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

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.

ABSTRACT

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.

INTRODUCTION

Living organisms are constantly exposed to a myriad of environmental factors that are potentially cytotoxic and genotoxic. It has been reported that hexavalent chromium [Cr(VI)] has the ability to permeate the bacterial envelope and to promote distinct types of genetic lesions (1). Following interactions with cellular redox compounds, the conversion of Cr(VI) to Cr(III) generates reactive oxygen species (ROS) that are capable of attacking DNA and generating distinct types of lesions, including oxidized bases, apurinic/apyrimidinic (AP) sites, and strand breaks (2, 3). Because of its mutagenic character and potential lethality, the oxidized base guanine (8-oxoG) is considered one of the most detrimental types of genetic damage for cells (4). 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 (5–7). 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). DPCs can be generated through an oxidative free radical mechanism or indirectly elicited by chemical linkers or through coordination with a metal atom (11). Proteins irreversibly trapped on the DNA strand would block the progression of DNA and RNA polymerases and hence hamper the faithful replication and transcription of genetic information, exerting deleterious effects on cells (12, 13). Several chemical and physical agents, including aldehydes (14–16), anticancer drugs (17, 18), ionizing radiation (19, 20), and transition metals, including Cr(VI), promote the formation of DPCs (21, 22). Whereas oxidized bases, single-strand breaks, and AP sites are mainly processed by components of the base excision repair (BER) pathway, the repair mechanisms involved in processing DPCs are not completely understood (23, 24).

In distinct prokaryotes, RecA and components of the nucleotide excision repair (NER) pathway have been identified as part of the SOS regulon (25). In B. subtilis and Escherichia coli, this transcriptional response is triggered by DNA-damaging agents and is under the control of RecA and the repressor LexA (DinR in B. subtilis) (26, 27). When DNA damage levels overcome the repair capacity of the NER and RecA pathways, a mutagenic phase of the SOS system is triggered (28, 29). This phase is mediated by DNA polymerases that replicate past template lesions, in a process called DNA translesion synthesis (TLS) (30, 31). In B. subtilis, the global SOS response is composed of more than 60 genes, including those encoding proteins required for DNA repair as well as for faithful and error-prone replication (32–34). In B. subtilis, the TLS performed by the Y-DNA polymerases YqjH and YqjW, homologous to E. coli PolIV (DinB) and PolV (UmuDC), respectively, is required for processing spontaneous and induced genetic damage in vegetative cells, as well as during spore morphogenesis (35, 36). In this microorganism, the yqjH gene is constitutively expressed during vegetative growth, and its transcription is not SOS dependent (35); in contrast, transcription of yqjW is highly repressed in vegetative cells but is induced by the SOS response (35). DNA polymerases involved in TLS are distributed across all domains of life, including the human homologs REV3, REV1, and DNA polymerases η, ι, and κ (37). Of note, when the NER and TCR pathways become insufficient for processing UV-promoted DNA lesions (pyrimidine dimers), these low-fidelity polymerases promote mutagenesis in B. subtilis sporangia (36, 38, 39).

Genetic studies have revealed that uvrA and recA mutants of E. coli that are deficient in NER or homologous recombination (HR) are hypersensitive to formaldehyde, a type of chemical agent that induces DPCs, suggesting that both pathways play a role in processing these types of lesions (15). The ability of Cr(VI) to induce the formation of DPCs has been demonstrated in vitro in eukaryotic cells (40–42); however, formation and processing of these replication- and transcription-interfering lesions in bacteria following exposure to Cr(VI) remains poorly understood. In this work, we demonstrated that Cr(VI) promoted the in vivo synthesis of DPCs in growing B. subtilis cells and activated the SOS response. In agreement with these observations, (i) the NER and HR pathways were found to be required to contend with the noxious effects of Cr(VI) and (ii) the mutagenic effects promoted by this oxyanion were mediated by the TLS polymerases YqjH and YqjW.

RESULTS

Hexavalent chromium activates the SOS response in B. subtilis cells.

