Function of c-di-AMP in Archaea

环核苷酸及其作为细胞内信号转导的第二信使的作用已经在细菌中得到了很好的描述。最近，环状二磷酸腺苷被发现是细菌的第二信使，在细菌的正常生长条件下被证明是必不可少的。与细菌相比，我们对古细菌中的第二信使知之甚少。我们已经证明c- 2 - amp存在于极端嗜盐的火山盐古菌中。其基因组编码一种二腺苷酸环化酶（DacZ），我们对其进行了表达和纯化，并在体外证明了该酶能产生c-二- amp。与细菌一样，在正常生长条件下，火山嗜血杆菌也需要dacZ基因，过表达是致命的。这表明c- 2 - amp水平在火山螺中受到严格调节。用表达较低水平dacZ基因的H. volcanii突变体进行的实验表明，该突变体在低盐浓度下细胞体积增加，这表明c-di-AMP在调节渗透稳态中起重要作用。为了了解C -di- amp在H. volcanii中的作用，我们将(A)进一步表征DacZ并确定n端丙酮酸激酶效应结合域的作用，(B)表征C -di- amp在H. volcanii中调节渗透稳态中的作用，(C)鉴定C -di- amp结合蛋白和参与该生物C -di- amp调节网络的其他蛋白。

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.