טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNataf Yakir
SubjectRegulation and Functional Analysis of Cellulose
Utilization Elements in Clostridium Thermocellum
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham
Full Thesis textFull thesis text - English Version


Abstract

Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multi-enzyme complex, the cellulosome, which mediates the cell attachment to, and hydrolysis of crystalline cellulosic substrate. The objectives of this research were to identify and characterize regulatory mechanism controlling the utilization of cellulose by Clostridium thermocellum, and to identify and characterize the cellodextrins transport systems.

            Five sugar ABC transporter systems were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The ability of the five solute-binding proteins to interact with different sugars was examined by isothermal titration calorimetry (ITC). Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G2 to G5), One protein, CbpA, binds only cellotriose (G3), and another protein, Lbp, interacts with laminaribiose. The sugar specificity suggests that cellodextrins (G3 to G5) are assimilated faster than cellobiose. Since this bacterium employs phosphorolytic cleavage, it gains more ATP per glucose molecule by utilizing cellodextrins.

            C. thermocellum batch and continuous cultures coupled with DNA microarray, Real-time RT-PCR and SDS-PAGE analyses have indicated that cellulosomal genes expression is affected by the cellobiose availability, growth rate and by the presence of extracellular polysaccharides. Set of putative σ and anti-σ factors that include extracellular polysaccharide-sensing module were identified. These factor-encoding genes are homologous to the Bacillus subtilis bicistronic operon sigI-rsgI, which encodes for an alternative σI factor and its cognate anti-σI regulator RsgI that is functionally regulated by an extracytoplasmic signal. The binding specificity of C. thermocellum putative anti-σI factors to their corresponding σ factors was demonstrated by ITC. Quantitative Real-time RT-PCR measurements revealed three- to 30-fold up-expression of the σ factor genes in the presence of cellulose and xylan, thus connecting their expression to direct detection of their extracellular polysaccharide substrates. Cellulosomal genes that are putatively regulated by two of these σ factors, σI1 or σI6, were identified based on the sequence similarity of their promoters. The ability of σI1 to direct transcription from the sigI1 promoter and from the promoter of celS was demonstrated in vitro by runoff transcription assays. Taken together, the results reveal a novel regulatory mechanism in which alternative σ factors are involved in regulating the cellulosomal genes.