Transcription Regulation of Cellulase Genes in Clostridium thermocellu

Clostridium thermocellum is an anaerobic, cellulolytic thermophile that degrades polysaccharides from plant biomass mainly through its cellulosome, a multi-enzyme complex. Understanding of gene expression regulation of cellulosomal components is the foundation for genetically engineering C. thermocellum as an industrial candidate for cellulosic biofuel production. Studies have suggested that cellulases expression are affected by carbon source and growth rate. However, knowledge of the molecular mechanism behind these phenomena remains limited. We cloned and expressed one of the C. thermocellum alternative sigmaI factors, SigI4. In vitro transcription assay indicated that SigI4 activated transcription of its own operon. Primer extension determined the putative -10 and -35 regions for SigI4 recognition. Genomic search using SigI4 recognition sites revealed a glycosyl hydrolase gene, celW, as a potential downstream target of SigI4. Transcription start site of celW was determined by primer extension, and SigI4 was shown to activate celW transcription by in vitro transcription assay. The results prove that SigI4 upregulates its own gene and celW. In this work, we also identified an AbrB-family transcription regulator and studied its function in celS transcription. We showed that the recombinant protein rCthe_2100 binds to 5' regulatory region of celS DNA. The least-required binding site was determined to be -240 bp ~ -210 bp (relative to translation start site) using electrophoretic mobility shift assay (EMSA) with nested probes. In vitro transcription assay showed rCthe_2100 repressed celS transcription, and such repression was abolished when Cthe_2100 binding site was swapped with a random sequence. In addition, we showed that rCthe_2100 binds to the 5' regulatory region of its own gene. EMSA using nested probes revealed -70 bp ~ -40 bp (relative to translation start site) as Cthe_2100 least-required binding site upstream of its own gene. Primer extension was used to determine the transcription start site of Cthe_2100. In vitro transcription assay showed that rCthe_2100 repressed its own gene transcription. These results suggest that Cthe_2100 is self-regulated. In summary, this thesis investigated the cellulase transcription regulation controlled by SigI4 and Cthe_2100 in C. thermocellum..