Activation of global stress responses following exposure to Cr(VI) has been documented in distinct bacteria (43–45). In B. subtilis, it has been shown that damaging factors that promote bulky DNA lesions activate the SOS response, resulting in derepression of genes encoding NER proteins, Y-DNA polymerases, and RecA, among others (33, 34, 36, 38). 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 (LD₅₀) of this oxyanion (8). As shown in Fig. 1A, the alkylating agent mitomycin C (M-C), which was employed as a positive control, dramatically increased the expression levels of the P_recA-gfp fusion. Importantly, the expression levels of green fluorescent protein (GFP) in a culture of B. subtilis supplemented with 22 ppm of chromate were increased ∼3.3-fold with respect to an untreated control (Fig. 1A). In line with these results, fluorescence microscopy analysis of cell samples obtained from cultures of the P_recA-gfp strain confirmed that both M-C and Cr(VI) were able to activate the synthesis of the chimeric RecA-GFP protein (Fig. 1C to G). We further corroborated the capacity of Cr(VI) to activate the SOS response by treating B. subtilis strains carrying the recA-lacZ and uvrA-lacZ fusions with an LD₅₀ of this heavy metal. As shown in Fig. 1H, compared to untreated controls, the presence of Cr(VI) in the cultures significantly increased the expression levels of both the recA-lacZ and uvrA-lacZ fusions in B. subtilis.

FIG 1.

Monitoring of activation of the SOS response induced by DNA damage using a translation in-frame P_recA-gfp-mut3a fusion. (A) B. subtilis strain PERM1238 (recA-GFP) was grown at 37°C in LB medium to an OD₆₀₀ of 0.5; at that point, the culture was divided into three subcultures. One of the subcultures was left as an untreated control (U), whereas the other two were supplemented with LD₅₀s of Cr(VI) or mitomycin C (M-C) and shaken for an additional period of 30 min at 37°C. Samples collected from the control and treated cultures were washed and suspended in PBS, and the GFP fluorescence of the samples was measured as described in Materials and Methods. (B to G) Samples collected from the untreated culture (B and C) or from the cultures treated with Cr(VI) (D and E) or M-C (F and G) were processed and photographed as described in Materials and Methods. (B, D, and F) Bright field; (C, E, and G) GFP channel. Scale bar, 2 μM. (H) B. subtilis strains YB3001 (recA-lacZ) and PERM1126 (uvrA-lacZ) were grown at 37°C in LB medium to an OD₆₀₀ of 0.5; at that point, the cultures were divided into two subcultures. One of the subcultures was left as an untreated control (−), whereas the other was supplemented with an LD₅₀ of Cr(VI) (+) and shaken for an additional period of 90 min at 37°C. Samples collected from the control and treated cultures were processed to determine β-galactosidase activity as described in Materials and Methods. In panels A and H, each bar represents the mean of data collected from three independent experiments performed in triplicate, and the error bars represent SEMs. The asterisks indicate values that were significantly different (*, P < 0.05).

RecA and the NER pathway protect B. subtilis from the noxious effects of chromate.

As noted above, B. subtilis cells treated with Cr(VI) showed induced expression of recA and uvrA, whose encoded products (RecA and UvrA) play pivotal roles in the NER and HR pathways. Therefore, we explored whether these proteins are necessary to contend with the genotoxic effects of Cr(VI). To this end, B. subtilis cells of the wild-type (WT) strain, as well as growing cultures of strains deficient in UvrA, RecA, or both proteins, were challenged with increasing amounts of hexavalent chromium. The results of these experiments, reported as inactivation curves and 90% lethal dose (LD₉₀) values (Fig. 2), revealed that the lack of RecA or UvrA significantly increased the susceptibility of B. subtilis to treatment with Cr(VI). Interestingly, the absence of both UvrA and RecA sensitized B. subtilis cells even more to the noxious effects of the Cr(VI) oxyanion (Fig. 2). Together, these results strongly suggest that hexavalent chromium promotes some type of DNA damage that is processed by the RecA and NER pathways.

FIG 2.

Roles of RecA and UvrA in survival of B. subtilis cells exposed to hexavalent chromium. B. subtilis WT, PERM1030 (ΔrecA), PERM985 (ΔuvrA), and PERM1038 (ΔrecA ΔuvrA) strains were grown in LB medium at 37°C to an OD₆₀₀ of 1.0. At that point, the strains were treated with different concentrations of Cr(VI), and the cultures were grown for 3 h at 37°C with shaking. The LD₉₀ values (B) were calculated for each strain from the dose-response curves (A). The results represent the means of data collected from at least three independent experiments performed in triplicate for each sample, and the error bars represent SEMs.

