Signal transduction by the essential signalling molecule cyclic di-AMP in Bacillus subtilis

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Bacteria use second messengers for signal transduction in order to achieve rapid adaptation to changing environmental conditions. Cyclic nucleotides and dinucleotides are among the most common second messengers. The Gram-positive soil bacterium Bacillus subtilis forms c-di-GMP and c-di-AMP. For both second messengers, multiple synthesizing enzymes are present in B. subtilis. While c-di-GMP is involved in the control of motility, c-di-AMP seems to play a role in several signal transduction processes. So far, functions in the control of cell wall homeostasis and cell division as well as in the regulation of potassium uptake have been described. We have studied the activities of the diadenylate cyclases CdaA and CdaS, that have not been studied before. We observed that c-di-AMP is essential for the growth of B. subtilis, but that accumulation of the second messenger does also compromise the growth of the cells. In order to get molecular insights into the signal transduction processes we have identified the PII-like signal transduction protein DarA as a c-di-AMP binding protein. With this proposal we want to study the control of c-di-AMP synthesis in B. subtilis, in particular by the diadenylate cyclase CdaA, which seems to be causative for the essential function of c-di-AMP. In this context, we will study the molecular details of the interaction between CdaA, its modulator protein CdaR, and the essential phosphoglucosamine mutase GlmM. These three proteins are all encoded in a conserved operon in Gram-positive bacteria with low GC content. In the second step, we wish to unravel the function of DarA. For this purpose, we will co-purify DarA with its interaction partners, and identify them by mass spectrometry. It is well established that PII proteins can interact with a variety of other proteins to control their activity, and we think this may also be the case for DarA. Finally, the major question of this project will be the elucidation of the reason for the essentiality of c-di-AMP. Based on our previous results, we think that c-di-AMP is required for the activity of an essential protein of cell wall biosynthesis. We have developed experimental strategies to identify this protein. We are confident that this experimental program at the interface of molecular genetics and proteomics will provide major novel insights into the function of this unique essential second messenger.