Figure 3.
Changes in supercoiling levels affect the transcription of topA as measured by qRT-PCR. (A) Cultures of the R6 strain at an OD620nm of 0.4 were treated with 6 or 8 μM of SCN. (B) Cultures were grown at an OD620nm of 0.4 and diluted 100-fold in medium containing either 6 μM or 8 μM of SCN. After 120 min of growth, cells were centrifuged, washed, and suspended either in medium alone (− SCN) or medium containing 6 or 8 μM of SCN (+ SCN). (C) Correlation between fold changes in supercoiling levels and changes in the transcription of topA. The data corresponds to samples treated with either SCN or NOV at concentrations that allowed cellular growth and recovery of DNA supercoiling: 4 and 6 μM (0.25× MIC and 0.375× MIC) for SCN; 0.5 and 1 μg/ml (0.5× MIC, and 1× MIC) for NOV. In both cases, time points of 15 and 30 minutes were used. The conditions of the qRT−PCR experiments and the normalization of the data are described in the Materials and Methods. In (A) and (B), values from three independent replicates (mean ± SEM, n = 3) were normalized against values obtained at time 0 min.