Role of the Nfo and ExoA Apurinic/Apyrimidinic Endonucleases in Radiation Resistance and Radiation-Induced Mutagenesis of Bacillus subtilis Spores

Abstract

The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/β-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/β-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.

TEXT

Bacterial endospores of Bacillus species are among the most durable forms of life, and they owe much of their longevity to mechanisms that (i) protect DNA from damage during spore dormancy and (ii) repair DNA damage upon spore germination (31, 32, 40). DNA lesions are produced by artificial and environmental DNA-damaging agents. One predominant type of DNA damage produced by radiation is the modification or loss of a base; thus, apurinic/apyrimidinic (AP) sites are expected to be a frequent lesion in DNA exposed to ionizing radiation (11, 12, 20, 22), mainly due to DNA's reaction with reactive oxygen species (ROS) (2). AP sites block DNA replication and lead to increased rates of mutagenesis (9, 23). Two AP endonucleases, ExoA and Nfo (38, 43), have been implicated in DNA repair in germinating Bacillus subtilis spores (1, 21, 37). However, possible roles of ExoA and Nfo alone and together with DNA protection by α/β-type small, acid-soluble spore proteins (SASP) in spore resistance to and mutagenesis by different types of radiation (35, 36, 44, 45) have not been explored.

In this communication, we have examined the roles of ExoA, Nfo, and SASP in spore resistance to germicidal UV (254 nm) and environmentally relevant polychromatic (280 to 400 nm) UV radiation, as well as to sparsely ionizing (X rays) and densely ionizing (high-energy charged [HZE] particle) radiation. Since AP sites can cause errors in DNA replication (i.e., are mutagenic), we also determined the frequency of resistance to nalidixic acid (Nal^r) of irradiated AP repair-deficient, SASP-deficient, and wild-type spores to obtain a better understanding of the mechanisms involved in spore resistance to UV and ionizing radiation (4, 19, 32, 40).

Strains used in this work are listed in Table 1, and all are isogenic with the wild-type strain PS832. Spores were obtained by cultivation under vigorous aeration in double-strength liquid Schaeffer sporulation medium (39), and spores were purified and stored as described previously (27–29). When appropriate, chloramphenicol (5 μg/ml), neomycin (10 μg/ml), or tetracycline (10 μg/ml) was added to the medium. Spore preparations consisted of single spores with no detectable clumps and were free (>99%) of growing cells, germinated spores, and cell debris, as seen with a phase-contrast microscope (27–29, 41).

Table 1.

B. subtilis strains used in this study^a

Strain	Genotype and phenotype	Source and reference
PS832	Trp+ revertant of strain 168 (wild type)	P. Setlow (13)
PS356	ΔsspA ΔsspB α−β− Cmr	P. Setlow (29)
PERM450	ΔsspA ΔsspB ΔexoA::tet Δnfo::neo α−β− Cmr Tetr Neor	M. Pedraza-Reyes (21)
PERM452	ΔexoA::tet Tetr	M. Pedraza-Reyes (21)
PERM453	Δnfo::neo Neor	M. Pedraza-Reyes (21)
PERM454	ΔexoA::tet Δnfo::neo Tetr Neor	M. Pedraza-Reyes (21)

^aCm^r, resistant to chloramphenicol (5 μg/ml); Tet^r, resistant to tetracycline (10 μg/ml); Neo^r, resistant to neomycin (10 μg/ml).

Preparation of spore samples for radiation exposure has been described in detail previously (28, 30). Spores were exposed to monochromatic 254-nm UV-C, UV-A plus UV-B [UV-(A+B)] (280 to 400 nm), UV-A (320 to 400 nm), or X rays (150 keV/14 mA) as described previously (30). HZE particle irradiation was performed at the Heavy Ion Medical Accelerator (HIMAC) at the National Institute for Radiological Sciences (NIRS) in Chiba, Japan, under the aegis of HIMAC research project 20B463. The following two ion species were applied (energies [in MeV/nucleon {n}] and linear energy transfer [LET] rates [in keV/μm] are in parentheses): helium (He; 150 MeV/n and 2.2 keV/μm) and iron (Fe; 500 MeV/n and 200 keV/μm). Further details on the irradiation geometry of the HIMAC facility, beam monitoring, dosimetry, and dose calculations have been described previously (27, 28, 34). Spore recovery and viability assays were performed as described previously (27–30). The surviving fraction of B. subtilis spores was determined from the ratio N/N₀, with N being the number of CFU of the irradiated sample and N₀ that of the nonirradiated controls. Spore inactivation curves, representing fluence- or dose-effect correlations, were obtained as described previously (28, 30). Data are reported as F₁₀ or D₁₀ values, the UV fluence or dose of ionizing radiation killing 90% of the initial spore population (29, 30, 41).

