Abstract
Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB–Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
Highlights
Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis
A L. monocytogenes strain lacking c-di-AMP is unable to grow on complex media, but suppressor mutants with the inactivated homolog of DarB (CbpB) were able to grow on complex medium[18]
Rel exhibited only background activity. These results demonstrate that the interaction between apo-DarB and Rel stimulates the synthesis of pppGpp and that this stimulation is prevented in the presence of c-di-AMP
Summary
Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. Of all known second messenger nucleotides c-diAMP is unique in binding and controlling both a protein and the mRNA molecule that encodes it This is the case for the Bacillus subtilis KtrA and KimA potassium transporters that are both bound and inhibited by c-di-AMP. In contrast to most other c-di-AMP-binding proteins, DarA and DarB do not contain any other domain that might be controlled by the binding of the second messenger. It is, likely that these proteins interact with other proteins in a c-di-AMP-dependent manner to control their activity. The integration of c-di-AMP and (p)ppGpp signaling allows a global cellular response to the availability of potassium
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