RecA fulfills a double function in bacteria, as a regulator of the SOS response and in HR (26, 32–34). To try to distinguish whether the noxious effects of Cr(VI) on the ΔrecA strain were due to single or combined defects in these functions, we employed a B. subtilis strain that was incapable of triggering the SOS response, expressing basal levels of RecA (46) and presumably of UvrA. Accordingly, the LAS523 strain, which carries a noncleavable form of the SOS repressor protein DinR [lexA(Ind⁻)] (47), and its parental genetic background strain were treated with increasing concentrations of Cr(VI). As shown in Fig. 3, the lexA(Ind⁻) strain was significantly more susceptible to the deleterious effects of the oxyanion chromate than was its parental strain. Furthermore, the LD₉₀ values for Cr(VI) susceptibility revealed that the LAS523 strain was more susceptible to Cr(VI) than the RecA- and UvrA-deficient strains but was significantly less susceptible than the double knockout ΔrecA ΔuvrA strain.

FIG 3.

Effects of hexavalent chromium on the survival of B. subtilis LAS600 (WT; black circles) and LAS523 {dinR3 [lexA(Ind⁻)]; gray circles} strains. The strains of interest were grown in LB medium at 37°C to an OD₆₀₀ of 1.0. At that point, the strains were treated with different concentrations of Cr(VI), and the cultures were grown for 3 h at 37°C, with shaking. The LD₉₀ values (B) were calculated for each strain from the dose-response curves (A). The results represent the means of data collected from at least three independent experiments performed in triplicate for each sample, and the error bars represent SEMs.

RecA and NER counteract the mutagenic effects of hexavalent chromium.

We next investigated whether the lesions inflicted by Cr(VI), which are putatively processed by the RecA and NER pathways, promote mutagenesis in B. subtilis. To accomplish this task, the frequency of mutation to rifampin resistance (Rif^r) in the presence or absence of Cr(VI) was determined in the WT, ΔrecA, ΔuvrA, and ΔrecA ΔuvrA strains. Our results revealed that the single absence of recA and the combined absence of recA and uvrA but not the individual disruption of uvrA promoted mutagenesis in B. subtilis. Thus, with respect to WT cells, the absence of RecA or of both RecA and UvrA increased ∼2-fold and 3.9-fold, respectively, the spontaneous Rif^r mutagenesis in this bacterium (Fig. 4). Notably, the presence of Cr(VI) in the cultures induced significant but similar increases in the frequency of mutation to Rif^r for the WT, ΔuvrA, and ΔrecA strains. However, the frequency of mutation to Rif^r in cultures of the ΔrecA ΔuvrA strain treated with Cr(VI) was significantly higher than that of the Cr(VI)-treated WT strain. Thus, in reference to untreated controls, amendment of cultures with an LD₅₀ of Cr(VI) increased by ∼3-, 2.3-, 2.1-, and 3.6-fold the Rif^r mutagenesis of the WT, ΔuvrA, ΔrecA, and ΔuvrA ΔrecA strains, respectively (Fig. 4). These results strongly suggest that lesions inflicted by chromate that activate the SOS response promote mutagenesis in B. subtilis. Furthermore, our results suggest that UvrA and RecA act independently to counteract the mutagenic effects of these lesions.

FIG 4.

Effects of Cr(VI) on the mutation frequencies of different B. subtilis strains. B. subtilis WT, PERM1030 (ΔrecA), PERM985 (ΔuvrA), and PERM1038 (ΔrecA ΔuvrA) strains were grown at 37°C in LB medium to an OD₆₀₀ of 1.0 and then were divided into two Erlenmeyer flasks; one of the flasks was left as an untreated control (gray bars), and the other was supplemented with an LD₅₀ of Cr(VI) (black bars). The cultures were shaken for an additional period of 16 h at 37°C and processed to calculate the frequencies of mutation to Rif^r, as described in Materials and Methods. Each bar represents the mean of data collected from five independent experiments performed in sextuplicate, and the error bars represent SEMs. The asterisks indicate values that were significantly different (*, P < 0.05).

Hexavalent chromium induces the synthesis of DNA-protein cross-links in B. subtilis.

