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Applied and Environmental Microbiology, April 2005, p. 1843-1849, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1843-1849.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Exposure to Cadmium Elevates Expression of Genes in the OxyR and OhrR Regulons and Induces Cross-Resistance to Peroxide Killing Treatment in Xanthomonas campestris

Peerakan Banjerdkij,^1,2 Paiboon Vattanaviboon,^1* and Skorn Mongkolsuk^1,3*

Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si,¹ Postgraduate Education, Training and Research Program in Environmental Science, Technology and Management, Asian Institute of Technology,² Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand³

Received 30 July 2004/ Accepted 22 October 2004

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of Xanthomonas campestris, a soil and plant pathogenic bacterium. The exposure of X. campestris to low concentrations of cadmium induces cross-protection against subsequent killing treatments with either H₂O₂ or the organic hydroperoxide tert-butyl hydroperoxide (tBOOH), but not against the superoxide generator menadione. The cadmium-induced resistance to peroxides is due to the metal's ability to induce increased levels of peroxide stress protective enzymes such as alkyl hydroperoxide reductase (AhpC), monofunctional catalase (KatA), and organic hydroperoxide resistance protein (Ohr). Cadmium-induced resistance to H₂O₂ is dependent on functional OxyR, a peroxide-sensing transcription regulator. Cadmium-induced resistance to tBOOH shows a more complex regulatory pattern. The inactivation of the two major sensor-regulators of organic hydroperoxide, OxyR and OhrR, only partially inhibited cadmium-induced protection against tBOOH, suggesting that these genes do have some role in the process. However, other, as yet unknown mechanisms are involved in inducible organic hydroperoxide protection. Furthermore, we show that the cadmium-induced peroxide stress response is mediated by the metal's ability to predominately cause an increase in intracellular concentrations of organic hydroperoxide and, in part, H₂O₂. Analyses of various mutants of peroxide-metabolizing enzymes suggested that this increase in organic hydroperoxide levels is, at least in part, responsible for cadmium toxicity in Xanthomonas.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Heavy metals are recognized as environmental pollutants and are released from both industrial and agricultural sources. The intensive use of high-phosphate fertilizers in agriculture leads to an increased accumulation of metal ions, especially cadmium, in the soil (34). Cadmium ions are highly toxic to ecosystems, even at very low concentrations. Although cadmium scores negative in the Ames Salmonella mutagenicity test, it is a carcinogen in laboratory animals and can induce DNA deletions in Saccharomyces cerevisiae (28). Cadmium is a non-redox-reactive heavy metal, and its toxicity is believed to be due to the depletion of glutathione and sulfhydryl groups in proteins (12, 31). Furthermore, cadmium is known to displace Zn and Fe ions from metalloproteins, resulting in their inactivation as well as the release of free Fe that can then catalyze the generation of reactive oxygen species via the Fenton reaction (30).

The effects of cadmium exposure have been examined in some microorganisms. Studies of S. cerevisiae indicated that cadmium increases oxidative stress, since strains deficient in antioxidant defense enzymes have a high sensitivity to cadmium and cells grown in the absence of oxygen are more tolerant to cadmium (5, 39). The exposure of bacteria to cadmium induces the expression of genes in many regulons, including genes involved in metal transport (1, 4, 27), DNA repair, the heat shock response, and the oxidative stress response (2, 10, 35).

Xanthomonas campestris pv. phaseoli is a soil bacterium and plant pathogen that often encounters reactive oxygen species (superoxide anions, H₂O₂, and organic hydroperoxides) that are generated either by other soil microorganisms or by host plants as a defense mechanism during microbial invasion (17). Thus, Xanthomonas uses both enzymatic and nonenzymatic strategies to survive and proliferate in the presence of these reactive oxygen species. It logically follows that any agents that affect the bacterial oxidative stress response may also alter the organism's ability to survive in the soil environment as well as affecting its pathogenicity. The peroxide-sensing transcription regulators OxyR and OhrR mediate the peroxide stress responses in bacteria (20). For Xanthomonas, exposure to hydroperoxide leads to OxyR oxidation, which in turn activates the expression of ahpC, ahpF, katA, and oxyR itself (21). OhrR is an organic hydroperoxide sensor and transcription repressor that regulates the expression of ohr, encoding a thiol peroxidase (22). These regulators and the products of the genes that they control play crucial roles in the adaptive and cross-protective responses to peroxide stress in Xanthomonas (20).

For this study, the effects of cadmium on the Xanthomonas peroxide stress protective response and the expression of genes in the oxyR and ohrR regulons were investigated. A possible mechanism for cadmium induction of peroxide stress-dependent gene expression is discussed.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains and growth conditions.
X. campestris pv. phaseoli was grown aerobically in Silva-Buddenhagen (SB) medium (0.5% sucrose, 0.5% yeast extract, 0.5% peptone, 0.1% glutamic acid; pH 7.0) at 28°C. Overnight cultures were inoculated into fresh SB medium to give an optical density at 600 nm (OD₆₀₀) of 0.1. Exponential-phase cells (OD₆₀₀ of 0.5 after 4 h) were used in all experiments.

Construction of X. campestris pv. phaseoli oxyR ohrR double mutant.
Genomic DNA extracted from X. campestris pv. phaseoli oxyR (Gen^r) (26) was transferred into X. campestris pv. phaseoli ohrR (Amp^r) (33) by electroporation as previously described (26). Extracts of the oxyR ohrR double mutant (Gen^r Amp^r) were analyzed by Western blotting to confirm the absence of expression of OhrR and OxyR (data not shown).

Determination of cadmium resistance levels.
Analyses of the killing effects of various reagents on Xanthomonas strains were performed by the use of inhibition zone assays (23). Exponential-phase cultures (1 ml) were mixed with 10.0 ml of molten top agar (SB medium containing 0.7% agar) held at 50°C and then overlaid onto SB plates (14-cm-diameter petri dishes containing 40 ml of SB agar). The plates were left at room temperature for 15 min to let the top agar solidify. Sterile 6-mm-diameter paper disks, soaked with 5 µl of CdCl₂ (0.2 M), were placed on top of the lawn of cells, and the zones of growth inhibition were measured after 24 h of incubation at 28°C.

Determination of the effect of cadmium on oxidant killing treatments.
Cadmium-induced cross-protection experiments were performed by adding 75 µM CdCl₂ to exponential-phase cultures. These cultures were grown for an additional 30 min before aliquots of cells were removed and treated with killing concentrations (10, 20, and 50 mM) of H₂O₂ or tert-butyl hydroperoxide (tBOOH) for 30 min. After treatment, the cells were removed and washed once with fresh SB medium before determinations of cell survival by plating appropriate dilutions on SB agar. Colonies were counted after 48 h of incubation at 28°C. The percent survival was defined as the number of CFU recovered after treatment divided by the number of CFU prior to treatment multiplied by 100.

Northern analysis of ahpC and ohr expression.
Exponential-phase cultures were induced with 75 µM CdCl₂ for 15 min. Cells were harvested by centrifugation at 4°C. Total RNAs were extracted from uninduced and cadmium-induced cultures by a modified hot acid phenol method (21). Purified RNAs (10 µg) were separated in a formaldehyde-agarose gel. After electrophoretic separation, the RNAs were transferred to a piece of nylon membrane and hybridized with a radioactively labeled ahpC or ohr DNA probe, prepared as previously described (23). Prehybridization, hybridization, and high-stringency washes were done according to the methods of Mongkolsuk et al. (21).

Western immunoblot analysis.
Western immunoblot analyses of AhpC levels, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotting to polyvinylidene difluoride membranes, and immunodetection with an anti-Escherichia coli AhpC polyclonal antibody (32), were performed as previously described (21). Antibody reactions were visualized by use of an alkaline phosphatase antibody detection kit from Promega.

