|M.Sc Student||Sand Andy|
|Subject||Regulation Analysis of Cellulose Utilization Genes by|
Alternative SIGMA Factors in Clostridium
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
Clostridium thermocellum is a gram positive, anaerobic, thermophilic soil bacterium that secretes a high molecular weight protein complex, the cellulosome, which is capable of hydrolyzing crystalline cellulose. C. thermocellum attracts much interest because of its ability to both hydrolyze cellulose and to produce ethanol and thus can be part of a consolidated bio-processing for bioethanol production. Recently, we identified a set of σ and anti- σ factors that play a role in the regulation of the cellulosomal genes in C. thermocellum. These σ-factors are located upstream to a gene encoding to a trans-membrane protein with an intracellular anti - σ domain at its N-terminus and an extracellular polysaccharide-related function module at its C-terminus. This arrangement provides a novel regulatory mechanism in which the expression of the cellulosomal genes can be controlled by the composition of the extracellular polysaccharides. In this study we attempted to link between the alternative σ factors and their corresponding genes. Based on the sequence similarity in their promoter regions, we identified 44 cellulosomal genes that are potentially regulated by the alternative σ factors. The proposed regulatory mechanism was directly challenged by deleting one of the rsgI genes, rsgI6, and the transcription level of the suspected genes was measured by real - time RT PCR. In total, 4 genes, including sigI6, exhibited increased transcript level in the rsgI6 deletion mutant compared to its parental strain. The results from this analysis enabled us to deterimine the consensus promoter sequence recognized by the alternative σ factor, SigI6. The rsgI6 - deleted strain also exhibited higher activity on two synthetic substrates: para-nitrophenyl- β-D-xylopyranoside (pNPX) and para-nitrophenyl- β-D-xylobioside (pNPX2), consistent with the up regulation of the SigI6-regulated genes.