Die stressprotektive Wirkung von Aminosäuren in Bacillus subtilis: Endogene Synthese von Prolin und die GltT - vermittelte Aufnahme von Glutamat und Aspartat zum Schutz vor Hyperosmolarität und Hitze

Under high-osmolarity conditions, B. subtilis accumulates the amino acid proline via de novo synthesis as a so-called compatible solute (Whatmore, 1990). This synthesis is provided by the enzymes ProJ, ProA and ProH. The structural genes proH and proJ are organized as an osmotically controlled operon (Brill, 2001). This work should identify the essential elements in cis for the osmoregulated transcription of proHJ. By using reporter gene constructs we could pinpoint a minimal regulatory fragment. After site-directed mutagenesis of this fragment we found evidence that the crucial cis elements are located in the 5´non-translated region of proH. Some of the mutagenized strains showed an overproduction of proline. In spite of their higher intracellular proline level these strains have no growth advantage under hypertonicity but are less sensitive to the toxic proline analogue 3,4 dehydro-DL-proline. Investigations on the effect of known and, due to their homology, putative transcriptional regulators from B. subtilis did not help to identify any regulation system involved in the osmotic induction of proHJ. Analysis on another proline accumulating Bacillus, B. licheniformis, discovered a great similarity of the proline synthesis genes of both Bacilli. The minimal regulatory fragment of proHJ, which has been identified in B. subtilis, could also be pointed out in B. licheniformis. Furthermore, as its homologue, this fragment was able to provide fully osmotic regulated transcription. Detailed examination showed that this regulatory fragment possesses homologues in more Bacilli (B. amyloliquefaciens, B. mojavensis and B. vallismortis). The amino acid glutamate is known as osmo- and thermoprotective substance in B. subtilis (Kerres, 2002; Holtmann and Bremer, 2004). It could be demonstrated here that the Na+/H+/symporter GltT, a member of the glutamate transporter family, is involved in glutamate uptake under the named conditions. Moreover, GltT is also the main high-affinity glutamate transporter under standard growth conditions. In this thesis, the thermo- and osmoprotective properties of the amino acid aspartate in B. subtilis have been described for the first time. Members of the glutamate transporter family are known to accept aspartate as substrate as well as glutamate. So, in addition to its role as glutamate uptake system, GltT was characterized as the main aspartate transporter in B. subtilis . With glutamate and aspartate, two proteinogen amino acids were shown to work as stress protectants. To elucidate if this is a commun property of all proteinogen amino acids, experiments under osmo-, heat- and chill stress have been performed. It could be elucidated that amino acids working as osmoprotectants are supposed to be precursors for proline synthesis. Whereas no real cryoprotective function of these amino acids could be detected, many of them could be identified as potent thermoprotectants. However, the exact mechanism of thermoprotection remains unknown.