Fig. 2: Copy number diversification after in vitro evolution.

a USA300 csa1::tetK was grown over three consecutive days in six parallel cultures in the presence or absence of ciprofloxacin (CIP). Each day, the copy number of up to 16 clones of each culture showing high Tc resistance was screened by qPCR. Upper and lower box limits and the horizontal lines within the boxes represent 25 and 75% percentiles and the medians, respectively. The whiskers of the plots indicate minimum and maximum range. All data points are shown. Data are derived from six independent experiments and represent: Day 1, n = 96 and n = 73 of “no Cip” and “1 µg/ml Cip”, respectively; Day 2, n = 93 and n = 96 of “no Cip” and “1 µg/ml Cip”, respectively; Day 3, n = 93 and n = 96 of “no Cip” and “1 µg/ml Cip”, respectively. Datasets were not normal distributed (D’Agostino & Pearson omnibus test <0.0001) and statistical analysis was performed using two-tailed Mann–Whitney test. Source data are provided as a Source Data file. b Growth curves of USA300 csa1::tetK in TSB containing various concentrations of the antibiotic ciprofloxacin (CIP), which is known to enhance RecA expression. As expected, at increasing inhibitory concentrations of CIP, the growth rate of USA300 csa1::tetK decreased as assessed by measuring OD₆₀₀ every 2 h. This was accompanied by an increase in RecA protein levels as assessed in mid-exponential cells by Western blot analysis using LI-COR infrared technology. A representative blot is shown wherein the lower and upper bands represent RecA and an unspecific signal, respectively. Mean and SEM of three independent experiments are shown. Source data are provided as a Source Data file. c Frequency of amplification (Tc₂₀-resistant clones showing at least a 2 fold increase in csa1 copy number compared to the parental strain by qPCR) within the total population of living cells. Shown are the results of six independent experiments. x indicates a culture in which amplification was not detected. Statistical analysis was performed using two-tailed Mann–Whitney test. Source data are provided as a Source Data file. d A USA300 csa1::tetK high copy number variant (harboring ~50 copies of csa1) was grown over two consecutive days in three parallel cultures in the presence or absence of CIP. Each day, the copy number of the strains of 17 randomly chosen clones was screened by qPCR (n = 51). Shown is a violin plot, length of the box indicates minimum and maximum range. Width of the bar indicates accumulation of data points. Horizontal lines within the boxes represent 25 and 75% percentiles and the medians, respectively. Statistical analysis was performed using two-tailed Mann–Whitney test. Source data are provided as a Source Data file.