FIGURE 4: Overnight dormant cells did not fully account for the ampicillin persistent cells of ppGpp⁰ strain. (A, B) The ampicillin (250 μg/ml) killing curves of the exponentially growing wt, ΔrelA and ppGpp⁰ strains. (A) 5x iOD, five times more overnight cells (initial OD_600nm = 0.05) were inoculated into fresh MOPSr-Lp medium and grew to the exponential phase before being killed with ampicillin. (B) 10x iOD, ten times more overnight cells (initial OD_600nm = 0.1) were inoculated into fresh MOPSr-Lp medium and grew to the exponential phase before being killed with ampicillin. Two biological replicates of both 5x iOD and 10x iOD were performed, and the averages and SDs are shown. (C) The ampicillin (250 μg/ml) killing curves of the wt, ΔrelA and ppGpp⁰ cells directly out of the 16-hour overnight cultures (indicated by O/N). Two biological replicates were performed, and the averages and SDs are shown. (D) The ampicillin (250 μg/ml) killing curves of the wt, ΔrelA and ppGpp⁰ cells after subculturing twice in fresh MOPSr-Lp medium (indicated by 2x). After 3 hours of first subculturing in fresh MOPSr-Lp medium, cells were subcultured again from OD_600nm 0.01 for 2.5 hrs before being subjected to killing. Two biological replicates were performed, and the averages and SDs are shown. For comparison, the same killing curves of the three strains from (Figure 2B) were included throughout.