Dokument

Summary:
Water availability is crucial for the development of all living cells. Various physical and chemical parameters such as desiccation and hyperosmotic stress generate cellular dehydration. The mechanisms of cellular adaptation preventing water loss under hyperosmotic conditions (osmoregulation) have been extensively studied in many organisms. In Bacillus subtilis the DegS-DegU two-component regulatory system controls various processes that characterize the transition form the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down regulation of the sigma D regulon, which encompasses the genes involved in motility, chemotaxis and autolysin production. Besides, the system is the only one up to now that is expressed under hyperosmotic conditions (Steil et al., 2003). In this work the role of the DegS-DegU system was further investigated with respect to its role upon salt induction. Northern blot experiments performed together with reporter-gene fusion studies confirmed that both degS and degU genes are transcribed together as an operon. Additionally, the presence of internal promoter upstream from the degU gene could be demonstrated. Involvement of the DegS-DegU system in the osmotic regulation was supported by the accumulation of mRNA transcripts from both genes when the cells were subjected to hypertonic conditions. The detected internal degU promoter was shown to be activated through a positive feedback mechanism from the phosphorylated DegU protein. Investigations of the main degS-degU promoter in the presence of different charged and non-charged compound, which raised the surrounding osmolarity to an equal extent, demonstrated that the activation of the system is rather an osmotic response and not consequence of salt-specific stimulation. Moreover, it seems that the DegS-DegU system does not sense the osmolarity per se since the addition of the potent osmoprotectant glycine betaine did not exhibit any influence and the corresponding structural genes were still induced. The cytoplasmic localisation of the DegS protein raised the following question: what are the molecular mechanisms underlying the perceiption of a certain stimuli from the environment and their transition to the sensor kinase. For this purpose both proteins have been purified and in vitro experiments were established for the detailed analysis of the DegS sensing properties. The in vitro investigations of the DegS autokinase activity in the presence of a variety of osmolytes with different chemical nature showed that the sensor kinase was specifically stimulated by the presence of glutamate. The putative role of the latter in activating the DegS autophosphorylation was supported also from the fact that osmotically challenged cells accumulated glutamate immediately after the addition of NaCl to the growth medium. In parallel to exploring the role of the DegS-DegU system at the transcriptional and at the protein level, some additional experiments were performed in order to identify possible downstream regulated targets of the system. The utilization of wild type (degS degU), hyper mutant [degU32(Hy)] and deletion mutant (degS degU::aphA3) strains led to the identification of nine genes whose transcription was increased when the cell were subjected to high osmotic environment and concomitantly this expression was dependent on the availability of degS and degU gene products. Taken together, the data from the present work shed a light on the transcriptional regulation of DegS-DegU two-component system in B. subtilis and implicated intracellular glutamate as a positive stimulus involved as a transducer of the information from the environment to the intracellular apparatus.

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