RESUMO
During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA- background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Recombinases Rec A/metabolismo , Transcrição Gênica , Bacillus subtilis/ultraestrutura , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Guanina/metabolismo , Mutação , Espécies Reativas de Oxigênio , Esporos BacterianosRESUMO
Bacteria deploy global programs of gene expression, including components of the SOS response, to counteract the cytotoxic and genotoxic effects of environmental DNA-damaging factors. Here we report that genetic damage promoted by hexavalent chromium elicited the SOS response in Bacillus subtilis, as evidenced by the induction of transcriptional uvrA-lacZ, recA-lacZ, and P recA-gfp fusions. Accordingly, B. subtilis strains deficient in homologous recombination (RecA) and nucleotide excision repair (NER) (UvrA), components of the SOS response, were significantly more sensitive to Cr(VI) treatment than were cells of the wild-type strain. These results strongly suggest that Cr(VI) induces the formation in growing B. subtilis cells of cytotoxic and genotoxic bulky DNA lesions that are processed by RecA and/or the NER pathways. In agreement with this notion, Cr(VI) significantly increased the formation of DNA-protein cross-links (DPCs) and induced mutagenesis in recA- and uvrA-deficient B. subtilis strains, through a pathway that required YqjH/YqjW-mediated translesion synthesis. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves the formation of DPCs and that such deleterious effects are counteracted by both the NER and homologous recombination pathways, belonging to the RecA-dependent SOS system.IMPORTANCE It has been shown that, following permeation of cell barriers, Cr(VI) kills B. subtilis cells following a mechanism of reactive oxygen species-promoted DNA damage, which is counteracted by the guanine oxidized repair system. Here we report a distinct mechanism of Cr(VI)-promoted DNA damage that involves production of DPCs capable of eliciting the bacterial SOS response. We also report that the NER and homologous recombination (RecA) repair pathways, as well as low-fidelity DNA polymerases, counteract this metal-induced mechanism of killing in B. subtilis Hence, our results contribute to an understanding of how environmental pollutants activate global programs of gene expression that allow bacteria to contend with the cytotoxic and genotoxic effects of heavy metals.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Cromo/toxicidade , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Mutagênicos/toxicidade , Recombinases Rec A/metabolismo , Bacillus subtilis/metabolismo , Resposta SOS em GenéticaRESUMO
The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain B. subtilis YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the hisC952 (amber), metB5 (ochre) and leuC425 (missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC-MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆nrdR strain. Overall, our results revealed that amino acid-starved cells of strain B. subtilis ∆nrdR that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteoma , Proteômica , Ribonucleotídeo Redutases/genética , Aminoácidos/metabolismo , Cromatografia Líquida , Mutagênese , Nucleotídeos/metabolismo , Proteômica/métodos , Estabilidade de RNA , Estresse Fisiológico , Espectrometria de Massas em TandemRESUMO
A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM). In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10-9. Genetic disruption of the canonical mismatch (MMR) and guanine oxidized (GO) repair pathways increased the Tmpr frequency of mutation by ~2-3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10-7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutagênese , Mutação , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Trimetoprima/farmacologiaRESUMO
UNLABELLED: Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rif(r) and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1'-dimethyl-4,4'-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σ(B) regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phase-associated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCE: The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases.