## 4 Discussion  
In our study, we identified and partly characterized the DAC DisA and the DhhP-type PDE PdeA of _C. glutamicum_ as enzymes that are involved in c-di-AMP metabolism. In particular, we verified c-di-AMP synthesis from ATP by DisA and showed degradation of c-di-AMP and its linear derivate pAp by PdeA. Several arguments support our hypothesis that those two enzymes are solely responsible for c-di-AMP homeostasis in _C. glutamicum_. As all hitherto characterized DACs contain the DisA/DAC domain [16], the presence of other DACs besides DisA is highly unlikely in _C. glutamicum_. Furthermore, we were not able to delete _disA_ using established procedures, indicating a putative essential role for the cell, at least under the tested conditions. It is thinkable that this apparent essentiality could be circumvented, as described for _B. subtilis_. Here, a c-di-AMP-free strain lacking all three DAC-encoding genes could only be obtained at low potassium concentrations as control of this ion is one important role of c-di-AMP in this organism [4]. However, this approach still has to be evaluated for _C. glutamicum_. Although we were not able to detect c-di-AMP in the _C. glutamicum_ wild-type strain, some c-di-AMP accumulation was observed for the _\(\Delta\)pdeA_ mutant, verifying DAC activity of DisA in vivo. Regarding PDEs, we showed that the _pdeA_ deletion mutant is not capable of c-di-AMP degradation anymore. Thus, although PdeA belongs to the class of DhhP-type PDEs, which have been described to be mainly responsible for pApA degradation, a two-step mechanism with two different enzymes for c-di-AMP degradation [69] is probably unlikely for _C. glutamicum_. In _S. aureus_, c-di-AMP is first linearized to pApA by the membrane-bound PDE GdpP, which is not capable of further pApA cleavage but is then hydrolyzed to AMP by the DhhP-type Pde2 [69]. The authors further suggested that pApA is additionally actively involved in feedback inhibition of the c-di-AMP hydrolyzing enzyme GdpP. However, recombinantly produced _C. glutamicum_ PdeA-Strep degraded  
Figure 4: The gene _nlpC_ encodes a strongly regulated, non-essential putative cell wall peptidase. (**a**) Schematic depiction of the reporter plasmid comprising the _nlpC_ 5′-UTR and the reporter gene _cgFbFP_ fused to the first 30 bp of _cg2404/nlpC_; (**b**) The promoter reporter plasmid was introduced into different _C. glutamicum_ strains. Strains were grown in CGXII minimal medium and relative reporter fluorescence was determined after 6 h during exponential growth; the wild type without plasmid served as background control. N = 3, shown as mean and standard deviation; (**c**) Fluorescence microscopy pictures of nile-red-stained _C. glutamicum_ WT, _ApdeA_ and _nlpC_. both pApA and c-di-AMP, whereas pApA was cleaved faster in all conditions tested, similar to Pde2 of _S. aureus_ or _Streptococcus pneumoniae_ or TmPDE from _T. martiima_[23; 69]. This suggests that the linear intermediate might always be immediately cleaved to AMP in _C. glutamicum_, which would imply that pApA does not fulfill a regulatory function in this organism. Thus, we conclude that c-di-AMP metabolism is most likely mediated solely by DisA and PdeA in _C. glutamicum_. However, it remains to be elucidated how c-di-AMP pools are controlled and adjusted. Additionally, nothing is known about the regulation of _disA_ and _pdeA_ expression; in addition, _disA_ was reported to be located in an operon together with the SOS-response-related gene _radA_[65]. However, an in-depth analysis of the SOS response in _C. glutamicum_ only revealed _radA_ to be affected by the LexA regulator [70]. As one of the possible targets of c-di-AMP-mediated regulation in _C. glutamicum_, we characterized a putative _ydaO_-type riboswitch located upstream of the cell wall peptidase gene _cg2402/nlpC_. Whereas the prototype c-di-AMP riboswitch from _B.