## 2 Results
### CrtR from C. glutamicum Binds to Heterologous crtR Promoter DNA Sequences  
The binding of the _C. glutamicum_ CrtR protein to heterologous _crtR_ promoter DNA sequences was studied in order to (i) test if this specific DNA binding is conserved across the actinobacteria analyzed, and, if it is, to (ii) identify the putative DNA sequence motif. The intergenic DNA sequences between the five orthologous _crtR_ genes and the respective divergently transcribed genes were used in a bandshift assay with His-tagged CrtR proteins from _C. glutamicum_ (Figure 3A). The strong binding of CrtR\({}_{Cg}\) to the _crtR_ promoter sequences from _C. callunae_ and _P. nicotinovorans_ was detected, whereas the interactions between CrtR\({}_{Cg}\) and the _crtR_ promoter sequences from _A. jensenii_, _M. luteus_ and _P. chlorophenolicus_ were weak (Figure 3A). Previously, we narrowed down the target DNA sequence, to which CrtR from _C. glutamicum_ binds, to 19 bp (5'-CCCATGAGAATTATTTTT-3'), and mutational analysis revealed that exchanging the central four nucleotides (**TTAA**) simultaneously interfered with binding [23]. Inspection of the DNA sequences upstream of the _crtR_ genes from _C. callunae_, _A. jensenii_, _P. nicotinovorans_, _M. luteus_ and _P. chlorophenolicus_ revealed that this motif was present in the intergenic DNA regions in these species (Figure 3B), and conserved to some extent (Figure 3C). It is evident that conservation of the central **TTAA** sequence is not sufficient to explain the observed binding preferences of CrtR\({}_{Cg}\) and the variuos _crtR_ promoter sequences studied. It remains to be elucidated how specific nucleotides of the 15 bp motif (other than the central **TTAA**) affect the binding of CrtR\({}_{Cg}\) protein to DNA. The derived consensus DNA binding motif of CrtR proteins from the studied species is depicted in Figure 3D, with sequence conservation and relative frequency for each nucleotide position. Figure 2: In vitro characterization of CrtR orthologs. (**A**) Bandshift assays of CrtR orthologs from _C. callunae_, _A. jensenii_, _P. nicotinovorans_, _M. luteus_ and _P. chlorophenolicus_ with their respective own putative promoter region and the inhibition of the binding by GGPP. (**B**) Bandshift assays of CrtR from _C. callunae_ with a putative _crtE_ promoter region and the inhibition of the binding by GGPP. The presense or absence of CrtR protein and GGPP are indicated by “+” and “\(-\)”, respectively. Figure 3: Characterization of CrtR from _C. glutamicum_ in vitro. (**A**) Bandshift assays of His-tagged CrtR protein from _C. glutamicum_ (CrtR\({}_{\text{Cg}}\)) and the intergenic DNA sequences between the _crtR_ orthologs from _C. callunae_, _A. jensenii_, _P. nicotinovorans_, _M. luteus_ and _P. chlorophenolicus_, and the respective divergently transcribed genes. (**B**) Putative -10 and -35 promoter DNA sequences (underlined), translation start codons (italics) and the putative conserved CrtR binding sequences (boxed). The mapped transcriptional start sites of _C. glutamicum crtR_ and _crtE_ are given in bold. (**C**) Putative conserved CrtR binding sequences (conserved nucleotides are given in yellow; the **TTAA** sequence that was shown previously to be required for _C. glutamicum_ CrtR binding by mutational analysis is depicted in bold face). (**D**) The graphical representation of the derived consensus DNA binding motif of the CrtR proteins from _C. glutamicum_, _C. callunae_, _A. jensenii_, _P. nicotinovorans_, _M. luteus_ and _P. chlorophenolicus_ (designed using WebLogo). CrtR orthologs from Actinobacteria Affected Carotenogenesis and Expression of a CrtE Transcriptional Fusion in C. glutamicum In Vivo  
To monitor the promoter activity of the carotenogenic gene cluster of _C. glutamicum_ in vivo, the promoter probe vector pEPR1 was used [37]. This reporter system was employed to determine the promoter activity of the carotenogenic promoter (_PcrtE_) from _C.