## 4 Discussion
subtilis_ has been shown to act via premature transcription termination [26], a c-di-AMP riboswitch in _Streptomyces coelicolor_ has been reported to lack a canonical terminator structure downstream of the riboswitch and that the regulation is also Rho-independent [28]. Our reporter experiments with the _C. glutamicum_ riboswitch RS_nlpC_ (which also lacks a canonical terminator sequence) indicated that the presence of c-di-AMP represses the synthesis of reporter fluorophores most likely on a translational level. In particular, in the RS_nlpC_ reporter strains, c-di-AMP did not affect reporter transcript levels and only affected the reporter fluorescence of the first gene of an artificial operon. However, [PERSON] et al. [28] found that a _Streptomyces_ strain lacking the riboswitch sequence exhibited increased transcript levels of the regulated gene. In our study, we observed a similar effect for the reporter transcript levels when comparing the construct with riboswitch against the one without at equal c-di-AMP levels (Figure 3d). It became clear that presence of the riboswitch sequence led to a decrease in transcript levels regardless of the c-di-AMP level, suggesting a c-di-AMP-independent transcription attenuation just by the presence of the riboswitch. In an additional study, [PERSON] and [PERSON] [71] reported the observation of terminated transcripts in the presence of c-di-AMP for the _S. coelicolor_ riboswitch in vitro using an _E. coli_ RNA polymerase. They further hypothesized that in presence of the _S. coelicolor_ RNA polymerase, this effect might be even more pronounced. Our results, in contrast, suggest that for _C. glutamicum_ at least, transcription might not be affected by c-di-AMP, at least under the tested conditions, and that RS_nlpC_ likely acts on translation. On the molecular level, it is thinkable that the CTCC sequence contained in RS_nlpC_ might be responsible for this process. This sequence is complementary to the GGAG region present in both the native ribosomal binding site (RBS) of _nlpC_, as well as the RBS in front of _mCherry_ on the reporter plasmid. In theory, the absence of c-di-AMP would lead to the formation of a stem loop, where the CTCC pairs with a GGAG present roughly 20 bases upstream. When c-di-AMP would bind to the riboswitch, this theoretical stem could be relaxed and the exposed CTCC sequence then could block translation by formation of a pseudoknot by pairing with the GGAG present in the RBS. Future investigations to reveal the exact mechanism should involve RNA biochemistry experiments, as well as the crystallization studies of the riboswitch in the presence and absence of c-di-AMP, as is already shown for the transcriptional c-di-AMP riboswitches of Firmicutes [26; 72; 73]. The _C. glutamicum_ gene regulated by the riboswitch, _cg2402/nlpC_, is part of an operon of seven genes, which comprises a second _nlpC_-type gene, and other genes encoding for a glycosyltransferase, a glucokinase, two acyltransferases and a small putative protein [65]. In addition to the two _nlpC_ genes in this operon, _C. glutamicum_ possesses two other genes belonging to the class of NlpC/P60 proteins. These enzymes are involved in peptidoglycan hydrolysis for cell wall remodeling [38]. However, [PERSON] et al. [40] could show that deletion of the _cg2402/nlpC_ homolog in _C. glutamicum_ R, cgR_2070, does not affect cell morphology, which is in line with our findings for the _nlpC_ mutant. In contrast, a defect in cell separation was observed in a mutant lacking another NlpC-like peptidase-encoding gene, cgR_1596 (also described for _M. smegmatis_[74], and _C. glutamicum_ MB001 [75]). Additionally, when cgR_2070 was deleted in this strain, severe defects were observed [40]. It is, therefore, conceivable that in the _C. glutamicum_ ATCC 13032 \(\Delta\)_nlpC_ mutant, NlpC function would be compensated by the cgR_1596 homolog encoded by _cg1735_, which also suggests a redundant role for _nlpC_. However, the whole _nlpC_-operon appears to be strongly controlled by several mechanisms.