Function of c-di-AMP in Archaea

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Cyclic nucleotides and their role as second messengers for intracellular signal transduction has been well described in bacteria. Recently cyclic di-AMP was discovered as bacterial second messenger, which has been demonstrated to be essential under normal growth conditions in bacteria. Compared to bacteria, very little is known about second messengers in archaea. We have demonstrated that c-di-AMP is present in the extremely halophilic eurayarchaeon Haloferax volcanii. Its genome encodes one diadenylate cyclase (DacZ) which we expressed and purified and demonstrated to produce c-di-AMP in vitro. Like in bacteria, the dacZ gene is essential in H. volcanii under normal growth conditions and overexpression is lethal. This indicates that c-di-AMP levels have to be tightly regulated in H. volcanii. Experiments with an H. volcanii mutant expressing lower levels of the dacZ gene showed that this mutant had an increased cell volume at low salt concentrations, suggesting that c-di-AMP is important for the regulation osmohomeostasis. To understand the role of c-di-AMP in H. volcanii, we will (A) characterize DacZ further and identify the role of the N-terminal pyruvate kinase effector binding domain, (B) characterize the role of c-di-AMP in the regulation of osmohomeostasis in H. volcanii and (C) identify c-di-AMP binding proteins and other proteins that are involved in the c-di-AMP regulatory network in this organism.