RESUMO
We previously demonstrated that reactive oxygen species (ROS) could be involved in ultraviolet-C (UVC)-induced DNA damage in Escherichia coli cells. In the present study, we evaluated the involvement of the GO system proteins in the repair of the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG, GO) lesion, which is ROS-induced oxidative damage. We first found that the mutant strain Δfur, which produces an accumulation of iron, and the cells treated with 2,2'-dipyridyl, a iron chelator, were both as resistant to UVC-induced lethality as the wild strain. The 8-oxoG could be mediated by singlet oxygen ((1)O(2)). The Fpg protein repaired this lesion when it was linked to C (cytosine), whereas the MutY protein repaired 8-oxoG when it was linked to A (adenine). The survival assay showed that the Fpg protein, but not the MutY protein, was important to UVC-induced lethality and interacted with the UvrA protein, a nucleotide excision repair (NER) protein involved in UVC repair. The GC-TA reversion assay in the mutant strains from the '8-oxoG-repair' GO system showed that UVC-induced mutagenesis in the fpg mutants, but not in the MutY strain. The transformation assay demonstrated that the Fpg protein is important in UVC repair. These results suggest that UVC could also cause indirect ROS-mediated DNA damage and the Fpg protein plays a predominant role in repairing this indirect damage.
Assuntos
Quebras de DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Raios Ultravioleta/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Bacteriano/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese , Plasmídeos/genética , Plasmídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/metabolismo , Transformação BacterianaRESUMO
We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.
Assuntos
Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Mutagênese/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta , Escherichia coli/genéticaRESUMO
AIMS: The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases. METHODS AND RESULTS: The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea. CONCLUSIONS: Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.
Assuntos
Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Ferro/metabolismo , 2,2'-Dipiridil/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Linhagem Celular , Quelantes/farmacologia , Humanos , Ferro/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacosRESUMO
The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (1O2) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.
Assuntos
Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Espécies Reativas de Oxigênio , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Carotenoides/química , Carotenoides/farmacologia , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli/química , Sequestradores de Radicais Livres/química , Radicais Livres , Oxigênio/química , Azida Sódica/química , Azida Sódica/farmacologia , Transativadores/química , Fatores de Transcrição/química , Raios Ultravioleta , Água/químicaRESUMO
Carotenoids are 40-carbon molecules with conjugated double bonds, making them particularly effective for quenching free radicals. They have always been believed to possess anticancer properties, which could be due to their antioxidant potential. Norbixin is an unusual dicarboxylic water-soluble carotenoid present as a component in the pericarp of the seeds of Bixa orellana L. (from the Bixaceae family), a tropical shrub commonly found in Brazil. The main carotenoids present in these seeds, bixin and norbixin, form a coloring material, known as annatto, which is mainly used in the food industry. As annatto is only used as a coloring material, most studies of annatto pigments have focused on the determination of annatto levels in food. However, little attention has been given to the biological properties of bixin and norbixin. We evaluated the effect of norbixin on the response of Escherichia coli cells to DNA damage induced by UV radiation, hydrogen peroxide (H2O2) and superoxide anions (O2*-)) and found that norbixin protects the cells against these agents. Norbixin enhanced survival at least 10 times. The SOS induction by UVC was inhibited 2.3 times more when cells were grown in the presence of norbixin. We also found that norbixin has antimutagenic properties, with a maximum inhibition of H2O2-induced mutagenic activity of 87%, based on the Salmonella mutagenicity test
Assuntos
Antimutagênicos/farmacologia , Carotenoides/farmacologia , Dano ao DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bixaceae/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/efeitos da radiação , Dano ao DNA/efeitos da radiação , Escherichia coli/citologia , Peróxido de Hidrogênio/toxicidade , Resposta SOS em Genética , Superóxidos/toxicidade , Testes de Mutagenicidade/métodos , Raios UltravioletaRESUMO
