Analysis of LOV-FPs-mediated stress response in E. coli.. 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. Overall design: 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).