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B. subtilis lives in the upper layers of the soil and must therefore adapt to strong variations of osmolarity and water content. The soil bacterium can adapt to high osmolarity growth conditions through the osmotically induced de novo synthesis or the uptake of the compatible solute and osmostress protectant proline. B. subtilis possesses interlinked pathways for the synthesis of proline. The anabolic ProB-ProA-ProI route provides proline for protein biosynthesis, whereas the ProJ-ProA-ProH route is responsible for the high-level production of proline as an osmostress protectant. The first step of both pathways is catalyzed by the γ-glutamyl kinases ProB and ProJ. These isoenzymes show essential differences regarding their regulation. The transcription of the proBA operon is controlled in response to intracellular proline levels via a T-box regulatory element, whereas the transcription of the proHJ gene cluster is up-regulated in response to increases in the external osmolarity. ProB is feedback regulated by proline, but such a post-transcriptional regulation is unlikely to control the enzyme activity of ProJ.
The enzyme-inhibitor-interaction is modulated by a flexible 16-residue loop in the active center of the γ-glutamyl kinase. ProB and ProJ from B. subtilis reveal a striking difference regarding the amino acid sequence of this loop. The feedback inhibited ProB enzyme possesses a negative glutamate residue at position 142 in the flexible loop, whereas the ProJ enzyme possesses a positively charged Arg residue at the corresponding region. Bioinformatic analysis revealed that the negative amino acid glutamate in enzymes of the ProB type and the positive amino acid arginine in enzymes of the ProJ type is highly conserved within the genus Bacillus. Based on these bioinformatic analysis we created B. subtilis mutants with a substitution of the negatively charged amino acid glutamate of the anabolic ProB enzyme against a positively charged amino acid arginine by site directed mutagenesis. Vice versa, we replaced arginine in the osmoadaptive ProJ enzyme by glutamate. The amino acid substitution in ProB caused a decreased allosteric regulation of the protein and led to an increase in the cellular proline pool and the osmotic tolerance. Conversely, the amino acid substitution in ProJ causes an increased allosteric regulation of the protein and led to a decreased proline accumulation in vivo and a reduced osmotic tolerance. Our data strongly suggest that the different allosteric controls of the ProB and ProJ are well integrated into the physiological functions of either the anabolic or osmostress adaptive proline synthesis routes of B. subtilis.
The anabolic and the osmoadaptive proline pathways in B. subtilis are connected via the shared ProA protein. Because no paralogous protein to ProA exists, the deletion of the proA gene leads to a perturbation in proline biosynthesis. Suppressor mutations within the ahrC region and the argCJBD-carAB-argF region recruit enzymes of the arginine metabolism for the synthesis of proline. The suppressor mutations were of two types: First were mutations in the promoter region of argCJBD-carAB-argF, which encodes for genes of the arginine biosynthetic pathway. Second were mutations in the regulator protein AhrC, which represses the transcription of the argC operon. Both types of mutants result in a diminished binding of the transcription repressor AhrC to its operator region and enhance the transcription of the argC operon leading to increased amounts of ornithine within the cell. Ornithine can be converted via the RocD enzyme. RocD as part of the arginine degradation pathway synthesizes the same reaction product as the ProA enzyme and thereby bypassing the ProA mediated enzyme reaction. Furthermore our investigations suggest that under certain conditions the amino acid arginine can also be used as an osmostress protectant by B. subtilis. The data acquired in this dissertation demonstrate how effective bacteria can adapt to limitations on their essential biosynthetic pathways.