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

Abstract

<i>Pediococcus parvulus</i> 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 <i>P. parvulus</i> 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (<i>gutFRMCBA</i>), 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) <i>P. parvulus</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 <i>gutFRMCBA</i>. The promoter-probe vector pRCR based on the <i>mrfp</i> gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (P <i>_gut</i> ) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: <i>Lactobacillus plantarum</i> and <i>Lactobacillus casei</i>. Upon growth in the presence of sorbitol, but not of glucose, only the presence of P <i>_gut</i> was required to support expression of <i>mrfp</i> in <i>L. plantarum</i>. In <i>L. casei</i> the presence of sorbitol in the growth medium and the pediococcal <i>gutR</i> or <i>gutR</i> plus <i>gutM</i> in the genome was required <i>for</i> P <i>_gut</i> functionality. This demonstrates that: (i) P <i>_gut</i> is required for expression of the <i>gut</i> cluster, (ii) P <i>_gut</i> is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, <i>trans</i>-complementation for activation of P <i>_gut</i> exists in <i>L. plantarum</i> but not in <i>L. casei</i>.