Mismatch repair modulation of MutY activity drives Bacillus subtilis stationary-phase mutagenesis

Abstract

Stress-promoted mutations that occur in nondividing cells (adaptive mutations) have been implicated strongly in causing genetic variability as well as in species survival and evolutionary processes. Oxidative stress-induced DNA damage has been associated with generation of adaptive His(+) and Met(+) but not Leu(+) revertants in strain Bacillus subtilis YB955 (hisC952 metB5 leuC427). Here we report that an interplay between MutY and MutSL (mismatch repair system [MMR]) plays a pivotal role in the production of adaptive Leu(+) revertants. Essentially, the genetic disruption of MutY dramatically reduced the reversion frequency to the leu allele in this model system. Moreover, the increased rate of adaptive Leu(+) revertants produced by a MutSL knockout strain was significantly diminished following mutY disruption. Interestingly, although the expression of mutY took place during growth and stationary phase and was not under the control of RecA, PerR, or σ(B), a null mutation in the mutSL operon increased the expression of mutY several times. Thus, in starved cells, saturation of the MMR system may induce the expression of mutY, disturbing the balance between MutY and MMR proteins and aiding in the production of types of mutations detected by reversion to leucine prototrophy. In conclusion, our results support the idea that MMR regulation of the mutagenic/antimutagenic properties of MutY promotes stationary-phase mutagenesis in B. subtilis cells.

FIG. 1.

Stationary-phase-induced reversions of the his (A), met (B), and leu (C) mutant alleles of the YB955 (○), PERM704 (mutY) (•), and PERM899 (mutY amyE::Phs-mutY) (▴) B. subtilis strains are described in Materials and Methods. The results are average numbers of accumulated revertants for six different selection plates. This experiment was performed at least three times.

FIG. 2.

Abilities of strains PERM704 (mutY) (A) and PERM899 (mutY amyE::Phs-mutY) (B) to survive histidine (black bars), methionine (gray bars), and leucine (white bars) starvation. Three plugs of bacteria containing agar were taken from selection plates each day for testing of viability of bacteria (see Materials and Methods for details). The experiments were repeated at least twice.

FIG. 3.

(A) Analysis of mutation rates. B. subtilis strains YB955 (parental strain), PERM704 (mutY), PERM899 (mutY amyE::Phs-mutY), PERM739 (mutSL), and PERM828 (mutY mutSL) were tested for the ability to produce Leu⁺ revertants during exponential growth as described in Materials and Methods. The mutation rates were calculated as previously described, with the formula m/2Nt. Results presented are average mutation rates for three individual fluctuation tests. Error bars represent 1 standard error. (B) Stationary-phase-induced reversion to Leu⁺ of the YB955 (○), PERM739 (mutSL) (•), and PERM828 (mutY mutSL) (▴) B. subtilis strains were determined as described in Materials and Methods. Results are average numbers of accumulated revertants for six different selection plates. This experiment was performed at least three times.

FIG. 4.

(A) Levels of β-galactosidase produced by B. subtilis strains PERM668 (YB955) (• and ○) and PERM896 (ΔmutSL) (▪ and □). Strains were grown in liquid antibiotic (A3) medium. Cell samples were collected at the indicated times and treated with lysozyme, and the extracts were assayed for β-galactosidase as described in Materials and Methods. The data shown are average values for triplicate independent experiments ± standard deviations (SD) for β-galactosidase specific activity in strains PERM668 (○) and PERM896 (□) and for A₆₀₀ values for strains PERM668 (•) and PERM896 (▪). (B) Levels of β-galactosidase in a mutY-lacZ transcriptional fusion during vegetative and stationary phases of growth. B. subtilis strain PERM668 was grown in liquid antibiotic (A3) medium. Cell samples were collected at the indicated times and treated with lysozyme, and the extracts were assayed for β-galactosidase as described in Materials and Methods. Data shown are average values for triplicate independent experiments ± SD for β-galactosidase specific activity (▴) and for A₆₀₀ values (•).

FIG. 5.

(A) RT-PCR analysis of mutY transcription during vegetative and stationary phases of growth. RNA samples (∼1 μg) isolated from a B. subtilis YB955 A3 culture, at the steps indicated, were processed for RT-PCR analysis as described in Materials and Methods. The arrow shows the size of the expected RT-PCR product. 16S and 23S rRNA bands are shown in the lower panel. (B) Western blot analysis of MutY-FLAG synthesis during vegetative and stationary phases of growth. B. subtilis strain YB955 was grown in liquid A3 medium. Cell extract samples (100 μg of protein; see Materials and Methods), harvested at the steps indicated, were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The blots were stained with Ponceau red (top), probed with a FLAG monoclonal antibody diluted 10,000-fold, and then processed with an ECL Western blot system (bottom). The positions of molecular size markers are indicated to the left of the stained membrane. T₀ is the time point in the culture when the slopes of the logarithmic and stationary phases of growth intercepted. T₉₀, T₁₈₀, and T₂₇₀ indicate the time in minutes after T₀. Veg., vegetative growth.

FIG. 6.

(A) Lack of induction of a mutY-lacZ fusion by mitomycin C (M-C), hydrogen peroxide, and sodium chloride. B. subtilis PERM668 was propagated in A3 medium, in three independent flasks, to an OD₆₀₀ of 0.5. At this point, each culture was divided equally into 2 flasks; one set of flasks was left untreated (a, d, and f), and the other set was treated with either mitomycin C (0.5 μg/ml) (b), H₂O₂ (200 μM (e), or NaCl (4% [wt/vol]) (g). (c) B. subtilis YB3001 containing a recA′-lacZ fusion was treated with mitomycin C (0.5 μg/ml). Cells harvested after 1 h of induction were assayed for β-galactosidase activity as described in Materials and Methods. Values represent averages for triplicate independent experiments ± SD. (B) Expression of a mutY-lacZ fusion in PerR and σ^B genetic backgrounds. Levels of β-galactosidase produced by B. subtilis strains PERM668 (YB955) (• and ○), PERM897 (perR) (▪ and □), and PERM898 (sigB) (▴ and ▵) are shown. Strains were grown in liquid antibiotic (A3) medium. Cell samples were collected at the indicated times and treated with lysozyme, and the extracts were assayed for β-galactosidase as described in Materials and Methods. The data shown are average values for triplicate independent experiments ± SD for β-galactosidase specific activity in strains PERM668 (○), PER897 (□), and PERM898 (▴) and for A₆₀₀ values for strains PERM668 (•), PERM897 (▪), and PER898 (▴).