Catalase activity gels.
Crude bacterial lysates were prepared by resuspending X. campestris cell pellets in 50 mM sodium phosphate buffer (PB), pH 7.0, containing 1 mM phenylmethylsulfonyl fluoride and exposing them to intermittent sonication until the suspensions became clear. The lysates were then centrifuged at 10,000 x g for 10 min, and the cleared supernatants (80 µg of protein) were separated in 7.5% native polyacrylamide gels (37). After electrophoresis, the gels were soaked in PB containing 50 µg of horseradish peroxidase/ml for 45 min at room temperature. The gels were then soaked in PB containing 5 mM H₂O₂ for 10 min. After being washed briefly twice with distilled water, they were immediately stained with freshly prepared PB containing 0.5 mg of diaminobenzidine/ml until the background became dark brown. Catalase activity appeared as a colorless band.

SOD assays.
The xanthine-xanthine oxidase-coupled reduction of cytochrome c was used to monitor total superoxide dismutase (SOD) activities (19) in crude lysates. One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of reduction of cytochrome c by 50%.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cadmium induces cross-protection from killing treatments with H₂O₂ and tBOOH.
Exposure to low levels of a stress or chemical can induce increased resistance to a subsequent exposure to the same (adaptive) or an unrelated (cross-protective) stress or chemical. Bacterial adaptive and cross-protective responses are an important physiological adaptation for survival under stressful conditions and have been shown to be crucial protective mechanisms during peroxide stress (25, 36). Thus, we tested the effect of exposure to low cadmium concentrations on the physiological responses of X. campestris pv. phaseoli to peroxide stress. First, the effect of cadmium on the H₂O₂ resistance level was determined. Xanthomonas cultures were preexposed to 75 µM CdCl₂, and the percent survival following a subsequent treatment with a lethal concentration (10, 20, or 50 mM) of H₂O₂ was determined. The results showed that for cadmium-pretreated cells, the percent survival upon exposure to lethal concentrations of H₂O₂ was >10⁵-fold higher than that for nontreated cells (Fig. 1A). Cadmium-induced cross-protection against H₂O₂ has been observed in E. coli, for which pretreatment of the bacterial culture with CdCl₂ increases resistance to multiple stresses (10). Further experiments were done to determine whether cadmium could induce resistance to other oxidative stress-inducing compounds, such as organic hydroperoxide and superoxide anions. Cadmium-pretreated and nontreated cultures were treated with lethal concentrations of the organic hydroperoxide tBOOH and the superoxide-generating agent menadione (MD), and the percentages of survival were determined. Interestingly, cadmium-pretreated cells were 10⁴-fold more resistant to subsequent lethal treatments with tBOOH (Fig. 1B) than nontreated cells, while no induced cross-protection from MD was observed (Fig. 1C).

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FIG. 1. Cadmium-induced cross-protection in X. campestris pv. phaseoli. The growth, induction, and lethal peroxide treatment of cultures were performed as described in Materials and Methods. Survival curves are shown for X. campestris pv. phaseoli cultures pretreated with CdCl2 (•) or left untreated () before being exposed to the indicated lethal concentrations of H2O2 (A), tBOOH (B), and MD (C). Values presented are the means and standard deviations of three replicates.

