Characterization of the Sorbitol Utilization Cluster of the Probiotic 2.6: Genetic, Functional and Complementation Studies in Heterologous Hosts.

2.6 secretes a 2-substituted (1,3)-β-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-β-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of . The promoter-probe vector pRCR based on the gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (P ) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: and . Upon growth in the presence of sorbitol, but not of glucose, only the presence of P was required to support expression of in . In the presence of sorbitol in the growth medium and the pediococcal or plus in the genome was required P functionality. This demonstrates that: (i) P is required for expression of the cluster, (ii) P is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, -complementation for activation of P exists in but not in .