Analysis of the alternative sigma factor system, σI5/RsgI5, in Clostridium thermocellum

MSc. Graduate Seminar; Sari Cohen

Clostridium (Ruminiclostridium) thermocellum is a Gram-positive, thermophilic, anaerobic bacterium that is capable of efficiently hydrolyzing crystalline cellulose via an extracellular multienzyme complex, termed the cellulosome. C. thermocellum regulates its cellulosomal genes in response to extracellular biomass composition via a unique σI/RsgI biomass sensing system. Unlike the previously characterized sigI-rsgI operons that contain two genes, the uncharacterized sigI5-rsgI5 operon is composed of three genes including a gene upstream to sigI5, for a putative arabinofuranosidase from glycoside hydrolase family 43 (GH43). Transcription analysis using RT-PCR confirmed that gh43, sigI5 and rsgI5 are co-transcribed. The apparent transcription start site of the gh43-rsgI5 operon was identified and conserved sequence in the promoter region was observed. The predicted σI5 promoter sequence was identified in three arabino-xylan utilization genes, suggesting that their expression is controlled by σI5. Interestingly, the predicted σI5 promoter sequence was also identified in the upstream region of rsgI5, suggesting a different type of gene organization and regulation of the gh43-rsgI5 operon compared to the other sigI-rsgI operons. Due to the limited genetic tools available for C. thermocellum, a heterologous B. subtilis 168 strain host system was applied to verify the functionality of the predicted promoters regulated by σI5. We were able to integrate C. thermocellum sigI5 gene and the putative σI5 regulated promoters (fused to a reporter gene) into B. subtilis at different chromosomal sites, and found that using multimers plasmids contributed to the success of the integration events. Unfortunately, we could not observe expression form the tested promoters. Real-time reverse transcription (RT) PCR revealed the lack of the sigI5 transcript in B. subtilis. We attempted to use a cell-free system, but failed to obtain a soluble of the σI5 protein in E. coli.