GbsR : Ein neuer transkriptioneller Repressor in Bacillus subtilis zur Regulation des Cholin zu Glycin Betain Biosynthesewegs

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. It is a facultative anaerobic and rod-shaped bacterium. A primary habitant of B. subtilis is the upper layer of the soil. Within this ecosystem, B. subtilis experiences a wide variety of environmental challenges and nutrient limitations that in extreme cases can induce the formation of a highly resistant endospore. Changes in the osmolarity constitute a key factor that influences cell growth and survival in the soil. Since proper maintenance of turgor is essential for cell division and survival, bacteria must have active mechanisms to respond to changes in the environmental osmolarity in order to compete successfully for their ecological niche. A common response of many Bacteria to high-osmolarity growth conditions is the high-level accumulation, either through synthesis or uptake from the environment, of organic osmolytes, the so-called compatible solutes. B. subtilis can synthesize the compatible solute glycine betaine, when the precursor choline is present in the environment. Choline is taken up by the cell via OpuB and OpuC ABC transport systems and the conversion of choline into glycine betaine is mediated by the two enzymes GbsA and GbsB. The structural genes (gbsAB) for these enzymes form an Operon whose transcription is enhanced by the presence of choline in the growth medium, but not by high osmolarity. This work shows, that the gene gbsR, upstream of the gbsAB-genes, encodes for a repressor that controls the transcription of the gbsAB-operon in response to the availability of choline. Choline is actively bound by the repressor GbsR. Also the transcription of the ABC transporter OpuB-genes are controlled by the repressor GbsR. This work shows that the product of the synthesis, glycine betaine, represses the transcription of the gbsAB-operon. This happens via negative feedback-loop from glycine betaine to GbsR. The control of GbsR activity by choline and glycine betaine prevents that the compatible solute glycine betaine is accumulated in excessive amounts. In this work the repressor GbsR is further characterized. Band shift analyses have shown that GbsR is really a DNA-binding protein, which binds a specific sequence in the intergenic region of gbsAB and opuB. A possible operator-DNA, which includes an inverted repeat, a typical DNA-binding motive for transcriptional repressors, is found through shorting the promoter region. Bioinformatics studies showed that GbsR exists in many Bacilli and Staphylococcae as a regulator near the glycine betaine synthesis genes. An existing crystal structure of a GbsR-related protein of Methanococcus jannaschii allowed the classification of the GbsR protein as a winged helix turn helix protein.