## 2 Results  
### CrtR Orthologs from Actinobacteria Showed Binding to Their Own Promoters and Derepression by GGPP  
Previously, the MarR-type regulator CrtR was identified as a GGPP-dependent repressor of the carotenogenic gene cluster _crtE-mmpl-crtBJYofEb_, and was shown to auto-regulate its own expression [23]. Moreover, 94 CrtR homologs with at least a 25% amino acid identity with CrtR from _C. glutamicum_ were identified [23]. In order to study if the function of these MarR-type regulators as GGPP-dependent transcriptional repressors of carotenogenesis is conserved, the orthologs of five actinobacteria, with increasing phylogenetic distances between themselves and _C. glutamicum_, were selected for further analysis. The _crtR_ genes from _C. callunae_, _A. jensenii_ and _P. nicotinovorans_, as well as _C. glutamicum_, have in common that they are co-localized with genes of carotenoid biosynthesis [23] (Figure 1). The phylogenetically more distant _crtR_ genes from _P. chlorophenolicus_ and _M. luteus_ co-localize only with a _mmpL_ gene. The genome of _P. chlorophenolicus_ lacks carotenogenesis genes except _crtR_, whereas the carotenogenic genes of _M. luteus_ are encoded in loci distant from _crtR_ (Figure 1). The CrtR orthologs from _C. callunae_ (CrtR\({}_{Cc}\)), _A. jensenii_ (CrtR\({}_{Aj}\)), _P. nicotinovorans_ (CrtR\({}_{Pn}\)), _P. chlorophenolicus_ (CrtR\({}_{Pc}\)) and _M. luteus_ (CrtR\({}_{Ml}\)) were fused with an N-terminal His-tag, and the proteins were purified by Ni-NTA affinity chromatography. In order to test if _crtR_ autoregulation--as observed for CrtR from _C. glutamicum_--is conserved, each protein was tested for binding to the intergenic DNA sequences between its own gene and the divergently transcribed gene (Figure 2). Indeed, all CrtR orthologs analyzed bound to the DNA sequences upstream of their own _crtR_ gene (Figure 2). This indicated _crtR_ autoregulation and/or regulation of the respective divergently transcribed genes (_mmpl_ for _C. callunae_, _M. luteus_ and _P. chlorophenolicus_, _crtE_ for _A. jensenii_ and _idi_ for _P. nicotinovorans_) (Figure 1). The finding that _A. jensenii_ CrtR, _P. nicotinovorans_ CrtR and _M. luteus_ CrtR did not shift all target DNA may either indicate that the CrtR protein-target DNA interaction is less tight in these bacteria, or it may be due to technical reasons, e.g., due to the purification of the tagged proteins that may differ between the five CrtR proteins analyzed. Since GGPP inhibits the binding of CrtR from _C. glutamicum_ to its target promoter [23], it was tested if GGPP could also inhibit the binding of the other CrtR orthologs to their respective target DNA. Indeed, GGPP inhibited the interaction between the tested CrtR proteins and their target DNA (Figure 2A). Thus, these in vitro results revealed that the binding of CrtR orthologs, from several actinobacteria, to their own upstream DNA sequences, and the inhibitory effect of GGPP, are conserved. For _C. callunae_, a second putative target DNA sequence was tested, namely the intergenic region between its carotenogenic gene cluster _crtEBIY\({}_{\textit{eff}}\)Eb_, which is located a few genes upstream of _crtR_ in this bacterium, and the divergently transcribed gene _epi_ (Figure 1). This intergenic DNA sequence from _C. callunae_ was bound by a CrtR protein from _C. callunae_ (CrtR\({}_{Cc}\)) unless GGPP was added (Figure 2B). Thus, the GGPP-dependent regulation of carotenoid biosynthesis genes by CrtR is conserved at least in the closely related _C. callunae_, and possibly in other actinobacteria. Figure 1: Genomic organization of _crtR_ from _C. glutamicum_ ATCC 13,032 and the _crtR_ orthologs from _C. callunae_ DSM 20,147, _A. jensenii_ DSM 20,535, _P. nicotinovorans_ Hce-1, _M. luteus_ NCTC 2665 and _P. chlorophenolicus_ A6. Boxed areas highlight the putative promoter regions tested in bandshift assays. The _crtR_ orthologs (given in red) are transcribed divergently either to _crt_ genes (given in yellow) or to _mmpl_ genes. _P.