The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

Clostridium thermocellum utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the celC operon and determined that the celC-glyR3-licA gene cluster and nearby manB-celT gene cluster are co-transcribed as polycistronic mRNA.

Together these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of β-1,3-polysaccharides in its environment.

In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivo transcription of the celC-glyR3-licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription. Conclusions: Together these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of β-1,3-polysaccharides in its environment.