A Novel Periplasmic Glycosyl Hydrolase PssW Involved in Rhizobium leguminosarum Acidic Exopolysaccharide Depolymerization

The paper presents a functional characterization of the rhizobial periplasmic glycosyl hydrolase encoded by the pssW gene, which is localized in a conserved chromosomal cluster of acidic exopolysaccharide biosynthesis. We showed that PssW is synthesized as a 39.9 kDa precursor, followed by its translocation across the cytoplasmic membrane and cleavage of a 24-amino acid signal peptide. The periplasmic localization of PssW suggests that it may be active toward nascent exopolysaccharide prior to its secretion outside the cell. Knock-out of the pssW gene resulted in an approximately 6-fold increase in the high-molecular-weight to low-molecular-weight exopolysaccharide ratio thereby increasing the viscosity of the culture supernatant. Complementation of the pssW mutation restored the wild-type phenotype and even increased the level of secreted low-molecular-weight polysaccharide. PssW purified from the periplasmic space did not degrade carboxymethyl cellulose and succinoglycan and showed a two-fold decrease in the hydrolysis of partially pyruvated EPS compared to EPS isolated from the wild-type strain. Mass spectrometric analysis of oligosaccharide fractions obtained after exopolysaccharide hydrolysis showed that cleavage of the polysaccharide by PssW leads to the formation of mono-, di-, and trimers of octasaccharide repeat subunits which contain the different number of acetyl and hydroxybutanoyl substituents. PssW releases exopolysaccharide repeating units by hydrolyzing the β-1,4 linkage between GlcA(c)-OAc and Glc(b) residues and, to a lesser extent, the β-1,4 linkage between GlcA(с)-OAc and GlcA(d) of the main chain. Knock-out of pssW does not significantly affect the formation of pea nodules.