Proteomic Study of the Survival and Resuscitation Mechanisms of Filamentous Persisters in an Evolved Escherichia coli Population from Cyclic Ampicillin Treatment

Microscopy of persisters from E. coli K-12 and Evo3A during resuscitation after ampicillin treatment. (a) Cells exposed to ampicillin (∼15× MIC) for 5 h were resuspended in fresh LB medium and observed by epifluorescence microscopy at indicated time points during resuscitation. Green cells are live cells while red cells are dead cells (defined as those with compromised membrane). (b) Time-lapse microscopy of Evo3A during resuscitation on LB pads. Figures are representative phase-contrast images of recovering ampicillin-treated Evo3A cells on LB agar pads, and the panel at t = 180 min shows the incipient colony formation from a filament. Red arrow shows a lysed filament, and blue arrows indicate cell division events that are slower than the other cells. Bars, 50 μm.

Time-series analysis of the protein expression profile on Evo3A during antibiotic treatment. (a) Venn diagram for proteome comparison of Evo3A before and after 1 h, 3 h, and 5 h of ampicillin treatment (125 μg/ml). (b) Volcano plots of the protein expression profile on Evo3A from 1 h to 5 h of ampicillin treatment. Fold change values of the proteins after 1 h, 3 h, and 5 h of treatment are fitted to a linear regression model, and the slopes of the fitted curve were shown with the respective P value for each protein. Slopes with a P value below 0.05 or an absolute log₂(P value) above 4.32 are considered significant. Proteins with increasing expression profiles are marked by the blue dots, while proteins with decreasing expression profiles are marked by the red dots. (c) Protein-protein interaction network of the proteins with increasing and decreasing expression profiles throughout the antibiotic treatment as predicted by STRING v10.5. The lines represent protein interaction (thicker lines mean higher confidence), and the dots in different colors represent different biological processes involved. Nodes with black outlines are proteins with increasing expression profile, and nodes with no outlines are proteins with decreasing expression profile. Nodes without function enrichment are hidden from the network. (d) Linear model fitting of the 18 proteins with increasing expression profile during antibiotic treatment from 3 biological replicates of Evo3A. Red lines represent the fitted linear models with the 95% confidence bounds shown as red dotted curves.

Proteome comparison of Evo3A after 5 h of ampicillin treatment to that before treatment. (a) Venn diagram for proteome comparison of Evo3A before and after 5 h of ampicillin treatment (125 μg/ml). (b) Volcano plot for Evo3A after 5 h of ampicillin treatment compared with Evo3A before ampicillin treatment. Red dots are the downregulated proteins, and blue dots are the upregulated proteins. (c) Pathway enrichment study (KEGG) by DAVID of the differentially expressed proteins and newly detected proteins on Evo3A after 5 h of ampicillin treatment compared to those before treatment. Fold enrichment is defined as the ratio of proportion of the input information to the background information. (d) Functional classification of the differentially expressed and newly detected proteins on Evo3A after 5 h of ampicillin treatment compared to those before treatment by gene ontology (GO) analysis using DAVID, classified by biological process. (e) Total ROS generation in cultures treated with ampicillin (125 μg/ml) or 50 μM FeSO₄ and 10 mM H₂O₂ for 1 h was assayed using H₂DCFDA probe and normalized by CFU counts (mean ± SEM, n = 3). (f) Survival of cultures exposed to ampicillin (125 μg/ml) for 5 h in the presence or absence of 5% DMSO (an OH^· scavenger). Aliquots were washed and plated to determine survival (mean ± SEM, n = 3). Significance of difference: ***, P < 0.001; **, P < 0.01; ns, not significant (two-tailed t test with unequal variances of the log-transformed values). (g) Frequency of mutagenesis was assayed in cells previously exposed to ampicillin for 1 h. Cultures were washed, grown overnight, and plated on LB agar plates supplemented with rifampin (100 μg/ml). Changes in the frequency of generation of rifampin-resistant mutants were used as an indicator for DNA damage (mean ± SEM, n = 3). Significance of difference: ***, P < 0.001; *, P < 0.05; ns, not significant (two-tailed t test with unequal variances).

Separation of filaments from the new progeny after 3 h of resuspension in fresh medium. (a) Workflow for separation of filaments from the new progeny. A mid-exponential culture of Evo3A was treated with ampicillin (∼15× MIC) for 5 h, washed to remove antibiotic, and resuspended in fresh LB medium. After 3 h of resuspension, there was a mixture of filaments and newly divided cells. The mixture was filtered with a PVDF membrane (5-μm pore size, 25-mm diameter) to separate the filaments (residue) and the normal-sized new progeny (filtrate). (b) Epifluorescence microscopy of the cells before and after filtration. The top row shows filtration using one filter for 3 ml culture, and the bottom row shows filtration using 6 filters for 3 ml culture (one filter per 500 μl). (c and d) Venn diagrams for proteome comparison of the enriched filaments to those before resuspension (c) and the enriched new progeny to the enriched filaments (d). (e and f) Volcano plots of the enriched filaments compared to those before resuspension (e) and the enriched new progeny compared to the enriched filaments (f). Differentially expressed proteins are defined as those with Benjamini-Hochberg-corrected P values below 0.1, and fold change higher or lower than ±1.5, corresponding to the colored dots. Red dots are the downregulated proteins, and blue dots are the upregulated proteins. (g and h) Functional classification of the differentially expressed proteins identified in the enriched filaments compared to those before resuspension (g) and the enriched new progeny compared to the enriched filaments (h) by gene ontology (GO) analysis using DAVID, classified by biological process. Dot size is proportional to the y axis. (i) ATP level of Evo3A after 5 h of ampicillin treatment, Evo3A after 3 h of resuscitation, and WT after 3 h of resuscitation, compared to those before treatment, measured using the BacTiter-Glo assay. To calculate the abundance on a per-cell basis, the measured values were normalized by CFU counts by serially diluting and plating washed aliquots. Data are presented relative to the untreated cells (marked by the horizontal dashed line). Data represent three biological replicates, and each data point is denoted as mean ± SEM.

Importance of ElaB in WT E. coli. (a) Survival of wild-type E. coli BW25113 (WT) and ΔelaB cells after treatment with ampicillin (125 μg/ml) at the indicated time points (mean ± SEM, n = 3). Significance of difference with ancestral population: ***, P < 0.001; **, P < 0.01 (two-tailed t test with unequal variances of the log-transformed values). (b) Survival upon expression of elaB via pCA24N-elaB in the ΔelaB cells was determined with ampicillin (125 μg/ml) treatment for 3 h (mean ± SEM, n = 3). *, P < 0.05; ns, not significant (two-tailed t test with unequal variances of the log-transformed values). Overnight cultures at 1:1,000 were grown in fresh LB until the turbidity at 600 nm reached 0.3, and 1 mM IPTG was added for 2 h. The cells were then treated with ampicillin (125 μg/ml) for 3 h, and survival was measured. Empty plasmid pCA24N was used as a negative control. (c) Colony morphologies for WT and ΔelaB cells after 3 h of resuscitation. After treatment with ampicillin (125 μg/ml) for 3 h, cells were washed to remove antibiotics and resuspended in fresh LB for 3 h. After 3 h of growth, aliquots of both WT and ΔelaB strains were serially diluted and plated on LB agar (37°C, 20 h of incubation time). Images shown are representative images from 3 biological replicates.