## 3 Discussion  
This study revealed the conserved functions of CrtR orthologs in six actinobacteria with respect to GGPP-dependent regulation. The repression of carotenogenesis, and of the expression of a _crtE_ transcriptional fusion, in _C. glutamicum_ in vivo was highest for closely related species, and it decreased with phylogenetic distance. The _P_CrtE_gfpuv_ transcriptional fusion was suitable for monitoring intracellular GGPP concentrations in a strain with chromosomally encoded _crtR_ as a genetically encoded biosensor system. The conserved role of CrtR orthologs as GGPP-dependent transcriptional regulators suggested that they are relevant for the control of carotenogenesis and/or _mmpL_ genes. Although not tested, it is tempting to speculate that these proteins may be used to monitor intracellular GGPP concentrations  
Figure 7: GGPP concentration and GFPuv fluorescence during growth of GGPP accumulating _C. glutamicum_ strain GGPPB (pEPR1_P_CrtE). Intracellular GGPP concentration (blue triangles; in mM) and GFPuv signal (green circles; mean fluorescence intensities normalized to autofluorescence) were monitored during growth in CGXII (100 mM Gluc + 100 uM IPTG). Biomass concentrations are given in gCDW/L (empty squares). in their native hosts. _C. glutamicum_ and _C. callunae_ are close relatives, synthesizing the C50 carotenoid decaprenoxanthin and its glycosides as pigments [25; 27]. _P. nicotinovorans_ is pigmented most probably due to the accumulation of carotenoids [35]. _M. luteus_ is a yellow-pigmented bacterium due to the accumulation of the C50 carotenoid sarcinaxanthin and its glycosides [53]. By contrast, _A. jensenii_ does not synthesize a carotenoid, but the polyene pigment granadaene [54], while _P. chlorophenolicus_ is a non-pigmented soil bacterium [55]. Interestingly, relatives of _P. chlorophenolicus_, such as _Arthobacter arilaitensis_, are pigmented most probably due to the accumulation of C50 carotenoids, and are found on the surface of smear-ripened cheeses [55; 56]. Thus, carotenoids is not conserved in actinobacteria possessing CrtR orthologs that control the expression of their own gene and/or the divergently oriented gene(s) in a GGPP-dependent manner. Besides carotenogenesis genes, _mmpL_ genes are also transcribed divergently to _crtR_, and are presumably controlled by CrtR. MmpL transporters are considered candidate targets for the development of anti-tuberculosis drugs [57], as they couple lipid synthesis and the export of bulky, hydrophobic substrates [58]. Thus, MmpL proteins are essential for the cell envelope, and support the infectivity and persistence of _M. tuberculosis_ in its host [59]. Moreover, MmpL proteins of _M. tuberculosis_ are involved in the oxidative stress response [60]. MarR-type transcriptional regulators, including CrtR and its orthologs, are found in all bacteria, and are natural sensors that allow adaptation to environmental stresses such as ROS, toxic compounds or antibiotics (hence the name multiple antibiotic resistance regulators) [61]. As shown for the CrtR orthologs studied here (see Figures 1 and 2), MarR-type regulators typically repress genes in the close vicinity [34]. MarR-type regulators typically bind and respond to low-molecular-weight compounds [34; 62], such as GGPP for the CrtR orthologs. _C. glutamicum_ possesses nine MarR-type regulators, eight of which have been characterized in some detail. RosR [63], CosR [64], OhsR [65] and OsmC [66] play roles in the response to ROS stress, whereas CarR [67], MalR [68] and PhdR [69] deal with other environmental stresses, such as toxic compounds or cell-membrane associated stress. The GGPP-dependent control of carotenogenesis by CrtR from _C. glutamicum_ and its orthologs studied here may be considered a stress response, since the antioxidative properties of carotenoids counteract oxidative stress. This function would be in line with the CrtR-mediated control of _mmpL_ genes that are involved in the oxidative stress response (see above).