FIGURE 3.

Inhibition of DNA gyrase by ciprofloxacin/novobiocin and protection by QnrB1. A, QnrB1-mediated protection of gyrase (supercoiling activity) against ciprofloxacin. Lane 1, relaxed plasmid pBR322 alone; lane 2, relaxed pBR322 plus gyrase; lanes 3–7, relaxed pBR322, gyrase, and 2 μm ciprofloxacin in the presence of 20 nm QnrB1^WT (lane 4), 2 μm loop-AB double deletion (lane 5), loop B deletion (lane 6), and loop A deletion (lane 7) mutants. B, concentration-dependent inactivation of gyrase (supercoiling activity) by ciprofloxacin in the presence of QnrB1. Lane 1, relaxed plasmid pBR322; lane 2, relaxed pBR322 plus gyrase; lane 3, relaxed pBR322, gyrase, and 10 μm ciprofloxacin in the absence of QnrB1^WT; lanes 4–8, 2 (lane 4), 5 (lane 5), 10 (lane 6), 25 (lane 7), and 50 μm (lane 8) ciprofloxacin in the presence of 40 nm QnrB1^WT. C, effect of QnrB1 on ATPase inhibition and cleavage complex stabilization. Lane 1, relaxed plasmid pBR322; lane 2, relaxed pBR322 plus gyrase; lanes 3 and 4, relaxed pBR322, gyrase and 2 μm novobiocin in the absence of QnrB1^WT (lane 3) and in the presence of 200 nm QnrB1^WT (lane 4); lanes 5–8, cleavage complex stabilization assays using 4 μm (lanes 5 and 6) and 16 μm ciprofloxacin (lanes 7 and 8) in the absence of QnrB1 (lanes 5 and 7), and in the presence of 200 nm QnrB1 (lanes 6 and 8), respectively. nc, l, and sc indicate the nicked circular, linear, and supercoiled forms of pBR322, respectively.