UV-B-Induced DNA Repair Mechanisms and Their Effects on Mutagenesis and Culturability in Escherichia coli

Abstract

Ultraviolet-B (UV-B) radiation, intensified by ozone depletion, induces DNA damage and promotes mutagenesis, shaping evolution. While UV-induced SOS responses are well characterized in bacteria, the cellular consequences of prolonged UV-B exposure remain less clear. Prolonged UV-B exposure disrupts translation and RecA-mediated SOS induction without major changes in membrane permeability or reactive oxygen species. This impairs mutagenesis and induces a reversible loss of culturability. Genetic analysis reveals both redundant and differential roles for DNA repair pathways: homologous recombination (RecA, RecB), nucleotide excision repair (UvrA), and translesion synthesis (UmuC/D) are essential for maintaining mutagenesis and culturability, while others (RecN, RmuC) have limited impact. Notably, deletion of UvrD (a repair-associated helicase) intensifies transient non-culturability without affecting mutagenesis, underscoring complexity in repair networks. Overall, our findings reveal a dose-dependent trade-off: moderate UV-B promotes mutagenesis with minimal viability loss, whereas prolonged exposure suppresses mutagenesis via transient dormancy, reflecting an adaptive strategy with significant evolutionary implications.

Fig. 1:. Excessive UV exposure resulted in transient loss of culturability and decreased mutagenesis.

(a) The graph shows the relationship between UV-B exposure time (min) and calculated energy dosage (J/m²), based on measured irradiance (see Materials and Methods). (b) Exponential-phase E. coli MG1655 WT cells were exposed to UV-B light for 0, 2, 4, 8, 16, 24, and 32 minutes, followed by a 24-h recovery period. At specific time points during recovery (t = 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 24 h), cells were collected and plated to determine colony-forming units (CFU). (c) Levels of UV-induced rifampicin (RIF) resistance mutations were measured by counting RIF-resistant colonies (per 10⁸ cells) in the WT cultures after recovery for the indicated UV exposure times. (d) CFU counts and (e) mutation frequency of E. coli MG1655 following UV-B exposure were determined under dark conditions. The same experimental setup described above was used, but cultures were recovered in the absence of light to eliminate photoreactivation. (f) The temporal CFU profiles of E. coli MG1655 ΔrecA cells were monitored during recovery following UV treatment. n=4. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test, where *P < 0.05, **P < 0.001, ***P < 0.01, ****P < 0.0001. Data corresponding to each time point represent mean value ± standard deviation.

Fig. 2:. UV-induced upregulation of SOS genes and its impact on mutagenesis and cell culturability.

(a) A library of E. coli MG1655 strains with promoter reporters for SOS response genes was UV-treated during the mid-exponential phase and allowed to recover for 24 h. GFP levels were measured after recovery and normalized to untreated controls. WT cells without any promoter reporters were used as background controls. (b) Genes upregulated following UV exposure were individually or combinatorially deleted in E. coli MG1655, and a mutagenesis assay was performed. Mid-exponential phase cells were exposed to UV for 0, 16, and 32 min and recovered for 24 h. At specified time points during recovery (t = 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 24 h), cells were collected and plated to determine CFU. Note that only the 0 and 0.25 h time points are shown here; the full-time course is provided in Supplementary Fig. S3. (c) RIF-resistant cells were quantified by plating samples on RIF-agar plates after 24 h of recovery, with results reported as RIF-resistant colony counts per 10⁸ cells for different UV exposure durations. n=4. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test, where *P < 0.05, **P < 0.001, ***P < 0.01, ****P < 0.0001. Data corresponding to each time point represents mean value ± standard deviation.

Fig. 3:. Knockout library screening to elucidate their effects on mutagenesis and cell culturability.

(a) The E. coli BW25113 Keio knockout collection was screened following 16 min of UV treatment. RIF-resistant cells were quantified by spotting 10 μL samples from each well onto RIF-agar plates after a 24-hour recovery period. ΔlacI was used as the reference control (see Materials and Methods). (b) Selected genes were deleted in E. coli MG1655 and the mutagenesis assay was performed. Mid-exponential phase cells were exposed to UV for 0, 16 and 32 min and recovered for 24 h. At indicated time points (t = 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h and 24 h) during recovery, cells were collected and plated to enumerate CFU. Note that only the 0 and 0.25 h time points are shown here; the full-time course is provided in Supplementary Fig. S4. (c) RIF-resistant cells were quantified by plating the samples on RIF-agar plates after 24 h of recovery. The values were reported as RIF-resistant colony count per 10⁸ cell population for different UV exposure durations. n=4. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test, where *P < 0.05, **P < 0.001, ***P < 0.01. Data corresponding to each time point represents mean value ± standard deviation.

Fig. 4:. Perturbation of both SulA and TisB proteins moderately increased cell culturability during recovery.

(a-c) Exponential-phase E. coli MG1655 ΔsulA, ΔtisB, and ΔsulAΔtisB cells were exposed to UV-B light for 0, 2, 4, 8, 16, 24, and 32 minutes, followed by a 24-h recovery period. At specific time points during recovery, cells were collected and plated to determine their CFU levels. Note that only the 0 and 0.25 h time points are shown here; the full-time course is provided in Supplementary Fig. S5. (d-f) Levels of UV-induced RIF resistance mutations were measured by counting RIF-resistant colonies (per 10⁸ cells) in the cultures of three knockout strains after recovery for the indicated UV exposure times. n=4. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test, where *P < 0.05, **P < 0.001, ***P < 0.01, ****p < 0.0001. Data corresponding to each time point represents mean value ± standard deviation.

Fig. 5:. Excessive UV exposure did not compromise cell membrane integrity or increase hydrogen peroxide levels, but it did impair cellular translation.

(a) Membrane integrity of UV-treated cells (0, 16, and 32 min of UV exposure) was assessed via flow cytometry with PI staining (see Supplementary Fig. S8 for controls). FSC-H: Forward light scatter. (b) H₂O₂ concentrations were measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit. Reaction mixtures containing Amplex Red reagent, horseradish peroxidase, and UV-treated cells in sodium phosphate buffer (pH 7.4) were incubated for 30 minutes at room temperature, followed by fluorescence measurement with a microplate reader. H₂O₂ levels were calculated using a standard curve (Supplementary Fig. S9). (c) Expression of P_recA-gfp in UV-treated E. coli MG1655 cells was analyzed via flow cytometry for the indicated time points during recovery (P_recA : the recA promoter). (d) IPTG-inducible GFP expression in UV-treated E. coli MG1655 pUA66-gfp cells was also assessed by flow cytometry for the indicated time points during recovery. (e) GFP expression profiles of UV-treated cells harboring the recA promoter and (f) IPTG-inducible GFP expression systems were evaluated using mean GFP values from flow cytometry data. (g) Recovery (CFU counts) and mutation frequency of E. coli MG1655 ΔrecA carrying an IPTG-inducible recA overexpression plasmid were assessed following 16- and 32-minute UV-B exposures, shown for the indicated IPTG concentrations. n ≥ 4. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test, where *P < 0.05, **P < 0.001, ***P < 0.01, ****P < 0.0001. Data corresponding to each time point represent mean value ± standard deviation.