM1, screening on the indicative media, and measurement of β-galactosidase activity revealed an interaction of PssA with one GT involved in the octasaccharide backbone synthesis (PssC) and three GTs probably involved in the side chain synthesis (PssF, PssI, PssJ) ( Figure 5 ).…”

Rhizobial Exopolysaccharides: Genetic Regulation of Their Synthesis and Relevance in Symbiosis with Legumes

“…Based on the CAZy database (Carbohydrate Active Enzymes), some pss genes located in the Pss-I region are predicted to be engaged in this process (pssF, pssG, pssH, pssI) [63,64]. As recently evidenced, pssJ codes for a galactosyl transferase that is involved in the addition of the terminal sugar residue to the octasaccharide subunits [65]. A ∆pssJ deletion mutant produces EPS lacking terminal galactose in the side chain of the subunit (Figure 1).…”

Rhizobial Exopolysaccharides and Type VI Secretion Systems: A Promising Way to Improve Nitrogen Acquisition by Legumes

“…EPS contain mostly D-glucose and D-galactose (Castellane et al, 2014(Castellane et al, , 2017(Castellane et al, , 2018(Castellane et al, , 2019, uronic acids and non-sugar substitutions can also be very important (López-Baena et al, 2016). Enzymes such as glycosyltransferase (PssA) and galactosyltransferase (PssJ) are responsible for assembling EPS sub-units in R. leguminosarum (Marczak et al, 2020).…”