In Xanthomonas, there are at least two global peroxide-sensing transcription regulators, OxyR and OhrR, involved in mediating peroxide stress-inducible responses (20). OxyR is essential for most peroxide stress responses and is involved in induced adaptation against H₂O₂ killing, while OhrR regulates the expression of ohr in response to organic hydroperoxides (20). In order to test whether the cadmium-induced cross-protection against H₂O₂ and tBOOH killing was dependent on OxyR and/or OhrR, we determined the effect of a cadmium pretreatment on the resistance to H₂O₂ and tBOOH in an X. campestris pv. phaseoli oxyR mutant (24) and an X. campestris pv. phaseoli ohrR mutant (23). The results in Fig. 2A clearly show that cadmium-induced cross-protection from H₂O₂ was completely abolished in the oxyR mutant. In contrast, the induced cross-protection against tBOOH killing treatments was only partially lost, since cadmium-pretreated X. campestris pv. phaseoli oxyR was 100-fold more resistant to tBOOH than the nontreated control (Fig. 2B). The inactivation of ohrR had no effect on cadmium-induced H₂O₂ protection (data not shown). However, the loss of OhrR led to a reduction in the level of cadmium-induced protection against tBOOH (Fig. 2C). An analysis of the peroxide regulatory mutants clearly showed that the cadmium-induced cross-protection from H₂O₂ was wholly dependent on functional OxyR, while the cadmium-induced cross-protection from tBOOH was more complex and appears to involve genes in both the OxyR and OhrR regulons. These observations were extended by examining the cadmium-induced cross-protection from tBOOH of the oxyR ohrR double mutant (Fig. 2D). Cadmium-pretreated X. campestris pv. phaseoli oxyR ohrR cells were 50-fold more resistant to tBOOH than nontreated cells; however, the relative difference between the resistance levels of the pretreated and nontreated oxyR ohrR cells was less than that observed for either X. campestris pv. phaseoli oxyR or X. campestris pv. phaseoli ohrR (Fig. 2B, C, and D). These data reinforce the suggestion that genes in both the OxyR and OhrR regulons are required for high-level cadmium-induced resistance to tBOOH. Moreover, the fact that the oxyR ohrR double mutant could still mount a partial cadmium-induced cross-protective response to tBOOH indicates that other unknown gene systems participate in the process. Likely candidates for this role are genes encoding glutathione peroxidase-like and bactoferritin comigratory proteins (13, 15), which are found in the X. campestris genome and are known to be involved in organic hydroperoxide metabolism (8). These genes, in addition to genes in the OxyR and OhrR regulons, are likely to play some role in the detoxification of and protection against organic hydroperoxides. The regulatory mechanisms controlling these genes have not yet been characterized, so it is not possible to firmly assign them roles in the cadmium-induced cross-protective response to tBOOH.

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FIG. 2. Cadmium-induced cross-protection in various X. campestris pv. phaseoli mutant strains lacking peroxide stress response regulators. Survival curves are shown for exponential-phase cultures of the oxyR mutant (Xp oxyR) (A and B), the ohrR mutant (Xp ohrR) (C), and the oxyR ohrR double mutant (Xp oxyR ohrR) (D) pretreated with CdCl2 (•) or left untreated () prior to exposure to lethal concentrations of H2O2 or tBOOH at the indicated concentrations. The values presented are the means and standard deviations of three replicates.

