טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSand Andy
SubjectRegulation of Cellulolytic Genes in Clostridium Thermocellum
by Alternative Sigma Factors
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham
Full Thesis textFull thesis text - English Version


Abstract

Clostridium thermocellum is a Gram positive, anaerobic, thermophilic soil bacterium capable of utilizing cellulosic substrates. The bacterium 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. C. thermocellum possesses a high molecular weight protein complex, the cellulosome, which efficiently degrades cellulose and plant cell wall associated polysaccharides. Recently, a novel regulatory mechanism of cellulosomal genes was identified in C. thermocellum. This regulatory mechanism is based on a set of seven bi-cistronic operons that encode for alternative SigI-like sigma factors and a trans-membrane protein (RsgI) with an intracellular anti-sigma domain at its N-terminus, and an extracellular polysaccharide-related function module at its C-terminus. According to this regulatory mechanism, in the presence of a specific polysaccharide that can interact with the corresponding carbohydrate sensing domain of RsgI, the sigma factor is released from the anti-sigma domain, binds the RNA polymerase and promotes transcription. This arrangement provides a novel regulatory mechanism, in which the expression of the cellulosomal genes can be controlled by the composition of high molecular weight, extracellular polysaccharides.

The objective of this research was to assign between the alternative sigma factors and all of the cellulosomal genes. For this purpose, both bioinformatics and experimental tools were applied. Transcriptional start sites of sigI and cellulosomal genes were mapped with a modified primer extension reaction, in which the radioactive primer was replaced with a fluorophore labelled primer (6-FAM) and the exact size of the extension product was determined with capillary electrophoresis. Cellulosomal genes regulated by each sigma factor were identified by promoter sequence similarity to the sigI’s promoters and analysis of the expression levels of cellulosomal genes in sigI and rsgI knockout strains. These methods allowed us to refine the consensus promoter sequence recognized by each of the SigI sigma factors. Many cellulosomal genes were found to be transcribed from both alternative SigI and vegetative SigA promoters. These SigA promoters appeared to be less conserved compared to SigA promoters of sugar and amino acid metabolism related genes. The identification of SigA promoters upstream to cellulosomal genes suggest that cellulosomal genes are also expressed from weak vegetative SigA promoters that allow a basal expression level under all growth conditions.

Taken together, 58 cellulosomal genes were associated to their corresponding alternative sigma factors regulons. The alternative sigma factors SigI3, SigI4 and SigI6 were found to regulate the expression levels of pectinase, cellulase and xylanase genes respectively, and Sig1, SigI2, SigI5 and Sig24C were predicted to regulate cellulose and hemicellulose related genes.