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Abstract <p>The paper presents a functional characterization of the rhizobial periplasmic glycosyl hydrolase encoded by the <i>pssW</i> 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 <i>pssW</i> 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 <i>pssW</i> 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(<i>c</i>)-<i>O</i>Ac and Glc(<i>b</i>) residues and, to a lesser extent, the β-1,4 linkage between GlcA(<i>с</i>)-<i>O</i>Ac and GlcA(<i>d</i>) of the main chain. Knock-out of <i>pssW</i> does not significantly affect the formation of pea nodules.</p>

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

  • 期刊论文

  • 发表日期：

    2025年7月15日

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  • T. V. Ivashina,
  • A. S. Kanapina,
  • V. V. Marchenkov,
  • E. E. Fedorova,
  • A. K. Surin,
  • N. P. Ashina,
  • M. G. Shlyapnikov

摘要

Abstract

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.