To determine mutation frequencies to Nal^r, aliquots of spores of each strain were taken from UV after irradiation with fluences of 100 J/m² of UV-C (254 nm), 10 kJ/m² of UV-(A+B) (280 to 400 nm), and 250 kJ/m² of UV-A (320 to 400 nm) or 250 Gy of X rays or high-energy charged helium and iron ions, as well as untreated samples. Aliquots were spread on 2× LB medium agar plates with or without 20 μg nalidixic acid/ml (Sigma-Aldrich, Steinheim, Germany) and incubated for 24 to 48 h at 37°C, and the frequencies of mutation to Nal^r for spores of each strain were calculated as the number of Nal^r colonies/total survivors at the various radiation doses as described previously (10, 27).

All data are expressed as averages ± standard deviations (n = 3). Data were compared statistically using Student's t test. Values were analyzed in multigroup pairwise combinations, and differences with P values of ≤0.05 were considered statistically significant (27, 28, 30).

In order to assess the importance of the AP endonucleases ExoA and Nfo and the DNA-protective SASP in B. subtilis spore resistance to different types of radiation, wild-type, SASP-deficient (α⁻β⁻), and AP endonuclease-deficient spores, either air-dried monolayers or suspended in water, were irradiated with various UV wavelengths, X rays, and high-energy charged particles. α⁻β⁻ spores were significantly more sensitive to all UV wavelengths than were wild-type spores, in either the wet or dry state (Fig. 1), as expected (25, 29, 30). Spores lacking either ExoA or Nfo were not significantly more sensitive to 254-nm UV-C than were wild-type spores (Fig. 1A). However, single AP mutant spores were significantly more sensitive to environmentally relevant UV-(B+A) or UV-A when exposed in the dry state (Fig. 1B and C), but not in water (Fig. 1B and C). This finding suggests that there is a higher induction of AP sites by environmental UV radiation in dry spores than in spores in water.

Fig. 1.

Spore resistance to standard germicidal 254-nm UV-C (A), environmentally relevant 280- to 400-nm UV-(A+B) (B), and 320- to 400-nm UV-A (C) radiation. Spores were irradiated as air-dried monolayers (white bars) or in water (gray bars), and F₁₀ values are expressed as averages ± standard deviations (n = 3) as described in the text. Asterisks indicate F₁₀ values that were significantly different (P ≤ 0.05) from those for wild-type spores. Note the differences in the y axes between panels A, B, and C.

Spores of the exoA nfo mutant were significantly more sensitive to 254-nm UV-C than were wild-type spores in the dry state, but not in the wet state (Fig. 1A). However, spores of the double mutant were significantly more sensitive to environmentally relevant UV-(A+B) and UV-A when treated either wet or dry (Fig. 1B and C). α⁻β⁻ spores lacking ExoA and Nfo appeared slightly more sensitive than spores lacking only SASP to all UV wavelengths tested, in both the dry and the wet states, but these differences were not statistically significant (Fig. 1). Taken together, the results from previous (29, 31, 33, 40) and present experiments indicate that (i) SASP are important protectants of DNA against damage by germicidal and environmentally relevant UV radiation-induced damage, and (ii) both ExoA and Nfo play a role in spore resistance to DNA damage caused by environmentally relevant UV-B and UV-A wavelengths. It is well known that solar UV exerts its lethal effects through direct interaction with spore DNA and indirectly via the generation of ROS, such as peroxide, superoxide, or hydroxyl radicals (reviewed in references 5 and 6). Examples of direct UV damage include pyrimidine dimers, such as the “spore photoproduct” (SP) 5-thyminyl-5,6-dihydrothymine, cyclobutane pyrimidine dimers (CPDs), and 6-4 photoproducts (5, 13). These photoproducts are produced much more efficiently by 254-nm UV-C and the shorter-wavelength UV-B (290 to 320 nm) component of sunlight than they are by longer-wavelength UV-A (320 to 400 nm) (5, 19, 41). In contrast, nonspecific DNA damage, such as double-strand breaks (DSB), single-strand breaks (SSB), and AP sites are more likely to be produced indirectly from UV-A via ROS production (41, 42). Slieman and Nicholson (41) reported that CPDs were preferentially produced in spores exposed to the UV-B component of sunlight and that nonspecific DSB and SSB were formed mainly when spores were exposed to the UV-A component of sunlight. In their work, the assessment of the induction of UV-induced photoproducts relied on neutral agarose gel electrophoresis, which has limited sensitivity (8). Consequently, Slieman and Nicholson (41) did not detect AP sites in the DNA of UV-treated spores, whereas we observed clear differences in spore resistance to environmental UV radiation, depending on the presence or absence of AP endonucleases (Fig. 1B and C).

