Growth‐dependent heterogeneity in the DNA damage response in Escherichia coli

Abstract In natural environments, bacteria are frequently exposed to sub‐lethal levels of DNA damage, which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria, which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub‐lethal DNA damage in E. coli. We found heterogeneous expression of the SOS regulon at the single‐cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS‐induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. The result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrate how cell division and physiology come together to produce growth‐dependent heterogeneity in the DNA damage response.

For all plots, growth conditions are: M9-glycerol (blue), M9-glucose (green), and M9-glucose+amino-acids (red). Solid lines represent the average and shaded area the standard error from three biological repeats. Inset: A magnification of the second peak at high SOS expression.
A Steady-state distribution of GFP intensity from cell auto-fluorescence in different growth conditions. B Steady-state distribution of GFP intensity from SOS reporter PsulA-mGFP for cells unable to induce SOS (SOS-off, lexA3 background) in different growth conditions. For all plots, growth conditions are: M9-glycerol (blue), M9-glucose (green), and M9-glucose+amino-acids (red). Points (dots WT strain, 6-points-stars SOS-off, lexA3 background, 4-point-stars, autofluorescence) represent the average and bars the standard error from at least three biological repeats.
A Average GFP intensity from SOS reporter PsulA-mGFP for the bottom 99% of the population in different growth conditions. Inset: average GFP intensity in different growth conditions where SOS is de-repressed. B Average GFP intensity from SOS reporter PsulA-mGFP for the top 1% of the population in different growth conditions. Inset: average GFP intensity in different growth conditions where SOS is de-repressed. C Average mKate2 intensity from constitutive reporter PtetO-mKate2 for the bottom 99% of the population in different growth conditions. D Average mKate2 intensity from constitutive reporter PtetO-mKate2 for the top 1% of the population in different growth conditions.

Molecular Systems Biology
Sebasti 2 an Jaramillo-Riveri et al For all plots, growth conditions are: M9-glycerol (blue), M9-glucose (green), and M9-glucose+amino-acids (red). Points represent the average and bars the standard error from at least three replicates done in different days.
A Average GFP intensity from SOS reporter PsulA-mGFP for the whole population under replication-dependent DSBs as a function of growth rate. B Average GFP intensity from SOS reporter PsulA-mGFP for the whole population under ciprofloxacin as a function of growth rate. C Average GFP intensity from SOS reporter PsulA-mGFP for the bottom 85% of the population under replication-dependent DSBs as a function of growth rate. D Average GFP intensity from SOS reporter PsulA-mGFP for the bottom 85% of the population under ciprofloxacin as a function of growth rate. E Average GFP intensity from SOS reporter PsulA-mGFP for the top 1% of the population under replication-dependent DSBs as a function of growth rate. F Average GFP intensity from SOS reporter PsulA-mGFP for the top 1% of the population under ciprofloxacin as a function of growth rate. G Average mKate2 intensity from constitutive reporter PtetO-mKate2 for the whole population under replication-dependent DSBs as a function of growth rate. H Average mKate2 intensity from constitutive reporter PtetO-mKate2 for the whole population under ciprofloxacin as a function of growth rate. For every single-cell trajectory four metrics were computed as proxies for division and elongation rate reduction and its relation to GFP levels: the maximum observed time before a division or end of the tracking (t m ), the minimum and maximum cell lengths (l 0 and l m ), and the maximum GFP intensity registered for that lineage.
A, B These metrics are illustrated for a lineage with two single-cell trajectories. C-H The maximum observed time (t m ) was plotted against the maximum GFP intensity, comparing wild-type and cells undergoing replication-dependent DNA-damage (2-pal), where extreme values of GFP intensity correlate with significant delays in cell division only for the 2-pal strain. I-N We plot a proxy for the cell elongation rate computed as log(l m − l 0 )/t m against the maximum GFP intensity, where extreme values in GFP intensity correlate with a reduction in elongation rate as compared to the rest of the population.
Data information: Throughout panels C to N, colours represent the 2D density for each individual dataset, where yellow and blue denote high and low density, respectively. The vertical dashed line denotes the cutoff used throughout to discriminate high levels of SOS induction. Panels C-H contains data from 4,340, 1,016, 7,393, 2,572, 5,978, and 9,880 cell cycles, respectively.