FIG. 1.

Activation of the marRAB operon by aromatic acid metabolites in the superpathway of chorismate. (A) Chemical structure of metabolites selected based on their similarity to salicylate. (B) Observed transcriptional activation of the marR′-yfp promoter fusion in the presence of 5 mM concentrations of indicated inducers in wild-type (CR700) and ΔtolC mutant (CR703) backgrounds. Salicylate and IPTG served as positive and negative controls for activation, respectively. Cells were grown overnight in MOPS (morpholinepropanesulfonic acid) minimal medium (20 mM glucose, 0.2% Casamino Acids, pH 7.2) and subcultured 1:200 in fresh medium. Following dilution, 450 μl of culture was transferred to deep 96-well plates and grown at 37°C with aeration at 1,000 rpm to an OD of 0.5. At this time, 100 μl of medium containing dissolved inducer was added. Growth was continued for an additional 2 h prior to fluorescence and optical density measurements made with a Tecan Safire2 microplate reader. An expanded description of the experimental procedures is provided in the supplementary material. A.U., arbitrary units.