Exposure to cadmium induces high-level expression of peroxide-scavenging enzymes.
The ability of bacteria to protect themselves from peroxide stress is often associated with inducible increases in the levels of peroxide detoxification and protection enzymes (25, 36). Since the physiological data indicated that high-level cadmium-induced protection against peroxide depended on functional peroxide-sensing regulators, the effects of cadmium on the levels of peroxide and superoxide detoxification enzymes were examined. Catalases are the major protective enzymes against H₂O₂ toxicity. X. campestris possesses two monofunctional catalase isozymes, denoted KatA and KatE (37). The KatA catalase is peroxide inducible and is produced during all growth phases (S. Mongkolsuk, unpublished data). KatE is a growth-phase-dependent enzyme whose expression increases as cells enter stationary phase (37). The effect of cadmium exposure on catalase levels in X. campestris was determined by catalase activity staining of polyacrylamide gels, which can differentiate between the two isozymes. The results, shown in Fig. 3A, revealed that the exposure of X. campestris to 75 µM CdCl₂ for 30 min induced a 10-fold increase in the KatA level, as estimated by densitometry. The cadmium-dependent induction of KatA required functional oxyR since its expression was abolished in the Xanthomonas oxyR mutant (Fig. 3A). We have shown previously that in Xanthomonas the level of resistance to H₂O₂ is correlated with the catalase activity (38). The increased level of KatA resulting from exposure to cadmium likely accounts for the observed cadmium-induced cross-protection against H₂O₂ killing. Cadmium-induced protection from organic hydroperoxides showed complex physiological and regulatory patterns. Therefore, the effects of cadmium on the expression of the OhrR-regulated organic hydroperoxide detoxification enzymes alkyl hydroperoxide reductase and Ohr were also evaluated. Alkyl hydroperoxide reductase consists of two subunits, a catalytic subunit, AhpC, and a reductase subunit, AhpF. Western blot analysis with anti-E. coli AhpC was performed to determine the level of AhpC in lysates prepared from cadmium-pretreated and nontreated cultures. As shown in Fig. 3B, the level of AhpC increased 10-fold after the culture was exposed to 75 µM cadmium for 30 min, while no cadmium-dependent induction of AhpC was observed for the oxyR mutant (Fig. 3B). The effect of cadmium exposure on ohr expression was investigated. Northern blots of total RNAs, extracted from X. campestris cultures challenged with cadmium for 15 min, were probed with a ³²P-labeled ohr-specific DNA probe. Densitometer analysis of the Northern results showed an eightfold induction of ohr mRNA levels in cadmium-induced cultures compared to those in uninduced cultures (Fig. 3C). The cadmium-dependent induction of ohr expression was abolished in the ohrR mutant (data not shown). Clearly, cadmium is a potent inducer of peroxide-scavenging enzymes, and this effect is dependent on the functional global peroxide sensor-transcription regulators OxyR and OhrR. The data also indicated that the cadmium-induced cross-protection from peroxide killing was due to cadmium's ability to induce the high-level expression of these peroxide-scavenging enzymes.

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FIG. 3. Induction of peroxide-scavenging enzymes. The levels of KatA catalase (A), AhpC (B), Ohr (C), and SOD (D) in cultures of X. campestris pv. phaseoli (Xp) and its oxyR mutant (Xp oxyR), pretreated with the indicated concentrations of CdCl2, were determined by activity gel staining (for KatA and SOD) and Western blot analysis (for AhpC and Ohr) as described in Materials and Methods.

The inability of cadmium to induce a protective response against MD killing was surprising since we have previously shown that elevated levels of AhpCF can prevent MD toxicity in an X. campestris oxyR mutant (36). However, it has also been shown that residual organic peroxides in combination with MD can dramatically enhance MD's toxicity (29). It is possible that a pretreatment with cadmium at 75 µM continuously generates large quantities of organic hydroperoxides, as evident by its ability to deactivate the OhrR repressor, resulting in increased expression of ohr. Therefore, the increased AhpCF levels in cadmium-pretreated X. campestris cells may not be sufficient to confer additional protection against MD killing. In Xanthomonas, as in other bacteria, SODs play a crucial role in protecting cells against superoxide anion toxicity. A genome analysis of X. campestris revealed five putative open reading frames that have high homology scores to genes for known SODs (8). The effect of the cadmium pretreatment of X. campestris cultures on the total SOD activity was determined. As expected, the results revealed that a pretreatment with cadmium failed to increase the levels of total SOD activity (Fig. 3D). Thus, the inability of the metal to induce SODs may be responsible for the observed lack of cadmium-induced resistance to MD killing.

