Research ArticleCELL BIOLOGY

Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cells

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Science Advances  20 Jun 2018:
Vol. 4, no. 6, eaat1608
DOI: 10.1126/sciadv.aat1608
  • Fig. 1 Visualization and quantification of DNA replication errors at a single-cell level.

    (A) MutL foci in the mutH strain examined under a microscope during the exponential phase. Each fluorescent focus represents one unrepaired DNA replication error. (B) Growth kinetics of E. coli MG1655 cells. OD600 values of 0.2 (mid-exponential), 0.8 (late exponential), and 1.4 (early stationary) were selected for further analysis. (C) Quantification of DNA replication errors of mutH cells in different growth phases. Cells in the mid-exponential phase had statistically significantly more DNA replication errors than in two other growth phases (see table S3). (D) Quantification of DNA replication errors in different strains during the mid-exponential phase. All strains were statistically significant different from the mutH strain (see table S3).

  • Fig. 2 DNA replication errors in various subpopulations.

    We coupled the MutL-based assay with the following fluorescent transcriptional promoter reporters: P1rrnB (A to C), PkatG (D to F), PrecA (G to I), and PibpA (J to L). We plotted the mean fluorescence detected per cell against the number of foci per cell (A, D, G, and J). Heterogeneity of fluorescence detected in isogenic cells (B, E, H, and K). We further examined the cells having the strongest (top 10% of the population) and the weakest (the bottom 10% of the population) fluorescence for the distribution of the number of foci per cell (C, F, I, and L). We observed a statistically significant difference between cells with ≥1 and no MutL foci among all reporters (see Table 1 and table S3). AU, arbitrary units.

  • Fig. 3 Translation error reporter and DNA replication errors in cells with different levels of translation errors.

    (A) Construct of the chromosomal translation error reporter. We introduced two point mutations in the sfgfp gene, which is under control of the Ptet promoter, creating the UAG stop codon from the lysine AAA codon. As a control, a functional mCherry gene, which is also under control of the Ptet promoter, was added. (B to D) Validation of the translation error reporter. (B) Correlation between the fluorescence signals from mCherry and sfGFP. We found a significant but weak correlation, and we examined 334 cells. (C) The fluorescence signals increased in cells with an rpsD mutation, which is known to increase translation errors, and when cells were treated with sub-minimal inhibitory concentrations of streptomycin, an antibiotic known to increase mistranslation. (D) Representative flow cytometry histogram showing GFP intensity in cells after heat shock treatment from exponentially growing culture (OD600 = 0.2) to later growth phase (OD600 = 0.6). (E) The MutL-based assay was coupled with a translation error reporter in the mutH strain and the wild-type (WT) strain. The 5% of the cells with the strongest translation error reporter fluorescence (high translation error) had statistically significant more cells with ≥1 focus than the 5% of the cells with the weakest fluorescence (low translation error) in both strains (see table S3).

  • Table 1 Mean fluorescence of different transcriptional reporters among cells with ≥1 and no MutL focus.

    We observed a statistically significant difference in the fluorescence between cells with ≥1 and no MutL focus among all reporters except for P2rrnB-YFP.

    ReporterMean fluorescence per cell ±
    SEM (AU)
    Wilcoxon
    signed-rank
    test (P)
    Cells with no
    MutL focus
    Cells with ≥1
    MutL focus
    P1rrnB-GFP79 ± 1.088 ± 1.3<0.05
    P2rrnB-YFP180 ± 1.9188 ± 2.2>0.05
    PkatG-GFP3.2 ± 0.033.4 ± 0.03<0.05
    PrecA-YFP7.3 ± 0.108.2 ± 0.20<0.05
    PibpA-GFP4.9 ± 0.055.2 ± 0.05<0.05

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaat1608/DC1

    fig. S1. MutL reporters, cell area, and number of foci per cell.

    fig. S2. DNA replication errors and transcriptional promoter reporter P2rrnB.

    fig. S3. Induction of fluorescent transcriptional promoter reporters.

    fig. S4. Determination of minimum inhibitory concentration (MIC) of aminoglycosides for the E. coli wild-type strain MG1655 and the impact of sub-MIC of aminoglycoside on DNA replication errors.

    fig. S5. DNA replication errors of different strains with sub-MIC of streptomycin.

    fig. S6. DNA replication errors and translation error reporter.

    fig. S7. DNA replication errors and translation errors in wild-type cells with excess MutS proteins.

    table S1. Bacterial strains and plasmids.

    table S2. The frequency of appearance of rifampicin mutants in mismatch repair–proficient and –deficient strain.

    table S3. Chi-square test results of MutL foci frequency in this study.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. MutL reporters, cell area, and number of foci per cell.
    • fig. S2. DNA replication errors and transcriptional promoter reporter P2rrnB.
    • fig. S3. Induction of fluorescent transcriptional promoter reporters.
    • fig. S4. Determination of minimum inhibitory concentration (MIC) of aminoglycosides for the E. coli wild-type strain MG1655 and the impact of sub-MIC of aminoglycoside on DNA replication errors.
    • fig. S5. DNA replication errors of different strains with sub-MIC of streptomycin.
    • fig. S6. DNA replication errors and translation error reporter.
    • fig. S7. DNA replication errors and translation errors in wild-type cells with excess MutS proteins.
    • table S1. Bacterial strains and plasmids.
    • table S2. The frequency of appearance of rifampicin mutants in mismatch repair–proficient and –deficient strain.
    • table S3. Chi-square test results of MutL foci frequency in this study.

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