## 3 Results
### The Gene nlpC Is Strongly Regulated but Neglectable for Maintenance of Cell Morphology  
As stated, the riboswitch-controlled _nlpC_ encodes for one of four putative NlpC/P60-type cell wall peptidases in _C. glutamicum_, and the whole operon seems to be strictly regulated by several regulatory systems, i.e., MtrAB, GlxR and EsrISR [39; 41; 42]. This multi-level regulation suggests an important role of the _nlpC_-operon for _C. glutamicum_. To investigate the effects of the different regulatory elements, a reporter plasmid was constructed as follows. As schematically depicted in Figure 4a, it comprises the _nlpC_ promoter region, including the first 30 bp of the _nlpC_ gene fused to a _C. glutamicum_ codon-optimized version of the reporter gene _ecFbFP_, designated as _cgFbFP_. This gene codes for a flavin-mononucleotide-based fluorescent protein [62], the codon-optimized sequence of _cgFbPF_ is listed in Table S2. The reporter plasmid was introduced into the _C. glutamicum_ wild type, as well as the mutant strains _\(\Delta\)mtrAB_[47], _OasrSR_[48] (designated as cgtSR7) and _\(\Delta\)pdeA_. The strains were then grown alongside a wild-type control strain in CGXII medium with 1% glucose and fluorescence was measured in the mid-exponential growth phase. Relative fluorescence for all strains is depicted in Figure 4b. Background fluorescence in the wild-type control was slightly above 2000 RFU, whereas the strain carrying the promoter reporter exhibited more than 6000 RFU. This result proved that the 10 amino-acid-NlpC'-FbFP fusion protein can be used as fluorescence reporter in _C. glutamicum_ (most likely also under oxygen-independent conditions). In comparison to the wild-type reporter strain, the _\(\Delta\)mtrAB_ reporter strain showed slightly increased fluorescence of 8000 RFU, indicating that the absence of the repressor MtrA led to a somewhat higher reporter gene transcription. In contrast, the absence of EsrR had no effect on reporter expression. Both results are in accordance with literature reports, where it has been shown that MtrAB is partially activated during growth in the CGXII minimal medium, probably due to its relatively high osmolarity [47]. On the other hand, EsrISR is only active during cell wall stress, e.g., induced by bactracin treatment [42]. Furthermore, a rather strong repression of the reporter in the _\(\Delta\)pdeA_ reporter strain due to its elevated c-di-AMP level was observed. Altogether, the experiments confirm the multi-level regulation of the _nlpC_-operon, whereas the results in Figure 3 suggest that the riboswitch here also only affects the first gene, _nlpC_, due to its assumed translational regulation mechanism.  
As the deletion of DhhP-type PDEs, as well as _nlpC_ genes, have been reported to also affect cell morphology [21; 40; 68], this was also considered for _C. glutamicum_. To further analyze the relevance of NlpC for cell morphology, a _\(\Delta\)nlpC_ mutant was constructed, and this strain, as well as _C. glutamicum_ wild type and _\(\Delta\)pdeA_, were analyzed via fluorescence microscopy. For the better discrimination of single cells and division septa, cell membranes were stained with nile red. Representative pictures of the three bacterial strains are shown in Figure 4c. As can be seen, neither the presumably low translation rate of _nlpC_ in the _\(\Delta\)pdeA_ mutant nor the complete absence of _nlpC_ led to visible morphological changes when compared to the wild type. Thus, the _nlpC_ gene itself, although it represents the leading gene of a strictly regulated operon, seems to be dispensable for _C. glutamicum_ at least under the given laboratory conditions.