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

谷氨酸棒杆菌中环状单磷酸二腺苷 (c-di-AMP) 依赖性信号传导：鉴定钾依赖性控制 c-di-AMP 水平的机制并分析第二信使 c-di-AMP 的调节靶点

• 批准号: 314826179
• 负责人: Professor Dr. Bernhard Eikmanns, since 9/2019
• 金额: --
• 依托单位: Institut für Mikrobiologie und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/314826179?language=en
• 关键词: Cyclic; diadenosine; monophosphate; di; AMP

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.

在革兰氏阳性菌中，核苷酸次级信使环二AMP(c-di-AMP)参与控制细菌生理的许多方面，如DNA修复、孢子形成、细胞壁代谢和钾稳态。非致病性谷氨酸棒杆菌作为研究细菌渗透调节和密切相关病原菌细胞壁合成的模式生物，已经鉴定和鉴定了合成和降解c-二-AMP的酶。对于c-二-AMP的合成，谷氨酸双腺苷环化酶DISA与枯草芽孢杆菌DISA有很高的相似性。这种二腺苷环化酶在与DNA损伤结合的反应中被抑制，从而导致枯草杆菌对DNA损伤的反应延迟产孢量。然而，在DNA损伤剂存在下培养的谷氨酸杆菌细胞中，c-di-AMP水平没有变化，细胞内c-di-AMP水平在低外钾浓度培养时被降低。这表明，谷氨酰胺体内钾的稳态可能与c-diAMP的调节有关。此外，钾通道CglK被认为是c-di-AMP依赖调控的靶点，并与c-di-AMP结合。利用本课题组建立的c-di-AMP的遗传报告系统，我们的目标是识别和表征参与体内c-di-AMP水平的钾依赖控制的因素。此外，c-diAMP依赖控制CglK门控的潜在机制将使用遗传学、生化和电生理方法进行研究。利用亲和层析，我们已经在谷氨酸杆菌的无细胞提取物中进一步鉴定了c-di-AMP结合蛋白，例如30S核糖体蛋白S。我们将通过体外翻译研究来测试c-di-AMP结合对这些蛋白活性的影响。为了确定c-di-AMP调控的新靶点，下一代测序技术将被应用于识别在体外进化实验中产生的突变，以克服c-di-AMP水平的积累，并分析转录组对c-di-AMP浓度变化的响应。这里开发的方法还将与其他研究小组合作，研究单核细胞增多性乳杆菌和枯草杆菌中c-diAMP依赖的调节。通过这种方法，我们希望有助于对细菌中c-di-AMP依赖的信号转导的一般理解。