Fig. 3.

Recovery from starvation is slow and dependent on carbon source quality. (A) Starvation did not reduce viable cell counts in either MG1655 or BW25113 cells, suggesting that all cells recovered from shrinkage and were able to regrow when exposed to fresh nutrient. The data points are mean ± SD with n = 3 replicates. (B) Propidium iodide staining was similarly low (n > 100 cells, two-tailed Student’s t test) in log-phase and suddenly starved cells as compared with cells killed and fixed with ethanol. (C) Cells starved in M9 salts for 4 h were spotted onto an agarose pad with M9 glucose and MitoTracker green and monitored using time-lapse microscopy. The cytoplasm began to re-expand almost immediately after cells were placed on the agarose pad. Initially, cells exhibited bright MitoTracker green foci (white arrows), suggesting membrane crumpling, and the foci appeared to dissolve as the cytoplasm re-expanded. (D) MitoTracker green signal was more uniformly distributed in log-phase cells compared with suddenly starved cells (two-tailed Student’s t test), consistent with the observation of inner membrane puncta in suddenly starved cells. (E) Typical single-cell traces of cytoplasmic area expansion after suddenly starved cells were exposed to fresh nutrient. The expansion rate was faster during the recovery phase when the inner membrane was shrunk away from the cell wall but slowed down after the inner membrane rejoined the cell wall. Individual round dots are raw data, solid lines are best fits to exponential curves, and squares are the best fit for the transition time from fast to slow expansion. Inset: area expansion rate during recovery was faster than postrecovery growth (two-tailed Student’s t test). (F) The recovery rate after starvation was dependent on the quality of carbon sources in the recovery media. Recovery was slower on carbon sources that support slower steady-state growth rates. Addition of the nonmetabolizable glucose analog 3-O-methyl-glucose did not stimulate recovery. n > 100 cells for each condition. P values are from two-tailed Student’s t tests. (G and H) In the presence of various antibiotics, suddenly starved cells still recovered when exposed to fresh glucose. The recovery (G) and growth (H) rates after starvation were slower (P < 10⁻⁴ in all conditions, Student’s t test, n > 100 cells) compared to the antibiotic-free control. In D and F–H, colored circles represent data from individual cells. The white circles are median values across all cells in that condition, and error bars are 1.5 times the interquartile range.