Figure 1.

The effect of iron metabolism on replication initiation is associated with oxidative stress. (A,C) DNA replication patterns of E. coli. Exponentially growing E. coli cells were cultured to OD₄₅₀ = 0.15–0.2 in ABTG-CAA medium at 37 °C and treated with rifampicin and cephalexin for 3–5 doubling times. E. coli cells were fixed with 70% ethanol and then incubated with Hoechst 33,258 fluorescent dye; 10,000 cells were analyzed for DNA replication patterns using flow cytometry. The number of chromosome equivalents contained per cell is indicated on the X–axis, and the number of cells is shown on the Y–axis. In panel (A), doubling times are labeled in boxes and indicated by D.T. In panel (C), the indicated concentrations of ATP were added at around OD₄₅₀ = 0.04 and co-incubated with the culture for about two doubling times to grow to OD₄₅₀ = 0.15–0.2. (B,D) The average number of replication origins contained per bacterial cell in the respective category in panel (A,C). The average number of replication origins per cell (A.O.) represents the sum of the products of the number of chromosomes and the percentage of related cells. Data are the average of three independent biological replicates (marked above the corresponding column plot and showing one valid digit after the decimal point), and error bars represent standard deviations. Data significance analysis was performed using a t-test (two-tailed, sample-paired method). *: 0.01 < p-value < 0.05; **: 0.001 < p-value < 0.01; ***: p-value < 0.001.