Fluoroquinolones inhibit preferentially Streptococcus pneumoniae DNA topoisomerase IV than DNA gyrase native proteins

The genes encoding the subunits of DNA topoisomerase IV (parC and parE) and DNA gyrase (gyrA and gyrB) of Streptococcus pneumoniae were cloned and ove rproduced in Escherichia coli by using the T7promoter-T7 RNA polymerase sys tem. The four subunits were separately purified to near homogeneity by colu mn chromatography. Protein purification was achieved by DEAE-sepharose, hep arin-agarose, and hydroxylapatite chromatography. DNA topoisomerase IV was reconstituted when ParC and ParE were combined at a 3.8-fold excess of ParE . The reconstituted topoisomerase IV showed to generate efficient ATP-depen dent DNA decatenation activity. The DNA gyrase ATP-dependent supercoiling a ctivity was reconstituted by mixing equimolar amounts of the two gyrase sub units, The inhibitory effects of four representative fluoroquinolones on th e DNA decatenation activity of topoisomerase IV and DNA supercoiling of gyr ase have been examined and compared. All four compounds were more active in inhibiting topoisomerase IV than gyrase, Moreover, there was a positive co rrelation between the inhibitory activity against topoisomerase IV decatena tion and DNA gyrase supercoiling. The classification of the four fluoroquin olones, considering their inhibitory activities in decatenation, supercoili ng and growth was the following: clinafloxacin > trovafloxacin > sparfloxac in > ciprofloxacin. These results suggest these drugs primarily target topo isomerase IV of S. pneumoniae, and gyrase secondarily, in agreement with ge netic data.