Known binding sites for CsrA were sought based upon data described by Kulkarni et al., (10 (link)). RNA transcripts corresponding to the promoter regions of acrAB (including wild-type and those harboring site directed mutations), and ramA were synthesized in vitro using the AmpliScribe™ T7-Flash™ Transcription Kit (Cambio Ltd, UK) and purified by ammonium acetate precipitation. Concentrations of transcripts were determined with the Qubit™ fluorometric quantitation system (Life Technologies, UK). The electrophoretic mobility shift assay was done using an electrophoretic mobility shift assay (EMSA) kit (E33075; Thermo Fisher, UK) according to the manufacturer's instructions. Briefly, 0.5 nM target RNA was incubated with CsrA-HisX6 protein for 20 min at room temperature in a 15 μl reaction mixture containing 1 × binding buffer (750 mM KCl, 0.5 mM dithiothreitol, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris–HCl, pH 7.4). Following addition of EMSA gel-loading solution, mixtures were separated by electrophoresis on a non-denaturing 8% (wt/vol) polyacrylamide gel in 0.5 × TBE buffer (22 mM Tris-HCl, 22 mM boric acid, 0.5 mM EDTA, pH 8.0). Gels were stained with 1 × SYBR Green EMSA nucleic acid stain. DNA was then visualized using a G:BOX F3 gel documentation system (Syngene, UK).