טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShner Itzhak
SubjectL-Arabinose Sensing and Utilization Systems in Geobacillus
stearothermophilus T-6
DepartmentDepartment of Biotechnology
Supervisor Professor Yuval Shoham
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The purpose of this research was to characterize, at the physiological and molecular levels, two new arabinose related gene clusters, araPST and araJKLMN in Geobacillus stearothermophilus T-6. These gene clusters were identified following the partial genome sequencing of the bacterium and are located within the hemicellulolytic system for arabinan utilization. G. stearothermophilus is a thermophilic, Gram positive, aerobic soil bacterium which employs an efficient hemicellulolytic system that allows the cell to utilize hemicellulose polysaccharides. Hemicellulose polysaccharides comprise the plant cell wall and are highly variable in their structure, thus their efficient degradation requires the synergistic concerted action of several enzymes.

The araPST gene products show similarities to two-component regulatory systems. However, this cluster is unique because it contains an additional protein, AraP, instead of the usual two components. By using isothermal titration calorimetry (ITC) AraP was shown to have high affinity to L-arabinose (Kd=1/Kb=0.6µM), but failed to bind xylose, D- arabinose, galactose or oligomers of L-arabinose. AraT shows similarity to response regulator proteins and its conserved aspartic acid residue was suggested to be at position 59. In addition, the C-terminus domain resembles DNA binding domains. Gel retardation assay indicates that AraT binds specifically to the promoter region of araEGH, the L-arabinose transport system.

The araJKLMN gene cluster is located between the hemicellulolytic system for arabinan utilization and the hemicellulolytic system for xylan utilization. Real-time RT-PCR assays performed on mRNA samples from cultures grown on glucose, arabinose or xylose as carbon source has shown that the expression of the gene cluster is induced in the presence of L-arabinose and undergoes catabolic repression in the presence of glucose. Amino acid sequence analysis indicates that AraJ and AraM show homology to dehydrogenases, AraK to epimerase and AraL to a phosphatase. AraN did not show any similarities to known proteins. Secondary and tertiary structure analysis of AraN has indicated that it might be a glycoside hydrolase or a glycoside transferase.

AraJ shows 20% identity to an arabinose dehydrogenase from Caulobacter crescentus, which constitutes the first enzyme from an alternative arabinose utilization pathway. Activity assays of AraJ resulted in similar activity to the arabinose dehydrogenase from C. crescentus. It is likely that the gene cluster represents a new alternative pathway for L-arabinose utilization.