Interaction of the plasmid-encoded quinolone resistance protein QnrA with Escherichia coli topoisomerase IV

Abstract

Purified QnrA blocked ciprofloxacin inhibition of topoisomerase IV, just as QnrA was previously found to prevent quinolone inhibition of DNA gyrase. With a gel displacement assay, tagged QnrA was shown to bind to topoisomerase IV and its subunits in a reaction that did not depend on the presence of DNA, quinolone, or ATP.

FIG. 1.

Tag-100-QnrA-His₆ fusion protein protects topo IV from quinolone inhibition of decatenation. Reactions of 40 μl were analyzed by agarose gel electrophoresis. Reaction mixtures contained 0.5 μg catenated kinetoplast DNA (lanes 1 to 5), 3.5 nM topo IV (lanes 1 to 5), 18 μM ciprofloxacin (lanes 3 to 5), and purified QnrA at 0.4 μM (lane 3), 0.2 μM (lane 4), or 0.1 μM (lane 5).

FIG. 2.

Interaction of QnrA and ParE subunit does not depend on supercoiled plasmid DNA or ciprofloxacin. Reactions of 80 μl (40 μl per gel) were analyzed by native gel electrophoresis (A) and by Western blotting with Tag-100 antibody (QIAGEN) (B). Reaction mixtures contained 2 μM ParE subunit, 0.4 μM QnrA, 4.3 nM supercoiled pBR322 plasmid DNA, 18 μM ciprofloxacin, and 1 mM ADPNP where indicated in the figure. Prestained broad-range molecular weight markers (Bio-Rad) were run in lane 1 to allow comparison of the two gels and not for molecular sizing.

FIG. 3.

Interaction of QnrA and DNA ParE subunit does not depend on relaxed plasmid DNA, ciprofloxacin, and ADPNP. Reactions of 80 μl (40 μl per gel) were analyzed by native gel electrophoresis (A) and by Western blotting with Tag-100 antibody (B). Reaction mixtures contained 2 μM ParE subunit, 4.3 nM supercoiled pBR322 plasmid DNA, 18 μM ciprofloxacin, and 1 mM ADPNP where indicated in the figure. Prestained broad-range molecular weight markers were run in lane 1 to allow comparison of the two gels and not for molecular sizing.

FIG. 4.

Interaction of QnrA and topo IV and its subunits. Reactions of 80 μl (40 μl per gel) were analyzed by native gel electrophoresis (A) and by Western blotting with Tag-100 antibody (B). Reaction mixtures contained 3 μM ParE (lanes 3, 4, 6, and 7), 4 μM ParC (lanes 2, 4, 5, and 7), and 0.4 μM QnrA (lanes 5 to 8). Prestained broad-range molecular weight markers were run in lane 1 to allow comparison of the two gels and not for molecular sizing.