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

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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.