Article Figures & Data
Figures
- FIG. 1.
MfpAMt inhibits the catalytic activity of M. tuberculosis DNA gyrase in topoisomerase assays. (A) Concentration-dependent inhibitory effect of MfpAMt on supercoiling activity of M. tuberculosis DNA gyrase. Supercoiling assays were performed using relaxed pBR322 as the substrate (TR) and final concentrations of MfpAMt (μM) as indicated. R, relaxed pBR322; S, supercoiled pBR322. (B) Concentration-dependent inhibitory effect of MfpAMt on ATP-independent relaxation of supercoiled DNA. Supercoiled pBR322 (TS) was the substrate. (C) Concentration-dependent inhibitory effect of MfpAMt on decatenation activity of M. tuberculosis DNA gyrase. Interlinked kDNA (kDNA) was used as the substrate (TI). Decatenated minicircles were visualized in three forms: relaxed (R), linearized (L), and supercoiled (S).
- FIG. 2.
The direct effect of QnrB4 on E. coli DNA gyrase (A) and M. tuberculosis gyrase (B) supercoiling is minimal and is observed only at high concentrations. Supercoiling assays were carried out with 400 ng of relaxed pBR322 as the substrate (pBR) and 1 U of E. coli DNA gyrase or 1 U of M. tuberculosis DNA gyrase with increasing concentrations of QnrB4-His6 (μM) as indicated. R, relaxed pBR322; S, supercoiled pBR322. QC, QnrB4 with pBR322 without gyrase.
- FIG. 3.
MfpAMt inhibits E. coli DNA gyrase supercoiling activity with an IC50 of 3 μM. Supercoiling assays were performed with 400 ng of relaxed pBR322 as the substrate (pBR) and 1 U of E. coli DNA gyrase with increasing concentrations of MfpAMt (μM) as indicated. R, Relaxed pBR322; S, supercoiled pBR322.
- FIG. 4.
The MfpAMt effect on DNA gyrase catalysis is abolished with M. tuberculosis DNA gyrase harboring the D87G but not the A83S substitution of GyrA. (A) Supercoiling assays were performed with wild-type M. tuberculosis gyrase and relaxed pBR322 as a substrate. R, relaxed pBR322; S, supercoiled pBR322. Reactions were carried out without MfpAMt (lane 0) and with increasing concentrations of MfpAMt (final concentrations in μM are indicated above the gel). MfpAMt inhibited gyrase supercoiling in a concentration-dependent manner. (B) Same experiment with an altered M. tuberculosis DNA gyrase composed of GyrA A83S and wild-type GyrB subunit, which efficiently supercoiled pBR322 (lane 0). MfpAMt inhibited altered gyrase supercoiling in a concentration-dependent manner, as for the wild-type gyrase. (C) Same experiment with an altered M. tuberculosis DNA gyrase composed of GyrA D87G and the wild-type GyrB subunit, which efficiently supercoiled pBR322 (lane 0). No modification of this supercoiling activity in the presence of MfpAMt (from 0.5 to 5 μM) was observed.
- FIG. 5.
MfpAMt does not modify ciprofloxacin inhibition of M. tuberculosis gyrase. (A) Supercoiling assays were performed with relaxed pBR322 as a substrate (pBR). In the absence of fluoroquinolone, M. tuberculosis DNA gyrase efficiently supercoiled pBR322 (SC). In the presence of 50 μM ciprofloxacin, supercoiling activity was inhibited (lane 0). Increasing concentrations of MfpAMt (final concentrations in μM are indicated above the gel) were added to reaction mixtures containing M. tuberculosis DNA gyrase and ciprofloxacin. (B) Determination of the ciprofloxacin IC50 in the absence and presence of MfpAMt. The IC50 was measured by comparing the intensities of the bands corresponding to supercoiled pBR322.
- FIG. 6.
QnrB4 has a protective effect on fluoroquinolone inhibition of E. coli DNA gyrase supercoiling activity but not toward M. tuberculosis gyrase. Supercoiling assays were performed with 400 ng of relaxed pBR322 (pBR) as the substrate and 1 U of E. coli DNA gyrase or 1 U of M. tuberculosis DNA gyrase in the presence of increasing concentrations (μM) of QnrB4. R, relaxed pBR322; S, supercoiled pBR322. (A) In the presence of 6 μM ciprofloxacin, supercoiling activity was inhibited (lane 0); pBR322 remained in a relaxed form (R). When QnrB4 was added (final concentrations in μM are indicated above the gel), the intensity of the band corresponding to the supercoiled form of pBR322 (S) increased (lanes 0.1 to 5), showing that the inhibition of E. coli DNA gyrase supercoiling activity by ciprofloxacin was diminished. (B) Same experiment with QnrB4, M. tuberculosis gyrase, and ciprofloxacin at 50 μM. When QnrB4 was added, the topoisomeres produced by gyrase inhibition by ciprofloxacin remained and no supercoiling form (result of gyrase protection) was observed, even at high concentrations (5 μM) of QnrB4.
- FIG. 7.
MfpAMt does not protect against E. coli gyrase inhibition by quinolones even in the presence of KGlu. (A) Comparison of inhibition by ciprofloxacin of pBR322 (pBR) supercoiling by E. coli gyrase in the presence of QnrB4 or MfpAMt at the same concentration (0.5 μM). R, relaxed pBR322; S, supercoiled pBR322. (B) Similar experiments with 100 mM KGlu in the assay buffer.
- FIG. 8.
Comparison of sequences of four PRPs shows major differences in the region including residue 115, which was demonstrated previously to be involved in gyrase protection for quinolones. An alignment of MfpA (29), MfpAMt (Rv 3361c), QnrB4 (AAZ04784), and VPA0085 is shown. Conserved residues in the four sequences are in black, and those in three sequences are in light gray. C115 is indicated.
Tables
- TABLE 1.
Effects of MfpAMt and QnrB4 on DNA gyrases
Type of effect Type II topoisomerase Effect of: MfpAMt QnrB4 Direct effect on catalysis M. tuberculosis DNA gyrase Inhibition of supercoiling activity (IC50, 2 μM) No effect until 20 μM E. coli DNA gyrase Inhibition of supercoiling activity (IC50, 3 μM) No effect until 30 μM Protection from fluoroquinolone inhibition M. tuberculosis DNA gyrase No protection, no modification of ciprofloxacin or moxifloxacin IC50 No protection, no modification of ciprofloxacin IC50 E. coli DNA gyrase No protection, no modification of ciprofloxacin IC50 Concentration-dependent protective effect, increase of ciprofloxacin and moxifloxacin IC50s in the presence of QnrB4
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Fig. S1, similarity (67%) between MfpAMt from M. tuberculosis and MfpA from M. smegmatis.
Zipped MS Word document, 869K.
- Supplemental file 1
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Fig. S1, similarity (67%) between MfpAMt from M. tuberculosis and MfpA from M. smegmatis.