As noted above, Cr(VI) promotes some type of DNA damage that activates the SOS response in B. subtilis. Previous studies showed that treatment of mammalian and human cell lines with Cr(VI) resulted in the formation of DPCs (22, 40–42). Because of its chemical properties, these types of complexes may represent obstacles for DNA replication, resulting in activation of the SOS response in B. subtilis. To investigate this notion, we determined the formation of DPCs in B. subtilis cells deficient in RecA, UvrA, or both proteins, which were grown in the presence or absence of hexavalent chromium. It must be emphasized that the protocol employed to detect DPCs in our study has been widely employed and validated not only in Bacillus subtilis but also in other biological systems (48–52). As shown in Fig. 5, when cells of the WT, ΔrecA, ΔuvrA, and ΔrecA ΔuvrA strains were treated with an LD₉₀ of Cr(VI), the DPC coefficients were increased ∼2.1-, 2.3-, and 4.4-fold, respectively, in reference to the WT strain (Fig. 5). Together, these results strongly suggest that Cr(VI) promotes the synthesis of DPCs in B. subtilis, resulting in upregulation of UvrA and RecA, which employ independent pathways to process these bulky DNA lesions.

FIG 5.

Formation of DPCs in genomic DNA samples isolated from different B. subtilis strains exposed to Cr(VI). B. subtilis WT, PERM1030 (ΔrecA), PERM985 (ΔuvrA), and PERM1038 (ΔrecA ΔuvrA) strains were grown at 37°C in LB medium to an OD₆₀₀ of 1.0 and then were exposed (gray bars) or not exposed (white bars) to an LD₉₀ of Cr(VI), calculated for each strain from dose-response curves. DPCs were determined using the SDS-KCl assay, as described in Materials and Methods. Each bar represents the mean of data collected from four independent experiments performed in triplicate, and the error bars represent SEMs. The DPC coefficients were determined as a ratio of the percentage of fluorometrically quantified DNA in supernatant obtained from Cr(VI)-treated bacteria to the percentage observed in untreated control bacteria. The asterisks indicate values that were significantly different (*, P < 0.05).

Role of YqjW and YqjH in Cr(VI)-induced mutagenesis.

As demonstrated in this work, in B. subtilis the damage promoted by Cr(VI) that was counteracted by RecA and the NER system promoted mutagenesis (Fig. 4). In this microorganism, the TLS polymerases YqjH and YqjW have been implicated in UV-induced mutagenesis, as well as important countermeasures to prevent genetic damage during the development of B. subtilis spores exposed to distinct DNA-damaging factors (30, 36, 38, 39). The cyclobutane-pyrimidine dimers produced by UV-C radiation promote distortions in the double helix that interfere with replication, leading to production of single-stranded DNA, which in turn activates the bacterial SOS response (25–28). In B. subtilis, the Y-DNA polymerases YqjH and YqjW together with YwjD and YpcP have been implicated in a novel error-prone repair pathway that processes UV-induced photodimers in sporulating B. subtilis cells (39). As noted above, DPCs may also interfere with replication; therefore, we investigated whether these Y-DNA polymerases counteract the noxious effects of Cr(VI) and promote mutagenesis in B. subtilis cells exposed to this oxyanion. The results of these analyses revealed that, in comparison with the WT parental strain, the lack of either YqjH or YqjW significantly decreased the resistance of B. subtilis cells to Cr(VI); however, the absence of both polymerases resulted in marked susceptibility of vegetative cells to this oxyanion (Fig. 6A and B). Interestingly, in reference to untreated controls, genetic inactivation of yqjH, YqjW, or both genes resulted in a significant decrease in the Rif^r mutagenesis promoted by Cr(VI) (Fig. 6C). Importantly, the simultaneous disruption of yqjH and yqjW did not increase the susceptibility of the ΔuvrA strain to hexavalent chromium; accordingly, no significant differences (P = 0.77) between the LD₉₀s of the two strains were observed (ΔuvrA strain, LD₉₀ = 36 ± 0.53 ppm; ΔuvrA ΔyqjH ΔyqjW strain, LD₉₀ = 36 ± 5.5 ppm). A similar result was obtained when both TLS polymerase-encoding genes were genetically inactivated in the RecA-deficient strain; thus, the LD₉₀ values of the ΔrecA (LD₉₀ = 28 ± 0.156 ppm) and ΔrecA ΔyqjH ΔyqjW (LD₉₀ = 26 ± 3.74 ppm) strains did not exhibit a significant difference (P = 0.89). Based on these results, it is not unreasonable to predict that both polymerases could work in common pathways with UvrA (NER) or RecA in growing B. subtilis cells to counteract the genotoxic effects of Cr(VI).

