Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction

Abstract

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

FIG. 1.

Ionizing radiation induces sulA promoter activity in E. coli. (A) Representative micrograph of E. coli RecA-GFP after ionizing radiation challenge. RecA-GFP is shown in green, and the cell membrane stained with FM4-64 is shown in red. White bar, 2 μm. (B) E. coli cells bearing the sulApΩgfp-mut2 promoter were used to monitor sulA promoter activity in response to DNA damage in single cells using fluorescence microscopy. The percentages of cells with sulApΩgfp-mut2 fluorescence are shown for untreated cells (n = 1,021) and cells after 50 Gy (n = 1,329) or 100 Gy (n = 1,420) of ionizing radiation. Error bars indicate ± the 95% confidence interval (variance) between at least three independent experiments as follows: untreated, 1.2 ± 0.14; 50 Gy, 72 ± 0.94; and 100 Gy, 96 ± 0.11. (C) Killing curve of wild-type E. coli (strain MC4100) and an isogenic strain with recA deleted. The open symbols represent B. subtilis recA⁺ (recA_Bs⁺ [□]) and recA-deficient (ΔrecA_Bs [○]) strains, and the closed symbols are E. coli recA⁺ (recA_Ec⁺ [▪]) and recA-deficient (ΔrecA_Ec [•]) strains. The error bars represent the standard errors from at least three independent experiments.

FIG. 2.

Visualization of RecA-GFP foci, SOS induction, and PBSX expression after induction of double-strand breaks in B. subtilis. (A to D) Representative micrographs of RecA-GFP foci in B. subtilis. (A) Untreated; (B) I-SceI expression; (C) 100 Gy of ionizing radiation (IR); (D) MMC (1 μg/ml). RecA-GFP foci are shown in green overlaid with the membrane stain FM4-64, which is shown in red. (E to G) Cells with TagC-GFP demonstrating SOS inductions. (E) I-SceI expression; (F) 100 Gy of ionizing radiation (IR), (G) MMC (1 μg/ml). TagC-GFP fluorescent cells are overlaid with the membrane (red). (H to J) Cells with XkdF-YFP representing PBSX expression. (H) I-SceI expression; (I) 100 Gy of ionizing radiation (IR); (J) MMC (1 μg/ml). XkdF-YFP images are overlaid with membrane shown in red. White bar, 2 μm. The numerical scoring of these images is presented in Table 2.

FIG. 3.

The viabilities of B. subtilis and E. coli bearing the lexA(Ind⁻) allele differ after ionizing-radiation-induced DNA damage. (A) Sensitivity to ionizing radiation of wild-type (▪), lexA(Ind⁻) (⧫), and recA::neo (ΔrecA [•]) B. subtilis strains after exposure to a 0 to 150 Gy. (B) Sensitivity to ionizing radiation of wild-type (▪), lexA(Ind⁻) (⧫), and ΔrecA::tc (ΔrecA [•]) E. coli strains. Each strain was irradiated in at least quadruplicate. Error bars indicate the standard error between samples. For both B. subtilis and E. coli the lexA(Ind⁻) strains encode a noncleavable form of LexA bearing a G92D missense mutation in B. subtilis and a G85D missense mutation in E. coli.