Clostridium thermocellum is an anaerobic, thermophilic, soil bacterium. It utilizes crystalline cellulose efficiently and ferments the cellodextrins to ethanol; therefore, it could potentially play a part in a low-cost conversion process of biomass to biofuel by a consolidated bioprocess. The hallmark of the cellulose degradation system is an extracellular, multi-enzyme complex termed the cellulosome. There are approximately 70 genes that encode cellulosomal catalytic subunits (mainly cellulases and hemicellulases). Interestingly, the composition of the cellulosome is variable and affected by the presence of various polysaccharides in the extracellular surroundings. Works by our group and others revealed that many cellulosomal genes are up-regulated during slow growth rates in continuous cultures under carbon limitation. One of these genes is the cellulosomal anchoring protein olpB. Real-time RT-PCR revealed a 100-fold increase in expression of this gene, when the growth-rate decreased from 0.2 hr-1 to 0.1 hr-1. DNA pull-down assay was performed to detect regulatory proteins that bind to olpB’s promoter region. The most common putative transcription factor detected was a 27kDa transcriptional regulator from the GntR family, designated CgrA (Cellulosomal Gene Regulator A). Members of this family, GntR-like bacterial transcription factors (PF00392), were found to be involved in carbohydrate utilization in Gram-positive bacteria like Geobacillus stearothermophilus, Bacillus subtilis and Streptococcus pneumonia. Electrophoresis mobility shift assay (EMSA) showed that GntR binds specifically to olpB’s promoter region. This protein is a potential novel regulator of the cellulosomal system, and may be the first trans acting cellulosomal transcriptional element identified in this intriguing bacterium.
