Bacterial exopolysaccharides (EPS) are carbohydrate polymers secreted into the environment. EPS produced by lactic acid bacteria have many valuable properties in the food and health sectors. In this study, we isolated spontaneous mutants of lactobacilli that overproduce EPS, using a selection method based on their slow sedimentation rate in a semi-liquid medium. In the mutants selected from several strains, we detected a missense mutation in epsD , which encodes a tyrosine kinase, or an insertion in epsC , which encodes its transmembrane modulator. Both genes were located within a gene cluster involved in Wzy-dependent polysaccharide biosynthesis. We then characterized selected Lacticaseibacillus rhamnosus mutants in detail to gain insights into the mechanisms involved in EPS overproduction. We demonstrated that the single mutation D94L in the EpsD catalytic site prevents EpsD autophosphorylation. The chemical structure of the overproduced EPS was established, and consists of heptasaccharide repeating units with pyruvate substituents. In the wild-type parental strain, a polysaccharide with an identical structure was found covalently bound to the cell wall (CW) and covering the bacterial surface. In conclusion, our results indicate that the switch from CW-bound polysaccharides to EPS released into the environment is associated with a defect in autophosphorylation of the EpsD tyrosine kinase.
Kulakauskas et al. (2026) studied this question.