Switching cell wall-bound polysaccharides to secreted polysaccharides in lactobacilli

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Bacterial exopolysaccharides (EPS) are valuable carbohydrate polymers with applications in food and health.
• Lactic acid bacteria produce EPS with beneficial properties.
• Understanding EPS production mechanisms is crucial for harnessing their potential.

Purpose of the Study:

• To identify genetic mutations leading to EPS overproduction in lactobacilli.
• To elucidate the molecular mechanisms underlying the switch from cell wall-bound polysaccharides to secreted EPS.
• To characterize the structure of overproduced EPS and its relationship to cell wall-associated polysaccharides.

Main Methods:

• Isolation of spontaneous lactobacilli mutants with high EPS production using a sedimentation rate selection method.
• Genetic analysis to identify mutations in genes within the Wzy-dependent polysaccharide biosynthesis cluster, specifically epsC and epsD.
• Detailed characterization of Lacticaseibacillus rhamnosus mutants, including analysis of the EpsD tyrosine kinase activity and EPS structure determination.

Main Results:

• Spontaneous mutants overproducing EPS were obtained, frequently exhibiting missense mutations in epsD (encoding tyrosine kinase) or insertions in epsC (encoding its modulator).
• A specific D94L mutation in the EpsD catalytic site was found to inhibit its autophosphorylation.
• The overproduced EPS consists of heptasaccharide repeating units with pyruvate substituents, structurally identical to the cell wall-bound polysaccharide found in wild-type strains.

Conclusions:

• A defect in the autophosphorylation of the EpsD tyrosine kinase is associated with the switch from cell wall-bound polysaccharides to the release of EPS into the environment.
• Mutations in epsD or epsC can lead to significant alterations in polysaccharide localization and production.
• This study provides insights into the regulation of EPS biosynthesis and secretion in Lacticaseibacillus rhamnosus.