Article Figures & Data
Figures
- FIG. 1.
Structure of SD8. Two functional groups, the aminocoumarin and the glycone, are shown. The calculated molecular mass of SD8 is 932.2169, whereas the molecular masses, determined by mass spectrometry, of the original and current preparations of SD8 are 932.2134 and 932.2139, respectively. Me, methyl.
- FIG. 2.
SD8 is a potent inhibitor of S. aureus gyrase. The supercoiling reaction mixtures containing the relaxed DNA, either 50 fmol of S. aureus (Sa) gyrase (A) or 10 fmol of E. coli (Ec) gyrase (B), and the indicated concentrations of either SD8 or ciprofloxacin (Cipro) were incubated, and the DNA products were analyzed as described in Materials and Methods. Lanes 1 and 2 contain controls with gyrase and drug present (+) or absent (−). The arrows in panels A and B show that gyrase was present in lanes 3 to 13.
- FIG. 3.
The presence of KGlu affects the apparent SD8 sensitivity of S. aureus gyrase in the DNA cleavage assay. The two-step DNA cleavage assay was performed as described in Materials and Methods. (A) For E. coli (Ec) gyrase, 50 fmol of E. coli gyrase, the supercoiled DNA, and the indicated concentrations of SD8 were first incubated, and then 10 μM of ciprofloxacin (Cipro) was added to the reaction mixtures. (B and C) For S. aureus (Sa) gyrase, 400 fmol of S. aureus gyrase, the supercoiled DNA, and the indicated concentrations of SD8 were first incubated in the absence (B) or presence (C) of 400 mM KGlu, and then 50 μM of ciprofloxacin was added to the reaction mixtures. No EtBr was included in the TAE buffer. Lanes 1 and 2 contain controls with gyrase and drug present (+) or absent (− and 0). The arrows in panels A, B, and C show that gyrase was present in lanes 3 to 9 or 10.
- FIG. 4.
Effects of SD8 on the decatenation activity of Topo IV. The decatenation reaction mixtures containing kinetoplast DNA, 50 fmol of either S. aureus (Sa) Topo IV (A) or E. coli (Ec) Topo IV (B), and the indicated concentrations of either SD8 or ciprofloxacin (Cipro) were incubated, and the DNA products were analyzed as described in Materials and Methods. Lanes 1 and 2 contain controls with Topo IV and drug present (+) or absent (− and 0). The arrows in panels A and B show that Topo IV was present in lanes 3 to 12.
- FIG. 5.
Effect of SD8 on growth of wild-type and ΔacrB strains. Absorbance (Abs) over time at 600 nm of wild-type (A) and ΔacrB (B) cultures treated with various concentrations of SD8 recorded in a 96-well format. The error bars represent 1 standard deviation of measurements done in triplicate. The cells were treated with SD8 (D8) concentrations ranging from 10 nM to 50 μM; however, only results from 1 μM to 50 μM are depicted, as there was little difference in the growth pattern of cells treated with <1 μM SD8.
- FIG. 6.
Transcriptional profile of SD8 treatment is most similar to the profile of gyrase inhibition. For the set of genes significantly affected by SD8 treatment, the Pearson correlation coefficients between the vector obtained in SD8 treatment and each of the 198 microarray conditions were calculated and plotted in descending order.
- FIG. 7.
Surface antigen and DNA replication genes are similarly upregulated by SD8 and novobiocin. Pairwise correlations between profiles (expressed as one-dimensional vectors of log-transformed ratios of transcript abundances) of genes encoding DNA replication proteins and of genes encoding surface antigens. The reference treatments are indicated along the x axes. In Fig. 7 to 9, only the genes that were identified as significantly affected at a median false discovery rate of 5% or less are labeled.
- FIG. 8.
The pattern of SD8 activity shares limited similarity with transcriptional patterns of the norfloxacin effect. The characteristic profiles of norfloxacin treatment, the induction of the SOS response, upregulation of the TCA cycle genes, and downregulation of genes encoding components of the citrate-dependent iron transport system, were compared with those of SD8 treatment. Pairwise correlations between profiles of gene classes are shown as described in the legend to Fig. 7.
- FIG. 9.
The pattern of SD8 activity shares limited similarity with transcriptional patterns of the novobiocin effect. The transcriptional signatures of the novobiocin effect, the induction of RpoH targets, upregulation of relaxation-sensitive genes, and downregulation of supercoiling-sensitive genes, were compared with those of the SD8 treatment. Pairwise correlations between profiles of gene classes are shown as described in the legend to Fig. 7.
- FIG. 10.
Effect of SD8 on nucleoid morphology. Exponentially grown ΔacrB cells were treated with either SD8 or DMSO, the solvent of SD8, stained with DAPI, and visualized by fluorescence (A, B, E, and F) and phase-contrast (C, D, G, and H) microscopy.
Tables
- TABLE 1.
Drug sensitivities of S. aureus and E. coli topoisomerasesa
Topoisomerase IC50 (μM) of topoisomerase SD8 Ciprofloxacin S. aureus gyrase 1.45 ± 0.35 48 ± 9 E. coli gyrase 0.41 ± 0.03 2.35 ± 0.55 S. aureus Topo IV 14.5 ± 0.5 8.4 ± 0.1 E. coli Topo IV 270 ± 10 34 ± 1 ↵ a The drug sensitivities of gyrases and Topo IVs were measured in the supercoiling and decatenation assays, respectively. Each assay was repeated two or three times, and the IC50s were determined based on the amounts of either the supercoiled DNA or decatenated DNA. Representative results are shown in Fig. 2 and 4.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
NMR studies of SD8.
TIFF file, 6.5MB. - Supplemental file 2
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Effects of SD8 on Ec gyrase.
TIFF file, 6.6MB. - Supplemental file 3
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Effect of KGlu on the apparent SD8 sensitivity of Sa Topo IV in the DNA cleavage assay.
TIFF file, 5.4MB. - Supplemental file 4
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Elongation of cells caused by SD8 treatment.
TIFF file, 5.2MB. - Supplemental file 5
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Effect of SD8 on the cleavage activity of DNA gyrase in vitro.
TIFF file, 4.75MB. - Supplemental file 6
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Text description of the characterization of SD8, plus two supplemental tables.
MS Word document, 293K.
- Supplemental file 1
-
NMR studies of SD8.