FIG 6.

(A and B) Roles of YqjH and YqjW in the survival of B. subtilis cells exposed to hexavalent chromium. B. subtilis WT, PERM646 (ΔyqjH), PERM647 (ΔyqjW), and PERM715 (ΔyqjH ΔyqjW) strains were grown in LB medium at 37°C to an OD₆₀₀ of 1.0. At that point, the strains were treated with different concentrations of Cr(VI), and the cultures were grown for 3 h at 37°C with shaking. The LD₉₀ values (B) were calculated for each strain from the dose-response curves (A). The results represent the means of data collected from three independent experiments performed in triplicate for each sample, and the error bars represent SEMs. (C) Effects of Cr(VI) on the mutation frequencies of different B. subtilis strains. B. subtilis WT, PERM646 (ΔyqjH), PERM647 (ΔyqjW), and PERM715 (ΔyqjH ΔyqjW) strains were grown at 37°C in LB medium to an OD₆₀₀ of 1.0 and then were divided into two Erlenmeyer flasks; one of the flasks was left as an untreated control (gray bars), and the other was supplemented with an LD₅₀ of Cr(VI) (black bars). The cultures were shaken for an additional period of 16 h at 37°C and then were processed to calculate the frequencies of mutation to Rif^r, as described in Materials and Methods. Each bar represents the mean of data collected from three independent experiments performed in sextuplicate, and the error bars represent SEMs. The asterisks indicate values that were significantly different (*, P < 0.05).

DISCUSSION

Many bacterial species capable of proliferating in environments polluted with Cr(VI) have evolved strategies to cope with its toxicity, including decreased uptake, biosorption, chemical reduction, or extrusion of the metal from the cells (1). 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, 52–54). 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. 1). These results not only suggest a mechanism of DNA damage distinct from base oxidation but also indicate that repair proteins belonging to the SOS system are involved in counteracting such types of stress. Experimental evidence presented here confirmed this suggestion, since the absence of RecA and UvrA, an essential factor of the NER system, strongly sensitized B. subtilis to chromate treatment. In line with our results, it was reported that the absence of a functional HR system increased the sensitivity of Rhizobium etli to the noxious effects of chromate (55). Based on these observations, we postulate that RecA-dependent HR is required for processing of single- and double-strand breaks elicited directly or indirectly by Cr(VI) treatment (52, 53).

As shown in this work, B. subtilis cells exposed to Cr(VI) activate the SOS response; although such effects have also been reported for distinct bacterial species (43–45), the type of genetic damage that activates this transcriptional regulon is less well understood. 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. Our results confirmed this notion by showing the presence of DNA-protein adducts in B. subtilis cells treated with chromate, with the contents significantly increasing following disruption of the RecA and NER systems. These observations, together with the exacerbated susceptibility to the noxious effects of Cr(VI) exhibited by ΔrecA ΔuvrA mutants (Fig. 2), strongly suggest the existence of a DNA-damaging mechanism whose processing requires components of the SOS regulon in B. subtilis. Results from in vitro experiments that postulated a three-step process resulting in the formation of DNA-Cr(III)-protein cross-links provide support for this mechanism (58). Further studies have also proposed that Cr(VI) elicits a mechanism of ROS-induced protein carbonylation as a promoter of DPCs in eukaryotic cells (42). It must be also emphasized that UvrA (NER) and RecA most probably act in independent pathways, as suggested by the results of Cr(VI)-induced genotoxicity/cytotoxicity and the accumulation of DPCs in the mutant strains employed in this study.

