Fig. 3: Metabolic activity of dividing and non-growing cells.

a E. coli cultures were grown for 16, 24, or 48 h, transferred to fresh medium, and analyzed after 2 h using RSG staining. These samples correspond to the 2-h time point shown in Fig. 2. Dot plots show RSG versus mCherry fluorescence, separating dividing and non-growing cells. b Normalized histograms highlight reduced metabolic activity in most non-growing cells, with partial overlap with dividing cells. Note that flow cytometry diagrams are representative of four independent biological replicates, which yielded reproducible results (data shown in Supplementary Fig. 3). c E. coli cultures grown for 16, 24, or 48 h were transferred to fresh medium and assayed for antibiotic tolerance after 2 h. Persister levels were determined based on CFU counts. Note that CFU-based assays detect culturable persisters but not VBNC cells; therefore, the 48 h population does not necessarily exhibit higher survival, as the increased non-growing fraction is largely due to VBNC cells (Supplementary Fig. 4, and Supplementary Table 2). Data are shown as mean ± standard deviation. Number of biological replicates, n = 4. All raw data, including biological replicates, are available in the data repository Figshare¹⁰⁰.