## 3 Results
### C-di-AMP Regulates nlpC via a c-di-AMP Riboswitch  
One important regulatory feature of c-di-AMP in other bacteria is the control of gene expression via c-di-AMP-sensitive riboswitches. In an in silico analysis, [PERSON] et al. [26] predicted two putative candidates of these riboswitches in the genome of _C. glutamicum_, one upstream of the resuscitation-promoting factor gene _rpf1_ and another upstream of the cell wall peptidase gene _cg2402/nlpC_. As a putative target of c-di-AMP mediated regulation, we selected the riboswitch in front of _nlpC_ (RS_nlpC_) for further analysis. The riboswitch sequence was cloned together with the reporter gene _mCherry_ under the control of the IPTG-inducible P_lac_ promoter into the expression vector pXMJ19. A control plasmid with IPTG-inducible _mCherry_ expression without riboswitch sequence was also constructed (see Figure 3a). To analyze the influence of the riboswitch in the presence or absence of c-di-AMP in the c-di-AMP-negative host _E. coli_, two plasmids were constructed based on pACYC184 harboring either a full-length or a truncated _disA_ gene (_disA_', obtained by Pstl digestion and subsequent religion, resulting in the elimination of 414 bp covering parts of the DAC domain and the linker motif). _E. coli_ DH5\(\alpha\) was co-transformed with the _mCherry_ reporter plasmids together with the _disA_ plasmids. The resulting strains thus contained either a functional or disrupted _disA_ gene in combination with either a riboswitch-controlled or independent _mCherry_ gene. The four strains were grown in 2 xTY medium containing 1% arabinose for the induction of _disA/disA_' expression and after 7 h _mCherry_ expression was induced by addition of 1 mM IPTG. After another 15 h, mCherry fluorescence was determined. As shown in Figure 3b, mCherry fluorescence was independent on the functionality of the DisA protein for the control plasmid pXMJ19_mCherry_. In contrast, mCherry fluorescence differed strongly between the strains harboring the functional and disrupted _disA_ in case of the reporter plasmid comprising RS_nlpC_. Here, a strong reduction in fluorescence was observed for the strain expressing the functional _disA_. This implies that the combination of functional _disA_- and RS_nlpC_-mediated control leads to reduced gene expression, verifying that RS_nlpC_ is indeed a c-di-AMP-responsive 'OFF'-riboswitch. To evaluate the functionality of the riboswitch in the native host, we introduced the pXMJ19-based reporter plasmids into _C. glutamicum_ wild type and the _DpdeA_ mutant harboring elevated c-di-AMP levels because of its inability of c-di-AMP degradation (compare Figure 2d-f). As shown in Figure 3c, without riboswitch, reporter fluorescence did not differ between the wild-type-like and the _DpdeA_ reporter strains, whereas in the presence of RS_nlpC_, fluorescence was lower in the _DpdeA_ reporter strain compared to the strain with functioning PDE. This, on the one hand, verifies the functionality of the riboswitch in _C. glutamicum_ and, on the other hand, confirms that the _DpdeA_ strain indeed harbors elevated c-di-AMP levels. Furthermore, mutations, as described earlier for the _ydaO_ riboswitch [26], led to the loss of c-di-AMP responsiveness of RS_nlpC_ (Figure S2). To get insights into the mode of action of the riboswitch, we isolated total RNA from the reporter strains and determined relative _mCherry_ transcript levels via RT-qPCR. As shown in Figure 3d, the transcript levels did not differ between the wild-type-like and mutant reporter strains, indicating that the regulation by RS_nlpC_ most likely takes place at the level of translation. To further evaluate this finding, we introduced a second reporter gene, _mVenus_, together with a ribosomal binding site (RBS) downstream of the _mCherry_ in the riboswitch-containing reporter plasmid (Figure 3e). The plasmid was again introduced into the _C. glutamicum_ wild type, and the _DpdeA_ and fluorescence of both reporter proteins was determined.