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. In prokaryotes, lack of repair or ongoing repair of distorting DNA lesions, including thymine photodimers and DPCs, can lead to stalling of DNA replication at replication forks, to trigger the SOS response (27). RecA- and NER-dependent error-free DNA repair plays a prominent role during DNA replication (6, 26, 27, 46). However, if faithful repair pathways cannot efficiently process DNA damage to recommence replication, then error-prone DNA replication is triggered to bypass DNA lesions via TLS (31, 37, 59). Evidence presented here revealed that B. subtilis YqjH and YqjW could coordinately work with the NER and HR pathways to contend with the noxious effects of hexavalent chromium. These results strongly suggest that processing of DPCs proceeds through low-fidelity synthesis of DNA, with mutagenic consequences. Accordingly, Cr(VI)-induced mutagenesis was abolished in B. subtilis cells lacking YqjH, YqjW, or both Y-DNA polymerases. These results support the notion that YqjH and YqjW act in a common pathway, perhaps in coordination with the proofreading-deficient DNA polymerase PolA. Accordingly, a previous study reported physical and functional interactions between PolA and the low-fidelity polymerases YqjH and YqjW (60). Consistent with these results, a previous study that employed a chicken DT40 cell line revealed that, in reference to the WT strain, the absence of REV1 and POLD3 (both involved in error-prone TLS) sensitized the cell line to the noxious effects of hexavalent chromium (61). Based on these observations, it is feasible to propose that TLS polymerases are critical in prokaryotic and eukaryotic cells for tolerating the DNA damage caused by Cr(VI). In summary, our results support the concept that, in B. subtilis, TLS and the NER and RecA systems, acting in independent pathways, play prominent roles in the removal of DPCs adducts promoted by Cr(VI), thus counteracting the cytotoxic and genotoxic effects of this metal oxyanion.

MATERIALS AND METHODS

Bacterial strains, culture conditions, and reagents.

All B. subtilis strains used in this work were derived from strain 168 and are listed in Table 1. The growth medium used routinely was Luria-Bertani (LB) medium. When required, neomycin (Neo) (10 μg ml⁻¹), chloramphenicol (Cm) (5 μg ml⁻¹), spectinomycin (Spc) (100 μg ml⁻¹), erythromycin (Ery) (5 μg ml⁻¹), or rifampin (Rif) (10 μg ml⁻¹) was added to this medium. Liquid cultures were incubated at 37°C with vigorous aeration (shaking at 250 rpm). Cultures on solid media were grown at 37°C. The optical density at 600 nm (OD₆₀₀) of liquid cultures was monitored with a Pharmacia Ultrospec 2000 spectrophotometer. To measure Cr(VI) susceptibility, a stock solution of potassium dichromate (K₂Cr₂O) (99.97% purity; JT Baker, Phillipsburg, NJ) at 10,000 ppm as Cr(VI) was prepared in sterile Milli-Q water. Working concentrations were prepared by diluting the stock solution with sterile Milli-Q water.

TABLE 1.

B. subtilis strains used in this study

Strain	Genotypea	Source or reference
168	trpC2	Laboratory stock
PERM1030	ΔrecA::neo Neor	38
PERM985	ΔuvrA::cm Cmr	46
PERM1038	ΔrecA::neo ΔuvrA::cm Neor Cmr	Laboratory stock
PERM646	ΔyqjH::ery Eryr	36
PERM647	ΔyqjW::ery Eryr	36
PERM715	ΔyqjH::kan ΔyqjW::ery Kanr Eryr	36
PERM1719	ΔyqjH::kan ΔyqjW::ery ΔrecA::cm Kanr Eryr Cmr	This study
PERM1720	ΔyqjH::kan ΔyqjW::ery ΔuvrA::uvrA Kanr Eryr Cmr	This study
PERM1238	amyE::(PrecA-gfp spc) trpC2 pheA1 Spr	38
YB3001	amyE::recA-lacZ (Cm)	38
PERM1126	uvrA::lacZ (Ery)	38
LAS600	Δupp	47
LAS523	+dinR3 [lexA(Ind−)]	47

^aNeo^r, neomycin resistance; Cm^r, chloramphenicol resistance; Ery^r, erythromycin resistance; Kan^r, kanamycin resistance; Sp^r, spectinomycin resistance.

Construction of B. subtilis ΔrecA ΔyqjH ΔyqjW and ΔuvrA ΔyqjH ΔyqjW strains.

To generate a B. subtilis strain deficient in UvrA, YqjH, and YqjW, chromosomal DNA isolated from B. subtilis strain PERM688 (ΔrecA Cm^r) was used to transform competent cells of B. subtilis strain PERM715 (ΔyqjH ΔyqjW Kan^r Ery^r) to generate B. subtilis strain PERM1719 (ΔyqjH ΔyqjW ΔrecA Kan^r Ery^r Cm^r). A B. subtilis strain lacking RecA, YqjH, and YqjW was constructed by transforming competent cells of B. subtilis strain PERM715 with the insertional plasmid pPERM972 (pCP115 carrying an 867-bp EcoRI-BamHI fragment of the internal region of the uvrA open reading frame; ampicillin resistance [Amp^r] Cm^r), thus generating strain PERM1720 (ΔyqjH ΔyqjW ΔuvrA Kan^r Ery^r Cm^r).

