Untersuchung der Kälteschockantwort von Bacillus subtilis - σL-abhängige Transkriptionsaktivatoren und Identifizierung des kältespezifischen Wachstumsdefizits des Stammes JH642

For the investigation of the cold shock response the model organism B. subtilis is used. It is a gram positive mesophilic soil bacterium and its genome is completely known. A cold shock is defined as a temperature down shift from 37 °C to 15 °C. B. subtilis reacts to a cold shock with a change in gene expression, which leads to an adaptation of the complete cell physiology. This adaptation enables the survival at low temperatures due to the induction of cold protective proteins, which protect cold sensitive systems. These systems are the hindered initiation of translation and the decreased fluidity of the membrane. In the current work new cold induced genes and proteins were identified by DNA micro arrays and two dimensional gel electrophoresis. These candidates were tested for their cold sensitivity. In this and former investigations the gene yplP was found cold induced. It was shown, that a gene deletion results in a cold sensitive phenotype, which indicates a cold relevant role in the cold shock response. YplP shows significant homologies to σL-dependent transcriptional activators AcoR, BkdR, LevR and RocR. Via gene deletions and growth experiments the cold relevance of YplP, BkdR and the sigma factor SigL was observed. By electrophoretic mobility shift assays the interaction between SigL and BkdR was shown. They interact each alone and together with the known target-DNA. The identified target genes of YplP showed no interactions with SigL and YplP. Therefore, the regulated genes of YplP are still unknown. Furthermore, the reason for the cold sensitive phenotype of the strain JH642 was identified and characterized in the biosynthesis of the branched chain fatty acids. The strain JH642 shows a slower growth after cold shock in comparison to the strain MR168. In the current work the defect was identified by DNA sequencing in the gene ilvB. Via genetic complementation experiments and a biochemical approach the mutation in ilvB was found to be responsible for the growth defect of strain JH642. IlvB is involved in the degradation of threonine to isoleucine. Isoleucine serves as substrate for the synthesis of branched chain fatty acids that are essential for the membrane adaptation after cold shock.