Osmotisch bedingter Stress in Bacillus subtilis - C-di-AMP, der Regulator für die Aufnahme osmotischer Schutzsubstanzen?

Due to its natural habitet in the upper layers of the soil, Bacillus subtilis has to cope with permanent changing environmental conditions. B. subtilis utilizes the so-called salt-out strategy to adapt to a hyperosmotic environment with high salt concentrations. For this, the accumulation of so-called compatible solutes (little water-soluble compunds) is of particular importance. For the uptake, B. subtilis posseses different transporters, three of them belonging to the substrate binding protein dependent ABC transporters (OpuA, B and C). A special feature of these three transporters is the regulatory CBS domain at the C terminus of the nucleotide-binding domain, which catalyzes the transport by hydrolysis of ATP. In homologue transporters of other organisms there has been shown before, that these CBS domains interact with the second messenger cyclic di-AMP. Probably, this binding results in a switch-off oft he transport activity. Cyclic di-AMP is of particular interest because it is in B. subtilis as well as in other organisms involved in the uptake of potassium, the first step of the salt-out strategy. During this work, the CBS domains of the three transporters could successfully be produced in E. coli and also the subsequent purification was successful. With the help of two different methods it could be shown in vitro that c-di-AMP binds to the CBS domains of OpuB and OpuC but not to this of OpuA. The determination of the affinity of the CBS domains towards c-di-AMP resulted in values within the lower micromolar range. Using site-directed mutagenesis, there could amino acids be detected which seem to be essential for binding. Some interesting mutants have been found which do not bind c-di-AMP anymore. During osmoprotection experiments it was shown, that the in vitro tested mutants have no influence of the functionality of the transporter in comparison to the wild type. Also a strain which expresses either OpuB or OpuC without a CBS domain did not exhibit an alternated growth phenotype. An analysis of the minimal medium, which is usually used for B. subtilis, revealed that the high potassium concentration might be a problem for the growth experiments mentioned above. During this work it was not possible to find a medium composition with a low potassium concentration which is usable for growth of B. subtilis under high omolarity. In a second part of this work it could be shown that the c-di-AMP concentration in a B. subtilis cell is within the lower micromolar range. Furthermore, there is no significant change of the concentration during growth under high osmolarity. Combinations of knock-outs of the enzymes synthezising c-di-AMP showed reduced c-di-AMP concentrations. But it could not be revealed if one of the enzyme has a particular role during adaption to hyperosmotic conditions. In the end, this work gives a first hint, how c-di-AMP is involved in the regulation of the uptake of compatible solutes. Additionally, it is shown that during adaption to salt stress many factors are involved and are working together. For a little side project of this work, the cytoplasmatic volume of B. subtilis has been determined using different microscopy techniques. Interestingly, salt stress with 1.2 molar NaCl resulted in much smaller cells but also in a swelling of the cell envelope. This effect could be avoided if glycine betaine as compatible solute was used. These results are of particular importance for single cells. For example, the concentrations of c-di-AMP and other compounds are much higher than with the assumption that the cytoplasmatic volume remains similar unindependently of the external osmolarity.