Figure 1.

Hibernation factors only moderately affect long-term survival, but are essential for efficient regrowth. (A, B) Long-term viability of E. coli K-12 MG1655 (WT) and a hibernation factor-deficient mutant (ΔHF) in different starvation conditions. Cells were grown in batch culture in MOPS MM until growth arrest and incubation continued for 10 days (A). Exponentially growing cells were washed and transferred to phosphate-buffered saline (PBS) and incubation continued for ten days (B). At indicated time points the number of colony-forming units per ml of culture (CFU/ml) was determined by spot-plating of serial dilutions. Data points represent mean values of at least three independent biological replicates. Error bars represent the standard error of the mean (SEM). *P< 0.05 (two-tailed Student's t-test assuming unequal variances). The maximum fold difference in CFU/ml between WT and ΔHF at t = 120 h is indicated with a bracket. (C) Heterogeneity of colonies arising from cultures after prolonged starvation in (A). At the indicated time points appropriate dilutions of WT and ΔHF cultures in MOPS MM were plated on nutrient agar (NA) plates and incubated overnight. Images shown are representative of three independent biological replicates. (D–F) Regrowth of WT and ΔHF cells following nutrient replenishment after incubation in MOPS MM for 24, 72 and 120 h (D, E and F, respectively). Cells were collected at indicated time points, diluted into fresh MOPS MM, and regrowth was followed by optical density measurements at 600 nm (OD₆₀₀). Data points represent mean values of at least four independent biological replicates. Error bars represent SEM. Lag times were calculated using the tangent method (50) on independent regrowth curves. Indicated values correspond to the mean lag times and the SEM of at least four independent biological replicates. *P< 0.05 (two-tailed Student's t-test assuming unequal variances).