טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZaide Galia
SubjectRegulation of the Xylanolytic System from
Geobacillus stearothermophilus T-6
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham


Abstract

The purpose of this research was to characterize, at the molecular level, genetic elements that are involved in regulation and utilization of hemicellulose by Geobacillus stearothermophilus T-6. G. stearothermophilus T-6 is an aerobic thermophilic bacterium that possesses an extensive and highly regulated hemicellulolytic system which enables it to grow on hemicellulose as a sole carbon source.  A PCR-based chromosome walking method was applied, and a 14.3kb chromosomal segment flanking the previously characterized xylan utilization genes was cloned and sequenced.  The gene cluster encodes 13 putative open reading frames that are organized, based on Northern blotting and primer extension analyses, in five transcriptional units.  These units include the xylose operon encoding for xylose isomerase and xylulose kinase, and a transcriptional unit encoding for an intracellular xylanase and a xylosidase.  Both units are induced by xylose and appear to be repressed by glucose.  In addition, a two-component signal transduction system that regulates a xylosaccharide ATP dependent transport system (ABC) was revealed.  By using gel retardation and DNase I footprinting, the response regulator XynC, was shown to bind the promoter region of the ABC transport system.  Thus, it is proposed that upon sensing extracellular xylosaccharides, the sensor histidine kinase phosphorylates the response regulator, XynC, which in turn activates the transport system.  In addition, a reliable protoplast transformation procedure was developed for G. stearothermophilus T-6 resulting in transformation yields of over 102 transformants per microgram plasmid DNA.  This procedure may allow now the application of modern genetic tools for studying gene regulation in G. stearothermophilus.