P48: The role of GlyR3 in regulating expression of cellulolytic activity in Clostridium thermocellum

Cellulosic bio-ethanol is regarded as a promising transportation fuel since it can be used in mixtures with gasoline in conventional engines and does not use food-based starches such as corn for a feedstock. Clostridium thermocellum, a gram-positive bacteria, is considered to be a model organism for consolidated bioprocessing since it is capable of both hydrolyzing plant biomass and fermenting the hydrolysis products into ethanol.

We are investigating regulatory mechanisms of cellulolytic genes in C. thermocellum. The celC operon encodes for the cellulolytic genes celC and licA, in addition to the LacI family regulator GlyR3. The latter protein autorepresses the operon by binding to a repressor site located downstream of the celC promoter. The repression is relieved in the presence of laminaribiose. (Newcomb et al, Proc Natl Acad Sci U S A 104(10): 3747–3752, 2007).

We are investigating the possibility of GlyR3 regulating other cellulolytic genes in C. thermocellum. We compare the similarity of GlyR3 with CcpA, a well-characterized LacI family protein in B. subtlis and found strong similarity in the DNA-binding domains of the two proteins. Further, a position specific scoring matrix (PSSM) of DNA binding sites for CcpA showed similarity to the CelC binding site in C. thermocellum. Based on these similarities, we used the PSSM to look for novel binding sites of GlyR3 in C. thermocellum. The binding site candidates that were found by information content were tested by electrophoretic mobility shift assay (EMSA), in vivo RT-PCR, and in vitro transcriptional assay.