Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

Abstract

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 ⌬GO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ⌬GO 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.

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

“…Cr(VI) promotes mutagenesis in eukaryotic and prokaryotic organisms presumably through an indirect mechanism of radical oxygen attack of DNA (2,8). Results described in this work revealed that DNA-protein adducts presumably generated by partially reduced species of hexavalent chromium and processed by components of the SOS system also induced mutagenesis in B. subtilis.…”

“…Oxygen radicals generated after reaction of Cr(V) with cellular redox compounds have the ability to affect DNA and to produce distinct types of lesions, including 8-oxoG and AP sites, which are common substrates for the BER pathway (2,9,(52)(53)(54). In support of this notion, recent results revealed a ROS-dependent mechanism of DNA damage that is counteracted by the GO prevention/repair system in B. subtilis (8). The ability of chromate to induce the expression of recA-gfp, recA-lacZ, and uvrA-lacZ fusions revealed that this oxyanion is capable of activating the SOS response in B. subtilis (Fig.…”

“…In B. subtilis, factors that elicit the formation of strand breaks and chemical adducts, causing distortion of the DNA helix and replication arrest, are capable of triggering the SOS response (26,34,47). As noted above, the genotoxicity promoted by Cr(VI) in this microorganism has been associated with production of 8-oxoG and AP sites, which are counteracted by the BER pathway (more specifically, by the GO system) (8). Since these genetic lesions do not apparently activate the SOS regulon (56,57), we predicted that Cr(VI) produced a bulky lesion capable of triggering this transcriptional response in B. subtilis.…”

“…Therefore, we first investigated whether Cr(VI) or reduced species derived from this metal were able to target DNA directly and to activate the SOS response. To this end, B. subtilis cells bearing a chromosomal copy of a translational P recA -gfp fusion were treated with 22 ppm of Cr(VI), equivalent to the 50% lethal dose (LD 50 ) of this oxyanion (8). As shown in Fig.…”

“…To counteract the cytotoxic and genotoxic effects of this lesion, microorganisms from distinct species, including Bacillus subtilis, possess the guanine oxidized (GO) prevention/repair system, composed of the proteins MutT, MutM, and MutY (5)(6)(7). In line with these concepts, it was recently shown that the greater susceptibility to hexavalent chromium and the mutagenic effects promoted by this metal in a GO system-deficient strain of B. subtilis were associated with the formation of 8-oxoG in DNA (8). However, several in vitro and in vivo studies have shown that the repertoire of genetic lesions promoted by Cr(VI) can be even wider and can include DNA-protein cross-links (DPCs) (3,9,10).…”