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

细菌利用第二信使进行信号转导，以快速适应不断变化的环境条件。环核苷酸和二核苷酸是最常见的第二信使。革兰氏阳性土壤细菌枯草芽孢杆菌形成c-di-GMP和c-di-AMP。对于这两种第二信使，枯草芽孢杆菌中存在多种合成酶。c-di-GMP参与运动的控制，而c-di-AMP似乎在几个信号转导过程中发挥作用。到目前为止，已经描述了控制细胞壁稳态和细胞分裂以及调节钾摄取的功能。我们研究了二腺苷酸环化酶CdaA和CdaS的活性，这是以前没有研究过的。我们观察到c- 2 - amp对枯草芽孢杆菌的生长至关重要，但第二信使的积累也会损害细胞的生长。为了深入了解信号转导过程，我们鉴定了pii样信号转导蛋白DarA为c-二- amp结合蛋白。因此，我们希望研究枯草芽孢杆菌中c-二磷酸腺苷酸环化酶CdaA对c-二磷酸腺苷酸合成的控制，这似乎是c-二磷酸腺苷酸环化酶发挥其基本功能的原因。在这种情况下，我们将研究CdaA，其调节蛋白CdaR和必需磷酸氨基葡萄糖变化酶GlmM之间相互作用的分子细节。在GC含量低的革兰氏阳性菌中，这三种蛋白都编码在一个保守的操纵子中。在第二步中，我们希望揭示DarA的功能。为此，我们将与其相互作用伙伴共同纯化DarA，并通过质谱法对其进行鉴定。众所周知，PII蛋白可以与多种其他蛋白相互作用以控制其活性，我们认为DarA可能也是如此。最后，本课题的主要问题将是阐明c-di-AMP的必要性的原因。基于我们之前的结果，我们认为c-di-AMP是细胞壁生物合成必需蛋白活性所必需的。我们已经开发出实验策略来鉴定这种蛋白质。我们相信，这个结合分子遗传学和蛋白质组学的实验项目将为这种独特的第二信使的功能提供重要的新见解。

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.