The SoxRS regulon is induced when bacterial cells are exposed to redox-cycling agents such as menadione or paraquat. In this paper it is shown that a physical agent, such as ultraviolet radiation with a wavelength of 312 nm (UVB) can induce soxS gene expression. The results indicate that this induction involves the RpoS protein. Moreover, an unexpected increase of soxS gene expression independent of a functional soxR gene in UVB-irradiated cells has been verified. This increase could be explained by transcription of soxS gene in a rpoS-dependent pathway.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Fator sigma/metabolismo , Transativadores/metabolismo , Raios Ultravioleta , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Estresse Oxidativo/fisiologia , Doses de Radiação , Transativadores/genéticaRESUMO
The stannous ion, mainly the stannous chloride (SnCl(2)) salt form, is widely used as a reducing agent to label radiotracers with technetium-99m ((99m)Tc). These radiotracers can be employed as radiopharmaceuticals in nuclear medicine procedures. In this case, there is no doubt about absorption of this complex, because it is intravenously administered in humans, although biological effects of these agents have not been fully understood. In this work we used a bacterial system to study the cytotoxic potential of stannous chloride. It is known that SnCl(2) induces lesions that could be mediated by reactive oxygen species (ROS). We, thus, investigated the existence of cross-adaptive response between hydrogen peroxide (H(2)O(2)) and SnCl(2) and the role of the OxyR system known to promote cellular protection against oxidative damages. Here we describe the results obtained with prior treatment of different Escherichia coli strains with sub-lethal doses of H(2)O(2), followed by incubation with SnCl(2). Our data show that H(2)O(2) is capable of inducing cross-adaptive response against the lethality promoted by SnCl(2), suggesting the OxyR system participation through catalase, alkyl hydroperoxide reductase and superoxide dismutase enzymes
Assuntos
Adaptação Biológica/fisiologia , Proteínas de Ligação a DNA , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Repressoras/metabolismo , Compostos de Estanho/toxicidade , Fatores de Transcrição/metabolismo , Contagem de Células , Dano ao DNA , Escherichia coli/fisiologia , Proteínas de Escherichia coli , Genótipo , Oxirredução , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
In the present study, we evaluated the sensitivity of different Escherichia coli strains to Cumene hydroperoxide (CHP) treatment under distinct conditions of Fe2+ availability. Our results showed that the pretreatment with an iron chelator (dipyridyl) protects all the tested strains against CHP toxic effects, but it was not sufficient to abolish the CHP induced mutagenesis. On the other hand, simultaneous pretreatment with both dipyridyl and neocuproine (copper chelator) leads to a complete protection against CHP mutagenic effects. Our data suggest the participation of copper ion in the CHP mutagenesis induced in E. coli.
Assuntos
Derivados de Benzeno/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Ferro/farmacologia , Mutagênicos/farmacologiaRESUMO
We studied the participation of the stress-inducible systems, as the OxyR, SoxRS and SOS regulons in the protection of Escherichia coli cells against lethal effects of cumene hydroperoxide (CHP). Moreover, we evaluated the participation of BER and NER in the repair of the DNA damage produced by CHP. Our results suggest that the hypersensitivity observed in the oxyR mutants to the lethal effect of CHP does not appear to be due to SOS inducing DNA lesions, but rather to cell membrane damage. On the other hand, DNA damage induced by CHP appears to be repaired by enzymes involved in BER and NER pathways. In this case, Fpg protein and UvrABC complex could be involved cooperatively in the elimination of a specific DNA lesion. Finally, we have detected the requirement for the uvrA gene function in SOS induction by CHP treatment.
Assuntos
Derivados de Benzeno/efeitos adversos , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Oxidantes/efeitos adversos , Transativadores , Antimutagênicos , Proteínas de Bactérias , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Mutagênicos/farmacologia , Mutação , Proteínas Repressoras/genética , Resposta SOS em Genética , Superóxidos/farmacologia , Fatores de Transcrição/genéticaRESUMO
Pretreatment with 2.5 mM H2O2 protects E. coli cells against UV-C killing, a phenomenon independent of LexA cleavage. In this paper, we observe that this cross-protection response is neither dependent on the dinY gene product nor on the system that controls dinY, since H2O2 is able to induce cross-protection but not to induce the dinY gene in a lexA-noninducible strain [lexA (Ind-)]. Moreover, this response is not induced in a lexA (Def) background, suggesting that the expression of the SOS regulon may inhibit this cross-protection response.