Analysis of LOV-FPs-mediated stress response in E. coli.

Flavin-binding fluorescent proteins (FPs) are genetically encoded in vivo reporters, which are derived from microbial and plant LOV photoreceptors. In this study, we comparatively analyzed the ROS-induced stress responses of the two variants DsFbFP M49I and Pp2FbFP, exhibiting preferential photosensitization of superoxide and singlet oxygen, respectively, in E.coli. To analyze differences in global oxidative stress response in E. coli that were induced by Pp2FbFP and DsFbFP M49I, a DNA microarray-based analysis was conducted. E. coli cells expressing the respective photosensitizers were cultivated in LB media. LOV-FP expression was induced by addition of 0.4 mM IPTG during the logarithmic growth phase (~OD580 = 0.7). Three hours after induction, cultures were divided into two test cultures with a volume of 25 ml in 500 ml Erlenmeyer flaks and an OD580 of 0.5. For light treatment one of these cultures was placed between two blue-light emitting LED-panels, whereas the second culture was kept in the dark. Detailed transcriptome analyses of light-induced stress-response based on independent biological triplicates were finally carried out under best illumination conditions (i.e. Pp2FbFP: 5 min, DsFbFP M49I: 15 min).