High levels of peroxide-scavenging enzymes modulate the cadmium-induced stress response.
During the Xanthomonas peroxide stress response, the oxidation of OxyR by peroxides is responsible for the transcriptional activation and high-level expression of genes in its regulon, including ahpCF and katA (18, 21, 38). Furthermore, the oxidation of OhrR by organic hydroperoxides, such as tBOOH and cumene hydroperoxide, derepresses the expression of ohr (22). The data shown in Fig. 3 indicate that cadmium is a potent inducer of peroxide-scavenging enzymes. This probably results from the oxidation of OxyR and OhrR and the subsequent activation of genes in their respective regulons. The mechanisms by which cadmium treatment leads to the oxidation of OxyR and OhrR are not known. Cadmium is classified as a non-redox-reactive heavy metal; however, in eukaryotic cells it has been shown to generate intracellular oxidative stress (5, 30). Thus, cadmium may oxidize OxyR and OhrR, either indirectly or directly, through its ability to generate intracellular superoxide anions and/or peroxides (both H₂O₂ and organic hydroperoxides). For Xanthomonas, it was shown previously that the expression of ohr is specifically induced by exposure to organic hydroperoxides (23). We reasoned that if exposure to cadmium causes the in vivo generation of organic hydroperoxides that in turn oxidize OhrR, then the high-level expression of an organic hydroperoxide-scavenging enzyme such as AhpCF should reduce the in vivo concentration of organic hydroperoxide, leading to a reduction in the level of oxidized OhrR and a corresponding decrease in the level of ohr expression. Exponential-phase cultures of X. campestris harboring pAhpCF (an expression vector carrying ahpC and ahpF) (21) and control cultures harboring the empty vector were treated with 0, 50, and 75 µM CdCl₂ for 10 min before the total RNAs were extracted and Northern blot hybridization was performed with a ³²P-labeled ohr probe. The results shown in Fig. 4A indicate that cadmium induced a high-level expression of ohr in cells containing the vector alone. In contrast, in cells overexpressing AhpCF from a plasmid, the level of ohr mRNA, as determined by densitometry, was reduced fivefold (for 50 and 75 µM CdCl₂ treatments) relative to X. campestris containing the vector alone (Fig. 4A). These results strongly favor the hypothesis that the exposure of X. campestris to cadmium increases the generation of organic hydroperoxides. Using a similar rationale, we conducted further experiments to determine how cadmium treatment leads to the oxidation of OxyR. We reasoned that if a cadmium treatment results in the production of H₂O₂, then X. campestris harboring pKatA (carries katA) (6), which expresses high levels of the H₂O₂-metabolizing enzyme catalase, should have lower levels of cadmium-induced ahpC expression than a strain harboring the vector alone. However, if organic hydroperoxide alone is responsible for the oxidation of OxyR, then the magnitude of ahpC induction should not be affected. Cultures of X. campestris and X. campestris harboring pKatA were treated with various concentrations of cadmium. Northern blots generated with mRNAs isolated from these cultures were hybridized with an ahpC-specific DNA probe. Densitometer analysis of the ahpC-specific Northern blot shown in Fig. 4B indicated that the KatA-overexpressing strain contained 50% less ahpC mRNA than the wild-type strain after treatment with 75 µM cadmium. The Northern blot results suggest that cadmium-induced production from H₂O₂ contributes significantly to the oxidation of OxyR and the subsequent activation of katA and ahpC expression in Xanthomonas.

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FIG. 4. Effect of high levels of peroxide-scavenging enzymes on cadmium-induced ohr and ahpC expression. Northern blot results are shown for RNAs prepared from cultures of X. campestris pv. phaseoli harboring pBBR1MSC-4 (Xp), pAhpCF for the overexpression of AhpCF (Xp/pAhpCF), or pKat for the overexpression of KatA (Xp/pKatA) that had been pretreated with the indicated concentrations of CdCl2 and hybridized with radioactively labeled ohr (A)- and ahpC (B)-specific DNA probes. Arrowheads indicate ohr- and ahpC-specific mRNAs. BH represents induction with 100 µM tBOOH.

Taken together, these results indicate that both organic hydroperoxide and H₂O₂ resulting from cadmium treatment are responsible for the oxidation of OhrR and OxyR and the subsequent enhanced expression of genes in these regulons.

