|M.Sc Student||Langut Yael|
|Subject||Characterization of XylR, the Repressor of the Xylanolytic|
System in Geobacillus stearothermophilus T-6
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
The purpose of this research was to clone and sequence the xylose repressor (XylR) from Geobacillus stearothermophilus T-6, and to determine its role in regulating the hemicellulolytic system. G. stearothermophilus T-6 is an aerobic thermophilic bacterium that possesses an extensive hemicellulolytic system which enables it to grow on hemicellulose as a sole carbon source. The XylR gene, encoding a 385 amino acids protein, was cloned via PCR using primers based on the homologous xylR gene from G. stearothermophilus strain 10. XylR from strain T-6 shares homology to proteins of the ROK family which possess C-terminal sugar-binding domains with a high homology to glucokinase. The homology of XylR to the ROK family suggests that the XylR have an additional role in catabolic repression of the xylanolytic system. A His-tag fused XylR was successfully expressed in E. coli using the T7 RNA polymerase-based overexpression system. Expression was carried out by growing the cells for 24 hours at 18°C, and using low concentration of inducer (0.05mM IPTG). Gel retardation analysis demonstrated that XylR binds to the promoters of at least five transcriptional units: the xylose utilization operon (xylAB), the extracellular xylanase (xynA), the aldose-1-apimerase (xylM), the two-component regulatory system (xynDC) and the putative regulatory protein (xynX). A 25-base element containing an inverted repeat was identified in all of the promoters. The binding was efficiently prevented in vitro in the presence of xylose and xylosaccharides such as xylotriose and xylotetraose. These findings establish that XylR is a global regulatory protein of the xylanolytic system and xylosaccharides can act as molecular inducers, consistent with the notion that strain T-6 is capable of uptaking high molecular-weight xylooligossacrides from the environment.