Osmotisch bedingter Stress in Bacillus subtilis - C-di-AMP, der Regulator für die Aufnahme osmotischer Schutzsubstanzen?
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Philipps-Universität Marburg
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Abstract
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.
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Dates
Created: 2020Issued: 2020-11-10Updated: 2020-11-10
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
osmotischer StressSalzstressc-di-AMP
DFG-subjects
Bacillus subtilisPhysiologie
DDC-Numbers
570
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Hinkel, Doreen: Osmotisch bedingter Stress in Bacillus subtilis - C-di-AMP, der Regulator für die Aufnahme osmotischer Schutzsubstanzen?. : Philipps-Universität Marburg 2020-11-10. DOI: https://doi.org/10.17192/z2020.0515.
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