Cyclic diadenosine-monophosphate (c-di-AMP) dependent signaling in Corynebacterium glutamicum: Identification of mechanisms for potassium-dependent control of c-di-AMP levels and analysis of regulatory targets of the secondary messenger c-di-AMP

在革兰氏阳性菌中，核苷酸次级信使环二磷酸腺苷（c-二磷酸腺苷）参与控制细菌生理的许多不同方面，如DNA修复、孢子形成、细胞壁代谢和钾稳态。在非致病性谷氨棒状杆菌中，作为研究细菌渗透调节和密切相关的致病物种细胞壁合成的模式生物，合成和降解c-二磷酸腺苷的酶已经被鉴定和表征。c-二腺苷酸环化酶与枯草芽孢杆菌的二腺苷酸环化酶DisA具有高度的相似性。这种二腺苷酸环化酶在与DNA损伤结合的反应中被抑制，因此在DNA受损的情况下导致枯草芽孢杆菌延迟产孢。然而，在DNA损伤剂存在的情况下，c-二磷酸腺苷水平没有变化，细胞内c-二磷酸腺苷水平在低外部钾浓度的培养下被证明是降低的。这表明谷氨酸谷氨酸钾稳态可能与c-二磷酸腺苷的调控有关。此外，钾通道CglK被确定为c-di-AMP依赖控制的靶标，也被证明与c-di-AMP结合。利用我们小组建立的c-di-AMP遗传报告系统，我们在这里旨在确定和表征与体内c-di-AMP水平钾依赖性控制有关的因素。此外，c-二磷酸腺苷依赖控制CglK门控的潜在机制将通过遗传、生化和电生理方法进行研究。通过亲和层析，我们已经在谷氨酸丙氨酸的无细胞提取物中发现了更多的c-二磷酸腺苷结合蛋白，如30S核糖体蛋白s。c-二磷酸腺苷结合对这些蛋白活性的影响将被测试，例如通过体外翻译研究。为了确定c-di-AMP控制的新靶点，下一代测序技术将应用于鉴定体外进化实验中产生的突变，以克服高c-di-AMP水平的积累，并分析转录组对c-di-AMP浓度改变的响应变化。这里开发的方法也将用于与进一步的小组合作，以研究c-二磷酸腺苷在单核增生乳杆菌和枯草芽孢杆菌中的依赖调节。通过这种方式，我们希望有助于对细菌中c-二磷酸腺苷依赖的信号传导的一般理解。

The nucleotide secondary messenger cyclic di-AMP (c-di-AMP) is involved in the control of many diverse aspects of bacterial physiology in Gram-positive bacteria, such as DNA repair, sporulation, cell wall metabolism, and potassium homeostasis. In the non-pathogenic Corynebacterium glutamicum, which serves as a model organism to study osmoregulation in bacteria and cell wall synthesis in closely related pathogenic species, enzymes for synthesis and degradation of c-di-AMP have been identified and characterized. For c-di-AMP synthesis C. glutamicum possess only the di-adenylate cyclase DisA, which shares high similarities with DisA of Bacillus subtilis. This di-adenylate cyclase was shown to be inhibited in response to binding to DNA lesions and thus to be responsible for delayed sporulation of B. subtilis in response to presence of damaged DNA. However no alterations of c-di-AMP levels were detected in C. glutamicum cells cultivated in presence of DNA damaging agents, intracellular c-di-AMP levels were shown to be reduced in cultivations with low external potassium concentrations. This indicates that in C. glutamicum potassium homeostasis might be interconnected with c-di-AMP regulation. Moreover, the potassium channel CglK was identified as a target of c-di-AMP dependent control and also shown to bind c-di-AMP. Using a genetic reporter system for c-di-AMP, which is established in our group, we here aim to identify and characterize factors involved in the potassium dependent control of internal c-di-AMP levels. Furthermore the underlying mechanisms for the c-di-AMP dependent control of CglK gating will be studied using genetic, biochemical, and electrophysiological approaches. Using affinity chromatography, we already identified further c-di-AMP binding proteins in cell free extracts of C. glutamicum such as the 30S ribosomal protein S. Effects of c-di-AMP binding on the activity of these proteins will be tested e.g. using in vitro translation studies. To identify new targets of c-di-AMP control, next-generation sequencing techniques will be applied to identify mutations generated in vitro evolution experiments to overcome accumulation of high c-di-AMP levels and to analyze changes of the transcriptome in response to altered c-di-AMP concentrations. The here developed methods will also be used in collaboration with further groups to investigate c-di-AMP dependent regulation in L. monocytogenes and B. subtilis. By this means we hope to contribute to the general understanding of c-di-AMP dependent signaling in bacteria.