טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRam Nechooshtan
SubjectCharacterization of Regulatory Elements of the Cellulase
System in Clostridium thermocellum
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Shoham Yuval
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

            The purpose of this research was to identify elements involved in the regulation of cellulosomal components in Clostridium thermocellum.  The cellulosome is an extracellular enzymatic complex, composed of cellulolytic and hemicellulolytic enzymes, which degrades efficiently crystalline cellulose.  This complex can be used as a part of a biotechnological process to degrade cellulosic waste, and for utilizing cellulose as a renewable source of soluble sugars and fuel.  The cellulosome complex is composed of a central non-catalytic subunit; also known as scaffoldin, which contains Cellulose Binding Domain (CBD) and several other domains termed cohesins, which serve to bind the catalytic subunits. Each cellulosomal enzyme contains one or more catalytic modules and a single dockerin domain that mediates its interaction with the scaffoldin.

Based on the genome sequence of C. thermocellum, genes that are homologous to known regulators from other gram-positive bacteria were identified.  Two potential regulator genes were cloned, overproduced in Escherichia coli and their gene products were purified. The potential genes were celGR, a gene encoding a putative regulator located next to the celG gene, and ccpA ­­- a gene encoding for the putative global regulator mediating the carbon catabolite repression in gram-positive bacteria, CcpA. CcpA employs as an allosteric corepressor, the phosphoprotein HPr-Ser-P, which is formed under glucose-replete conditions. The CcpA-HPr complex binds specific catabolite responsive elements (cre), located usually at the operator and the upstream regions of catabolite repressed genes.

  Using electrophoretic mobility shift assay (EMSA), the ability of CelGR and CcpA to interact with potential regulatory DNA sequences was analyzed.  In our hands, no specific interaction was found between CelGR and the promoter region of celG.   We were able to demonstrate in vitro active phosphorylation of the HPr protein, the CcpA corepressor.  However, EMSAs did not reveal binding of CcpA with or without its corepressor, HPr-Ser46-P, to several different cre elements.  Based on the recent structure of the CcpA-HPr-Ser46-P-cre complex from B. megaterium, in-depth analysis of the amino acid sequence of the putative CcpA homologue was performed.  This analysis revealed that the putative CcpA protein lacks most of the conserved residues involved in the repressor-corepressor interaction. These results suggest that in C. thermocellum, carbon catabolite regulation is not mediated via the CcpA protein.