We next examined the roles of ExoA and Nfo and SASP in spore resistance to ionizing radiation from X rays and high-energy charged-particle beam irradiation (Fig. 2). Spores lacking both ExoA and Nfo, α⁻β⁻ spores, and α⁻β⁻ spores also lacking ExoA or Nfo were all significantly more sensitive than wild-type spores to ionizing radiation, whether exposed in the wet or dry state (Fig. 2). These results show that (i) both ExoA and Nfo play a role in the repair of X-ray and high-energy particle damage to spore DNA, and (ii) irradiation with X rays or HZE particles generates AP sites in spore DNA.

Fig. 2.

Spore resistance to X rays (A) and high-energy charged helium (B) and iron (C) ions. Spores were irradiated as air-dried monolayers (white bars) or in water (gray bars), and D₁₀ values are expressed as averages ± standard deviations (n = 3) as described in the text. Asterisks indicate D₁₀ values that were significantly different (P ≤ 0.05) compared to wild-type spores. Note the differences in the y axes between panels A, B, and C.

In addition to spore inactivation kinetics, we measured mutagenesis of AP repair-deficient, α⁻β⁻, and wild-type spores by germicidal and environmental UV (Fig. 3) and ionizing X rays and HZE particles (Fig. 4). Independent of the type and quality of radiation, α⁻β⁻ exoA nfo spores showed a significantly higher mutation frequency than α⁻β⁻, exoA nfo, or wild-type spores, with the order α⁻β⁻ exoA nfo > α⁻β⁻ > exoA nfo > wild-type spores (Fig. 3 and 4). Mutagenesis to Nal^r also exhibited wavelength dependence in the order 254 nm > 280 to 400 nm > 320 to 400 nm (Fig. 3A to C), in agreement with previous work (19, 35, 45). The level of mutagenesis to Nal^r also depended on the LET of the applied ion species and radiation, also in agreement with previous work (16, 18, 27, 45).

Fig. 3.

Mutation frequencies to Nal^r of dormant spores of different strains irradiated with 100 J/m² of 254-nm UV-C (A), 10 kJ/m² of UV-(A+B) (B), and 250 kJ/m² of UV-A (C) radiation. Spores were exposed as air-dried monolayers (white bars) or in water (gray bars), and values are averages ± standard deviations for triplicate determinations in three separate experiments as described in the text. One asterisk indicates a mutation frequency that was significantly different from that of wild-type spores, and two asterisks indicate a mutation frequency that was also significantly different from that of α⁻β⁻ spores (Student's t test; P ≤ 0.05). Note the differences in the y axes in the different panels. The spontaneous mutation frequency to Nal^r of the tested strains ranged from 1.6 × 10⁻⁷ to 8.3 × 10⁻⁷, in good agreement with previous data (10).

Fig. 4.

Mutation frequencies to Nal^r of dormant spores of different strains irradiated with 250 Gy of X rays (A) and high-energy helium ions and (B) and iron ions (C). Spores were exposed as air-dried monolayers (white bars) or in water (gray bars), and values are averages ± standard deviations for triplicate determinations in three separate experiments as described in the text. One asterisk indicates a mutation frequency that was significantly different from that of wild-type spores, and two asterisks indicate a mutation frequency that was also significantly different from that of α⁻β⁻ spores (Student's t test; P ≤ 0.05). Note the differences in the y axes in the different panels. The spontaneous mutation frequency to Nal^r of the tested strains ranged from 1.6 × 10⁻⁷ to 8.3 × 10⁻⁷, in good agreement with previous work (10).

Base excision repair (BER) is a major pathway involved in repair of a wide variety of DNA base damage resulting from exposure to various chemical (e.g., hydrogen peroxide) and physical (e.g., dry and wet heat, UV and ionizing radiation) insults (2, 3, 7, 15, 24, 26). In contrast to initial steps in BER, which require a large number of lesion-specific N-glycosylases that create AP sites, ExoA and Nfo are the major B. subtilis AP endonucleases that nick DNA at AP sites, leading to gap filling by DNA polymerase I (14). In contrast to the situation in vegetative cells, relatively little is known about how BER contributes to spore resistance to DNA-damaging agents (21, 37). This communication confirms and extends (i) the essential role of SASP as key spore DNA protectants against UV and ionizing radiation (17, 21, 28, 29), (ii) the important role of ExoA and Nfo as AP repair enzymes during spore germination and outgrowth (21, 37, 38), and (iii) the major roles that SASP and AP enconucleases play in the prevention of both lethal and mutagenic damage to spore DNA.