## 4 Discussion
Its promoter region comprises binding sites for the master regulator GlxR, as well as the response regulators MtrA and EsrR [39; 41; 42; 65] and the c-di-AMP riboswitch. GlxR is a cyclic AMP-dependent master regulator in _C. glutamicum_, which likely controls 14% of its annotated genes [76; 77]. The two-component system MtrAB has been shown to repress _nlpC_ and other cell-wall-related genes while activating the expression of genes encoding transporters for compatible solutes under hyperosmotic conditions [39; 47; 78]. Consequently, the three component system EsrISR also represses _nlpC_ but during cell envelope stress, e.g., bactircian treatment [42]. Whereas these transcriptional regulators most likely affect the whole operon, the supposed translational regulation via RS_nlpC_ indicates a modulatory fine-tuning role of c-di-AMP with regard to cell envelope biogenesis in _C. glutamicum_. In conclusion, we were able to identify c-di-AMP as a second messenger in _C. glutamicum_. The synthesis of the molecule is achieved by the diadenylate cyclase DisA under the consumption of two molecules' ATP while degradation to AMP is catalyzed by the soluble DhhP-type hydrolase PdeA. Furthermore, we could show that c-di-AMP is involved in the regulation of cell envelope homeostasis by interacting with a presumed translational riboswitch in front of the cell wall hydrolase gene _nlpC_. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/microorganisms11020296/s1](https://www.mdpi.com/article/10.3390/microorganisms11020296/s1)]([https://www.mdpi.com/article/10.3390/microorganisms11020296/s1](https://www.mdpi.com/article/10.3390/microorganisms11020296/s1)), Figure S1: PdeA-Strep-mediated degradation of a surplus of pApA in presence of c-di-AMP; Figure S2: Mutations in conserved regions of the riboswitch; Table S1: Oligonucleotides; Table S2: Synthesized Fragment. Supplementary references: [79; 80]  
Conceptualization, S.J.R. and O.G. ; methodology, S.J.R. and O.G. ; validation, O.G., T.L. and B.J.E. ; formal analysis, S.J.R. ; investigation, S.J.R., T.L., O.G., D.W. and T.W. ; resources, B.J.E. ; data curation, S.J.R. ; writing--original draft preparation, S.J.R. ; writing--review and editing, S.J.R., O.G and B.J.E. ; visualization, S.J.R. ; supervision, O.G. and B.J.E. ; project administration, B.J.E. All authors have read and agreed to the published version of the manuscript. This research was partially funded by the German Research Foundation (DFG) within the scope of the SPP 1879 (Nucleotide Second Messenger Signaling in Bacteria), grant number 314826179. T.L. received funding by the German Academic Exchange Service (DAAD). The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All data presented in this study are available in the main body or supplementary files of the manuscript. Parts of this work have been published in the doctoral thesis of [PERSON]. [81]. The authors would like to thank all former colleagues for their contributions in the realization of this study. The authors declare no conflict of interest.