Clostridium thermocellum is an anaerobic, thermophilic bacterium. It utilizes crystalline cellulose efficiently and ferments the cellodextrins to ethanol; therefore, it could potentially play a part in low-cost conversion of biomass to biofuel by a consolidated bioprocess. The hallmark of the cellulose degradation system is an extracellular, cell-wall bound, multi-enzyme complex termed the cellulosome. There are approximately 70 genes that encode cellulosomal catalytic subunits (both cellulases and hemicellulases). The enzymatic composition is variable and affected by the presence of various extracytoplasmic polysaccharides via alternative σ factors. Works by our group and others revealed that many cellulosomal genes are up-regulated during slow growth rates (generation time of 7-14 hours) 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 comparing doubling times of 7 vs. 3.5 hours, emphasizing the impact of growth-rate on transcription levels. DNA pull-down assay was performed to detect regulatory proteins that bind to olpB’s promoter sequence. Soluble protein extract from C. thermocellum was incubated with biotinylated DNA fragments containing olpB’s promoter region, and loaded on a streptavidin column. The bound proteins were eluted, analyzed by LC-MS/MS, and aligned against NCBI-nr database. One of the common putative transcription factors detected was homologous to Bacillus subtilis’s AbrB, a well characterized global transcription factor that binds several consensus sequences and regulates many genes during exponential growth. Aligning AbrBCthe with AbrBBsub revealed an 85% identity in the N-terminal DNA-binding domain and a complete identity in the DNA binding residues (identified by NMR resolved AbrBBsub-DNA structure); thus, suggesting binding to similar sequences in both bacteria. This proposes a putative role for AbrB in carbohydrate utilization regulation by this intriguing bacterium.
