Figures and data
![](https://prod--epp.elifesciences.org/iiif/2/99735%2Fv1%2Fcontent%2F598332v1_fig1.tif/full/max/0/default.jpg)
Crp/cAMP Regulation of Persister Cell Formation in the Stationary Phase.
E. coli K-12 MG1655 WT and mutant cells at early (t=5h) and late (t=24h) stationary phases were transferred to fresh medium with antibiotics for persister cell quantification. At time points 0, 2, 6, 8, and 20h, 1 mL of the treated culture underwent two washes with 1X phosphate-buffered saline (PBS) to remove antibiotics. It was then serially diluted and plated on an agar plate to count the colony-forming units (CFUs). (a) Persister levels of ampicillin-treated culture with an antibiotic concentration of 200 μg/mL. (b) Persister levels of ofloxacin-treated culture with an antibiotic concentration of 5 μg/mL. (c) Persister levels of gentamicin-treated culture with an antibiotic concentration of 50 μg/mL. The number of biological replicates is n=4 for all panels. Biphasic kill curves were generated using a non-linear model (see Materials and Methods). Statistical significance tests were conducted using F-statistics (*P < 0.05, **P < 0.01, ****P < 0.0001). The data for each time point represent the mean value ± standard deviation.
![](https://prod--epp.elifesciences.org/iiif/2/99735%2Fv1%2Fcontent%2F598332v1_fig2.tif/full/max/0/default.jpg)
The effect of Crp/cAMP on Persister Cell Metabolism during Stationary Phase.
(a) MS analysis of E. coli K-12 MG1655 WT, Δcrp, and ΔcyaA at early (t=5h) and late (t=24h) stationary phases. Unsupervised hierarchical clustering was applied to standardized metabolic data. Each column represents a biological replicate. n=4. (b) Pathway enrichment analysis was conducted using MetaboAnalyst47. Upregulated and downregulated pathways of the Δcrp strain compared to WT in the late-stationary growth phase were provided in this figure (refer to Supplementary Fig. 5, 6, and 7 for the other pairwise comparisons). (c, d) Pathway enrichment maps comparing metabolites of the TCA cycle, pentose phosphate metabolism, glycolysis, gluconeogenesis, and pyruvate metabolism in Δcrp versus WT for ESP and LSP conditions, respectively. Circle size corresponds to the ratio of normalized metabolite intensities between mutant and control cells. Blue (P ≤ 0.05 for dark blue; 0.05 < P < 0.10 for light blue) and red (P ≤ 0.05 for dark red; 0.05 < P < 0.10 for light red) indicate significantly downregulated or upregulated metabolites in the mutant compared to the control. White signifies no significant difference. n=4 for all panels. ESP: Early stationary phase, LSP: Late stationary phase.
![](https://prod--epp.elifesciences.org/iiif/2/99735%2Fv1%2Fcontent%2F598332v1_fig3.tif/full/max/0/default.jpg)
Validation of Crp/cAMP-Mediated Metabolic State in Persister Cells Through Proteomics Analysis.
Pathway enrichment analysis was conducted in STRING 48,49 for upregulated (panel a) and downregulated (panel b) proteins. Genes highlighted in red are linked with the upregulated protein networks, while genes in blue, gray, and purple correspond to those in the downregulated protein network. The visual network in STRING illustrates protein interactions. In evidence mode, color in the network represents the interaction evidence of data support, derived from curated databases, experimental data, gene neighborhood, gene fusions, co-occurrence, co-expression, protein homology, and text mining48,49.
![](https://prod--epp.elifesciences.org/iiif/2/99735%2Fv1%2Fcontent%2F598332v1_fig4.tif/full/max/0/default.jpg)
The Role of Crp/cAMP in Non-Growing Cell Formation.
(a, b) Flow cytometry histograms depict mCherry expression in E. coli K-12 MG1655 WT, Δcrp, and ΔcyaA at early (t=5h) and late (t=24h) stationary phases, respectively. Cells containing an IPTG-inducible mCherry expression system were cultivated with IPTG. After washing and dilution of early and late stationary phase cells in IPTG-free fresh media, fluorescence was tracked in non-growing and growing cells for 2.5 hours. The panel is a representative biological replicate. Consistent results were seen across all 3 biological replicates. (c) Growth curves of WT, Δcrp, and ΔcyaA cultures were determined using flow cytometry to calculate lag and doubling times. Lag times were calculated using the "Microbial lag phase duration calculator" 79. Doubling times were computed using the formula td=Δt/(3.3xLog10(N/No)). n=3. *Statistical significance observed between control and mutant strains (P < 0.05, 2-tailed t-test). The data for each time point represent the mean value ± standard deviation.
![](https://prod--epp.elifesciences.org/iiif/2/99735%2Fv1%2Fcontent%2F598332v1_fig5.tif/full/max/0/default.jpg)
Crp/cAMP-Mediated Metabolic State of Persister Cells.
(a) GFP reporter plasmid introduced into E. coli K-12 MG1655 WT, Δcrp, and ΔcyaA cells to monitor SQR gene activity. Flow cytometry was used to detect activity at early (t=5h) and late (t=24h) stationary phases. The panel on the left represents a biological replicate, and the results are consistent across all 3 replicates, as demonstrated in the panel on the right. Statistical significance observed between control and mutant groups (*P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed t-test). (b) Redox activities of E. coli K-12 MG1655 WT, Δcrp, and ΔcyaA cells were measured at early (t=5h) and late (t=24h) stationary phases by flow cytometry using a RSG dye. This dye fluoresces green after reduction by bacterial reductases. A representative biological replicate is shown (left), with consistent results across all 5 replicates (right). Statistical significance observed between control and mutant groups (*P < 0.05, **P < 0.01, 2-tailed t-test). (c) E. coli cells with integrated mCherry expression system used to validate cellular respiration. Cells were diluted into fresh media and treated with ampicillin (200 μg/mL) for 20 hours. Flow cytometry measured the red fluorescence of intact surviving cells. A representative biological replicate is shown, with consistent results across all 3 replicates. (d) RSG levels of cells (carrying the mCherry expression system) at exponential phase (t=3h); cells before ampicillin treatment; non-lysed (intact) cells after 20 hours of ampicillin treatment; and untreated cells after 20 hours of culturing. A representative biological replicate is shown (left), with consistent results across all 4 replicates (right). Statistical significance observed between intact antibiotic-treated cells and others (*P < 0.05, **P < 0.01, 2-tailed t-test). (e) High throughput screening of mutants from Keio collection. The mutant strains selected are associated with central metabolism. Stationary phase cells were diluted 100-fold in fresh medium and treated with ampicillin (200 μg/mL) or ofloxacin (5 μg/mL) for 20 hours. Treated cultures were washed, serially diluted, and plated on agar plates to quantify CFUs. (f) Genes related to the TCA cycle, ETC, ATP synthase, glycolysis, and pentose phosphate pathway (PPP) were knocked out and then treated with ampicillin (200 μg/mL) or ofloxacin (5 μg/mL) to enumerate CFUs. n=4. Biphasic kill curves were generated using a non-linear model. Statistical significance tests were conducted using F-statistics (*P < 0.05, and **P < 0.01). Each data point represents the mean value ± standard deviation.