Chromogenic Escherichia coli reporter strain for screening DNA damaging agents.

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

Roles of Bacillus subtilis RecA, Nucleotide Excision Repair, and Translesion Synthesis Polymerases in Counteracting Cr(VI)-Promoted DNA Damage.

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.

Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis.

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.

Role of Bacillus subtilis error prevention oxidized guanine system in counteracting hexavalent chromium-promoted oxidative DNA damage.

Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system.