Determination of Cr(VI) toxicity.

To determine the dose-response curve for the survival of B. subtilis cells following Cr(VI) exposure, WT and mutant strains deficient in DNA repair systems (Table 1) were grown at 37°C in LB medium to an OD₆₀₀ of 1.0 (0.3 × 10⁸ to 0.5 × 10⁸ cells ml⁻¹). At that point, cultures that were free of spores, as assessed by phase-contrast microscopy, were treated with different concentrations of Cr(VI) (0, 12, 24, 48, and 80 ppm) and incubated for 3 h at 37°C with shaking. After treatment, bacterial viability was then estimated by counting the CFU on LB agarose plates. To this end, samples of the bacterial suspensions were collected and serially diluted in 1× phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄), and aliquots were plated on LB agarose plates. Colonies were counted after overnight incubation at 37°C, and CFU were determined as described previously (8), in appropriately diluted plated samples (30 to 300 CFU). Data were reported as LD₉₀ values, namely, the concentrations of Cr(VI) that killed 90% of the bacterial population.

Induction experiments and microscopy analysis.

B. subtilis cells carrying a translational P_recA-gfp fusion (38) were grown in LB medium to an OD₆₀₀ of 0.5; at that point, the culture was split into three subcultures of equal volumes. One subculture was left untreated, one was supplemented with a dose of Cr(VI) necessary to reach the LD₅₀, and one was treated with M-C (0.5 μg ml⁻¹). The subcultures were shaken at 250 rpm and incubated at 37°C for 1 h. Subsequently, cells collected by centrifugation (15,000 × g for 10 min) were washed twice with 1× PBS and resuspended in 1 ml of that solution. Samples of the bacterial suspensions (100 μl) were serially diluted in 1× PBS and plated to determine CFU. In addition, 200-μl aliquots of the cell suspensions were used to measure the fluorescence intensity of the GFP in a multiwell fluorescence reader (Varioskan; Thermo Scientific, Pittsburgh, PA) at excitation and emission wavelengths of 490 nm and 510 nm, respectively. Emission values were standardized as viable cell counts (CFU). For epifluorescence microscopic analysis, cell samples (0.5 ml) collected from the cultures described above were centrifuged briefly (15,000 × g at 20°C) and resuspended in 50 μl of the solution. Samples spotted onto glass slides were mixed with a poly-l-lysine solution (Sigma-Aldrich, St. Louis, MO) and analyzed by fluorescence microscopy with a Zeiss Axioscope A1 microscope equipped with an AxioCam ICc1 camera. Fluorescence and phase-contrast images were acquired by using AxioVision v4.8.2 software, with adjustment only for brightness and contrast. The excitation and emission wavelengths employed for GFP were 490 and 510 nm, respectively.

β-Galactosidase assays.

Bacillus subtilis strains carrying recA-lacZ (YB3001) or uvrA-lacZ (PERM1126) fusions were grown in liquid LB medium at 37°C to an OD₆₀₀ of 0.5. At that point, the cultures were separated into equal volumes in two sterile flasks; one of the subcultures was left untreated and the other was mixed with a dose of Cr(VI) necessary to reach the LD₅₀. The subcultures were shaken at 37°C for an additional period of 90 min. Cells from 1-ml samples were harvested by centrifugation (15,000 × g for 1 min), and the pellets were washed twice with 50 mM Tris-HCl (pH 7.5). The levels of β-galactosidase in the distinct cell samples were measured using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate, as described by Nicholson and Setlow (62), and were expressed in Miller units (63). The endogenous ONPGase specific activity expressed by the WT strain without a lacZ fusion during growth was determined in parallel and subtracted from the data obtained for strains carrying the recA-lacZ and uvrA-lacZ fusions. These corrections were always ≤10%.

Analysis of mutagenesis induced by Cr(VI).

