Germination and Outgrowth of Bacillus subtilis Spores Deficient in BER and DisA Unveil Alternative Genetic Checkpoints

Abstract

During Bacillus subtilis spore germination/outgrowth, the rehydration of the spore core and activation of aerobic metabolism can generate reactive oxygen species (ROS)-promoted DNA lesions that are repaired via the base excision repair pathway (BER). Accordingly, spores deficient in the AP-endonucleases (APEs) Nfo and ExoA exhibit a delayed outgrowth that is suppressed following disruption of the checkpoint protein DisA. Here, we report that DisA-independent DNA damage checkpoints operate during B. subtilis spore outgrowth. Consistent with this notion, spores lacking Nfo, ExoA, and Nth, which functions as an APE, did not suppress delayed outgrowth following disA disruption. Furthermore, in reference to the ∆nfo ∆exoA ∆nth spores, spores deficient for these APEs and DisA displayed a significantly higher number of oxidative genetic lesions and failed to properly segregate its chromosome during the first round of replication in the outgrowth stage. Finally, we found that DisA promotes low-fidelity repair and replication events, as revealed by DNA-alkaline gel electrophoresis (AGE) as well as spontaneous and H₂O₂-promoted Rif^R mutagenesis. Overall, our results unveil the existence of DisA-independent DNA damage checkpoint(s) that are activated by genomic lesions of an oxidative nature during spore germination and outgrowth, ensuring a proper transition to vegetative growth.

Keywords: BER; Bacillus subtilis; DisA; checkpoint; germination/outgrowth.

Figure 1

Germination/outgrowth kinetics of different strains of B. subtilis spores. Dormant spores of wt (●), nfo exoA (▲), nfo exoA nth disA (◼), nfo exoA nth (◆), and nfo exoA nth disA (◯) strains were heat activated and germinated on 2 × SG medium. Germination and outgrowth were monitored by measuring the OD₆₀₀nm of the cultures as described in Section 2. The kinetics were performed with three different spore preparations and plotted as described in Section 2.

Figure 2

Germination/outgrowth kinetics of different strains of B. subtilis spores. Dormant spores of strains (A) wt (●) and wt H₂O₂ (◆), (B) nfo exoA nth (●) and nfo exoA nth H₂O₂ (◆), (C) nfo exoA nth disA (●) and nfo exoA nth disA H₂O₂ (◆) were heat shocked and germinated by the addition of L-alanine. Fifteen minutes after germination was initiated, H₂O₂ was added to the cultures to a final concentration of 0.5 mM. Germination and outgrowth were monitored by measuring the OD600nm of the cultures as described in Section 2. The kinetics were performed with three different spore preparations and plotted as described in Section 2.

Figure 3

Germination/outgrowth kinetics of different strains of B. subtilis spores. Dormant spores of strains (A) wt (●) and wt MMS (◆), (B) nfo exoA nth (●) and nfo exoA nth MMS (◆), (C) nfo exoA nth disA (●) and nfo exoA nth disA MMS (◆) were heat shocked and germinated by the addition of L-alanine. Fifteen minutes after germination was initiated, MMS was added to the cultures to a final concentration of 2 mM. Germination and outgrowth were monitored by measuring the OD600nm of the cultures as described in Section 2. The kinetics were performed with three different spore preparations and plotted as described in Section 2.

Figure 4

Frequency of spontaneous and induced mutation during germination/outgrowth of B. subtilis spores. Spore suspensions of the indicated strains were activated by heat shock and then supplemented with L-alanine to induce germination. In (A) 180 min after initiation of gemination, the mutation frequency to Rif^r was determined as described in Section 2. In (B,C), 15 min after germination initiation, H₂O₂ and MMS damage agents were added, respectively, and subsequently, at 180 min of incubation, the Rif^r mutation frequency was determined as described in Section 2. Values represent the mean of data collected from three independent experiments, and error bars represent the standard deviation. Letters above bars indicate statistical differences found by a Mann–Whitney U test (p < 0.001).

Figure 5

Determination of oxidative DNA lesions by alkaline gel electrophoresis (AGE). (A) Genomic DNA samples (3 µg) from dormant (DS) and outgrowing (OG) spores of wt (Left panel; lanes 1–4), nfo exoA nth (Middle panel; lanes 5–8), and nfo exoA nth disA (Right panel; lanes 9–12), were incubated without Fpg treatment (lanes 1, 3, 5, 7, 9, 11) or treated with 14 units of Fpg (lanes 2, 4, 6, 8, 10, 12). The reaction products were separated on 1% alkaline agarose gels and stained with ethidium bromide as described in Section 2. The data shown are representative of the results of two independent experiments. (B) Quantification of chromosomal DNA degradation from experiments shown in Figure 5A, were determined by densitometry using ImageJ 1.47n software. The analyses were performed with two alkaline gels (with different batches of outgrown DNA spores). Values represent the average of the two experiments ± standard deviations.

Figure 6

Microscopic analysis of chromosome replication status 90 min after the germination onset of spores with different phenotypes. Spores from wt, nfo exoA, nfo exoA nth, and nfo exoA nth disA strains were heat-shocked and germinated in 2 × SG medium supplemented with L-alanine. Cell samples were collected 90 min after the onset of spore germination, fixed, and stained as described in Section 2. The cells were analyzed by bright-field (BF) and fluorescence microscopy (FM4-64 and DAPI staining). Scale bar, 5 μm. (A–D) Bright field; (E–H) FM4-64 staining; (I–L) DAPI staining; (M–P); overlain images of FM4-64 and DAPI are depicted as MERGE. Arrowheads indicate septum stained with FM4-64 and chromosomes stained with DAPI, respectively.

Figure 7

Microscopic analysis of the DisA-Gfp foci synthesis. Outgrown spores of the strains wt (A) or nfo exoA nth (B) collected 90 min after the germination onset were fixed and stained as described in Section 2. The cells were analyzed by bright field (BF) and fluorescence microscopy (DAPI and Gfp staining). Scale bar, 5 μm. In (A,B); a–d: BF, bright field; Gfp, DisA-Gfp fluorescence; DAPI, chromosomal fluorescence; MG, overlain images of Gfp and DAPI. White arrowheads show replicated chromosomes; yellow arrowheads indicate DisA-Gfp foci.