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

• Published in: Frontiers in microbiology Vol. 8; p. 2393
• Main Authors: Pérez-Ramos, Adrian, Werning, Maria L, Prieto, Alicia, Russo, Pasquale, Spano, Giuseppe, Mohedano, Mari L, López, Paloma
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Media S.A 05-12-2017
• Subjects: exopolysaccharides; lactic acid bacteria; Microbiology; Pediococcus parvulus; probiotic; sorbitol; β-glucans; exopolysaccharides; probiotic; β-glucans; lactic acid bacteria; sorbitol; Pediococcus parvulus
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2017.02393

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
.