Inactivation of oxyR and ohrR increases cadmium sensitivity.
The ability of cadmium to generate reactive oxygen species (ROS), including organic hydroperoxides, suggests that oxidative stress may be one of the mechanisms responsible for cadmium toxicity in vivo. This notion is consistent with previous reports, mostly for eukaryotic systems, indicating that exposure to cadmium increases the levels of ROS (5). In order to examine this possibility in Xanthomonas, we determined the cadmium resistance levels in several peroxide stress response mutants by using an inhibition zone assay. The ohrR and oxyR mutants showed equal resistances to cadmium by giving inhibition zones of 25 mm in diameter, which were significantly larger (less resistant) than that of wild-type X. campestris pv. phaseoli (22 mm). Furthermore, the oxyR ohrR double mutant was more sensitive to cadmium than either the oxyR or ohrR single mutant, as judged by its inhibition zone of 27 mm. This evidence is consistent with investigations of E. coli and Salmonella enterica serovar Typhimurium (16) and indicates that genes in oxidative stress protective pathways contribute significantly to bacterial resistance to cadmium and that the mechanism of cadmium toxicity involves the production of toxic levels of peroxides. The data also indicate that the OhrR and OxyR regulons each function in a somewhat specialized, nonredundant manner to protect cells from cadmium toxicity. We extended the investigation to determine the cadmium resistance levels in ahpC, katA, and ohr mutant strains. Interestingly, the inactivation of ahpC and ohr, encoding organic hydroperoxidases, resulted in a significant reduction in the cadmium resistance levels, while the inactivation of katA, encoding catalase, had no effect (data not shown). This suggested that H₂O₂ has a minor role in the process and that organic hydroperoxides are the major ROS generated as a consequence of cadmium exposure. This idea is supported by the Northern hybridization results reported earlier showing that the overexpression of AhpCF had a larger negative effect on the cadmium-dependent induction of ohr than the overexpression of KatA had on the cadmium-dependent induction of ahpC (i.e., 10-fold versus 2-fold, respectively, with 75 µM CdCl₂). How then might exposure to cadmium lead to an increase in the levels of lipid hydroperoxides? Cadmium is classified as a non-redox-active metal, and thus the ability of the metal to directly cause peroxidation of membrane lipids resulting in the production of lipid hydroperoxides has only a minor role (14, 31). Cadmium is very reactive toward sulfhydryl groups and causes the depletion of glutathione and the inactivation of enzymes (3, 11). Thus, cadmium ions that enter the cytoplasm likely cause an increase in the level of lipid hydroperoxides by inhibiting enzymes involved in their metabolism, such as AhpC and Ohr. Both AhpC and Ohr have a cysteine residue at their active site, and mutations in these cysteine residues have been shown to inactivate these enzymes (7, 9). In addition, the depletion of glutathione, a common electron donor for oxidative stress protective enzymes, may lead to oxidative stress conditions, and the generated ROS may react directly with membrane lipids, resulting in the increased production of organic hydroperoxides. Clearly, the cadmium-dependent induction of oxidative stress is a complex process resulting from interactions of the metal with multiple enzyme systems. Ongoing efforts in our laboratory are focusing on identifying the ROS produced in response to cadmium exposure as well as more clearly defining the protective roles of enzymes within the OxyR and OhrR regulons.

 
We thank J. M. Dubbs for a critical reading of the manuscript.

This research was supported by a Research Team Strengthening Grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC) and senior research scholar grant RTA4580010 from the Thailand Research Fund to S.M. and by a grant from the ESTM under the Higher Education Development Project of the Ministry of University Affairs.

 
* Corresponding author. Mailing address: Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Phone: (662) 574-0630, ext. 3816. Fax: (662) 574-2027. E-mail for Paiboon Vattanaviboon: paiboon{at}tubtim.cri.or.th. E-mail for Skorn Mongkolsuk: skorn{at}tubtim.cri.or.th.

* Corresponding author. Mailing address: Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand. Phone: (662) 574-0630, ext. 3816. Fax: (662) 574-2027. E-mail for Paiboon Vattanaviboon: paiboon{at}tubtim.cri.or.th. E-mail for Skorn Mongkolsuk: skorn{at}tubtim.cri.or.th.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, April 2005, p. 1843-1849, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1843-1849.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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