The role of Fpg protein in UVC-induced DNA lesions.

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.

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement.

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.

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC).

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.

Reactive oxygen species mediate lethality induced by far-UV in Escherichia coli cells.

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.

Does UVB radiation induce SoxS gene expression in Escherichia coli cells?

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.

Adaptive response to H(2)O(2) protects against SnCl(2) damage: the OxyR system involvement.

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

Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli

The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture mediu. If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times. It is known that the Fe2+ -o-phenanthroline(I) and Fe2+ -o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe2+ -o-phenanthroline(III) complex is formed slowly, Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2. Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+