Supercoiling assay

To test gyrase activity, the M. tuberculosis and E. coli DNA gyrase assay kit from Inspiralis and TopoGEN, respectively, were used. The procedure adopted to monitor DNA gyrase dependent supercoiling followed kit instructions. Reaction was performed in 30µl volume by incubating at 37°C for 45 minutes approximately 250 ng of pHOT1 Relaxed DNA together with the purified gyrase protein. Moxifloxacin or the compound GSK1066288A was added to the reaction mixtures at different concentrations. Termination, detection, and readout were carried out as described in the kit protocol using agarose gel electrophoresis. Bands were quantified using imageJ. Raw gel pictures can be found in the Supplementary.