Kälteschockantwort von Bacillus subtilis - Supercoil-regulierte Kälteschockproteine und die kälteinduzierten DEAD-box RNA-Helikasen CshA und CshB

Decreasing environmental temperatures induce a cold shock response in microorganisms. To investigate this cold shock response in gram positive bacteria the mesophilic soil bacteria Bacillus subtilis is used as a model organism. A cold shock is defined as a temperature shift from 37 °C to 15 °C. After such a cold shock B. subtilis changes the gene expression to adapt its physiology to the new conditions. This adaptation facilitates the survival at low temperatures by protecting cold sensitive systems through the production of cold induced proteins. Among these cold sensitive systems are the fluidity of the membrane, the changed DNA topology and the hindered initiation of translation. After a cold shock the negative supercoil of the DNA is increased in B. subtilis. The increased negative supercoil results in a changed gene expression. In the current work the supercoil- dependent genes were identified by 2D-PAGE. To find these proteins the DNA gyrase was inhibited by the addition of Novobiocin and then the proteome of JH642 at 37 °C and 15 °C with and without Novobiocin was compared. By this approach eight genes were identified. The relevance of these genes was tested by gene deletions and growth experiments. A cold sensitive phenotype was observed for the fabI mutant. FabI is an enoyl-[acyl-carrier-protein] reductase, which is involved in the elongation cycle of the fatty acid biosynthesis. Here not only linear fatty acids are elongated, but also the branched fatty acids, which are involved in the adaptation of the membrane to low temperatures. In the second part of this work the cold induced proteins CshA and CshB were investigated, which show high sequence homology to DEAD box RNA helicases. Using an ATPase test and a RNA unwinding test the two proteins were characterized as ATP dependent DEAD box RNA helicases. Flourescence-microscopic measurements show the transcription dependent localization of the helicases CshA and CshB at the poles of the cell, what is also known for the CSPs. A cshB/cspD double deletion mutant shows an impaired growth at low temperatures compared to the wild type JH642. Furthermore the postulated interaction of the helicases and the CSPs could be shown by FRET measurements of the CspB-CFP and CshB YFP fusions in vivo. These results support the model, in which the RNA helicases unwind cold stabilized secondary structures of the mRNA. The CSPs can than bind the single stranded mRNA, which in turn facilitates the initiation of translation at low temperatures.