AUTHOR=Pérez-Ramos Adrian , Werning Maria L. , Prieto Alicia , Russo Pasquale , Spano Giuseppe , Mohedano Mari L. , López Paloma 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.02393

DOI=10.3389/fmicb.2017.02393

ISSN=1664-302X

ABSTRACT=<p><italic>Pediococcus parvulus</italic> 2.6 secretes a 2-substituted (1,3)-β-<sc>D</sc>-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the <italic>P. parvulus</italic> 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (<italic>gutFRMCBA</italic>), 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) <italic>P. parvulus</italic> 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-<sc>D</sc>-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 <italic>gutFRMCBA</italic>. The promoter-probe vector pRCR based on the <italic>mrfp</italic> gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (P<italic><sub>gut</sub></italic>) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: <italic>Lactobacillus plantarum</italic> and <italic>Lactobacillus casei</italic>. Upon growth in the presence of sorbitol, but not of glucose, only the presence of P<italic><sub>gut</sub></italic> was required to support expression of <italic>mrfp</italic> in <italic>L. plantarum</italic>. In <italic>L. casei</italic> the presence of sorbitol in the growth medium and the pediococcal <italic>gutR</italic> or <italic>gutR</italic> plus <italic>gutM</italic> in the genome was required <italic>for</italic> P<italic><sub>gut</sub></italic> functionality. This demonstrates that: (i) P<italic><sub>gut</sub></italic> is required for expression of the <italic>gut</italic> cluster, (ii) P<italic><sub>gut</sub></italic> is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, <italic>trans</italic>-complementation for activation of P<italic><sub>gut</sub></italic> exists in <italic>L. plantarum</italic> but not in <italic>L. casei</italic>.</p>