Construction and characterization of probiotic Lactobacillus rhamnosus GG deletion mutant for extracellular polysaccharide (EPS) biosynthesis regulator

Lactobacillus rhamnosus GG ATCC 53103 (LGG) is a probiotic microorganism producing a galactose-rich exopolysaccharide (EPS) surrounding the cell wall involved in adhesion and biofilm formation. The EPS gene cluster is composed by 16 genes associated to EPS biosynthesis. The wzb gene is an ortholog of the Streptococcus pneumoniae cpsB, encoding a protein phosphatase involved in the regulation of capsule production. In order to study the role of wzb gene in EPS biosynthesis, a L. rhamnosus GG wzb deletion mutant was constructed. Deletion of wzb produced a 12 minutes reduction of duplication time during growth. Deletion mutant strain produced smaller colonies than wild-type strain. Alcian Blue and India Ink staining denoted a strong reduction of the halo surrounding cells likely associated to EPS in Δwzb strain. Zeta potential analysis showed 2 bacterial populations in the Δwzb strain, while a single population was present in wild type indicating different Zeta potential values and consequently different electrokinetic properties of bacterial surfaces in the isogenic strains. Flow cytometry and dot blot analyses were used to assess the binding of Wheat Germ Agglutinin (WGA) and Chum Salmon Lectin 3 (CSL-3) lectins to the Nacetylglucosamine and rhamnose residues of EPS. In presence of WGA, 2 bacterial populations were observed in Δwzb strain, while a single population in wild type strain. In Δwzb strain a strong reduction of CSL-3 binding was observed. Furthermore, deletion of wzb is responsible of a significant reduction in biofilm production as estimated by crystal violet coloration. A general reduction of gene expression level of the EPS gene cluster was observed in Δwzb strain from the mid exponential to the early stationary growth phases. The present data reported the first inactivation of wzb gene in L. rhamnosus and the functional characterization of the Δwzb strain confirmed a role of Wzb phosphatase in EPS production