| Biomass conversion to ethanol as a liquid fuel may potentially offset some of the world's dependence on petroleum for energy. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic, and ethanogenic bacterium that holds much promise as an industrial ethanol producer. The organism is able to degrade cellulose into fermentable sugars by utilizing a complex cellulase system. Very little is known about how the bacterium regulates the expression of its cellulase system. We have identified glyR3 as the first transcriptional regulator of cellulase genes discovered in the organism. Northern blot showed that glyR3 is co-transcribed with cellulase/hemicellulase genes celC and licA. Using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR), it was shown that when the bacterium is grown on beta-1,3 linked glucans, the expression of celC, glyR3, and licA is increased when compared to growth on beta-1,4 linked glucans. Gel-shift assays revealed that recombinant GlyR3 bound specifically to the celC promoter region. GlyR3 was identified from cell lysate on lichenan-grown cells but could not be identified from cellobiose-grown cells. DNase I footprinting and competitive gel-shift proved the binding site was an 18-bp near perfect palindromic sequence. Gel-shift and in vitro transcription assays showed that the DNA-binding activity was specifically inhibited by laminaribiose, a beta-1,3 linked glucose dimer. The results prove that GlyR3 is a negative regulator of the celC operon with laminaribiose as an inducer. Also described in this work is an uncharacterized gene cluster of five cellulase system components, named Cluster X. Cluster X consists of five genes that encode proteins containing dockerin domains, meaning they are components of the cellulosome. The cellulosome is an extracellular multiprotein complex that efficiently degrades cellulose. Northern blot revealed that all five genes in Cluster X are co-transcribed and primer extension revealed two initiation sites for the transcript. RT-qPCR discovered that all of the genes have transcripts that are more highly expressed when the bacterium is grown on cellulose, as compared to cellobiose. Additionally, the highest point of expression, regardless of substrate, was found in the stationary growth phase. The results are a first step in understanding the regulation of the cellulase system in C. thermocellum. |