Mutations of B. subtilis strains to Rif^r in the absence or presence of Cr(VI) added to cultures to reach a LD₅₀ were determined as follows. Overnight LB cultures of each strain were inoculated into flasks containing fresh LB medium and grown to an OD₆₀₀ of 1.0; each culture was then split into two subcultures, which were transferred into different flasks. One of the subcultures was left untreated, and the other was amended with a Cr(VI) concentration necessary to reach the LD₅₀, as follows: WT, 22 ppm; ΔuvrA strain, 17 ppm; ΔyqjH strain, 15 ppm; ΔyqjW strain, 13 ppm; ΔrecA strain, 11 ppm; ΔyqjH ΔyqjW strain, 7 ppm; ΔrecA ΔuvrA strain, 3 ppm. The untreated and Cr(VI)-treated cultures were shaken at 37°C for 16 h. Mutation frequencies were determined with five independent cultures by plating aliquots of each culture, amended with Cr(VI) or not amended, onto six LB plates containing 10 μg ml⁻¹ Rif, as well as plating aliquots of appropriate dilutions onto LB plates without Rif. Rif^r colonies were counted after 24 h of incubation at 37°C.

Detection and quantitation of DNA-protein cross-links.

Induction and detection of DNA cross-linked to protein were performed according to a previously published procedure (3, 41), with modifications. Briefly, B. subtilis WT, PERM1038, PERM985, and PERM1030 strains were grown aerobically in LB medium to an OD₆₀₀ of 1.0, and then each culture was split into two subcultures; one subculture was left untreated, and the other was supplemented with an LD₉₀ of Cr(VI) and incubated for 3 h at 37°C with shaking. Aliquots of 2 ml were collected from the cell cultures and centrifuged at 12,000 × g for 1 min. The cell pellets were washed once with 10 mM Tris-HCl, 300 mM NaCl, 10 mM EDTA (pH 7.4), suspended in 300 μl of the same buffer containing 2.5 mg ml⁻¹ of lysozyme and 1 mM phenylmethylsulfonyl fluoride (PMSF), and incubated for 45 min at 37°C. To complete cell lysis, 0.4 ml of a 20 mM Tris-HCl (pH 7.5), 50 mM EDTA solution supplemented with 2% SDS and 1 mM PMSF was added; this solution was vigorously vortex mixed for 10 s and heated at 65°C for 8 min, and the mixture was stored overnight at −70°C. The next morning, the sample was thawed, vigorously vortex mixed for 10 s, and heated at 65°C for 8 min, followed by the addition of 0.5 ml of 20 mM Tris-HCl (pH 7.5), 200 mM KCl, 50 mM EDTA (buffer A). The suspension was chilled on ice for 8 min and centrifuged at 6,000 × g for 6 min at 4°C. The supernatant was saved and used for quantification of soluble DNA. This wash procedure was repeated three times prior to final resuspension in 0.5 ml of 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM EDTA. The proteins were digested by addition of 0.2 mg ml⁻¹ of proteinase K (Sigma-Aldrich) and incubation at 50°C for 3 h. After addition of 15 μl of ultrapure bovine serum albumin (10 mg ml⁻¹; Sigma-Aldrich), the solution was cooled on ice for 8 min, followed again by centrifugation (10,000 × g for 10 min at 4°C). The final supernatant, containing the DNA cross-linked to protein, and the supernatant from the first wash were treated with 0.2 mg ml⁻¹ of RNase-free DNase (Promega, Madison, WI) for 30 min at 30°C, to remove all RNAs. To determine the formation of DPCs, the DNA content of the first supernatant and the DNA precipitated with SDS-KCl and digested with proteinase K were measured in a multiwell fluorescence reader (Varioskan Flash multimode reader; Thermo Scientific) using Hoechst 33342 dye (Sigma-Aldrich), at excitation and emission wavelengths of 346 nm and 460 nm, respectively. DPC formation was calculated from a standard curve prepared with calf thymus DNA (Invitrogen, Carlsbad, CA). The amounts of DPCs in untreated and Cr(VI)-treated samples were calculated as the ratio of DNA precipitated by SDS-KCl to total DNA (SDS-KCl-precipitated DNA plus soluble DNA) (64).

Statistical analysis.

The results were reported as means ± standard errors of the mean (SEMs). For determination of Cr(VI) toxicity, differences between WT and mutant strains were calculated with the parametric Fisher least significant difference (LSD) test. Cr(VI)-induced mutagenesis and quantitation of DPCs were determined with a paired Wilcoxon signed-rank test. This parametric test was utilized because the data exhibited a normal distribution (using the Shapiro-Wilk test). All tests were performed with the program Statistica v12, and differences were considered statistically significant at P values of <0.05.