E. Fernandez-moreira et al., Fluoroquinolones inhibit preferentially Streptococcus pneumoniae DNA topoisomerase IV than DNA gyrase native proteins, MICROB DR R, 6(4), 2000, pp. 259-267
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.