Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand1
Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand2
Author for correspondence: Skorn Mongkolsuk. Tel: +662 574 0622 ext.1402. Fax: +662 574 2027. e-mail: skorn{at}tubtim.cri.or.th
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ABSTRACT |
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Keywords: methylglyoxal, N-ethylmaleimide, resistance, catalase
Abbreviations: MG, methylglyoxal; NEM, N-ethylmaleimide; SB, SilvaBuddenhagen
a Present address: Department of Biotechnology, Faculty of Engineering, Osaka University, Osaka, Japan.
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INTRODUCTION |
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In nature, bacteria are exposed to toxic electrophiles both from within the cell and from their environment. Methylglyoxal (MG) is the major electrophile produced intracellularly by bacterial cells. Millimolar concentrations of MG are produced during growth under certain conditions (Ferguson et al., 1998b , 1999
). In addition, electrophiles are released into the environment in the form of herbicides and chemicals used in the poultry industry (Stevens et al., 1995
; Zablotowicz et al., 1995
). The toxicities of these compounds are believed to stem from their interactions with nucleophilic centres of macromolecules. Electrophiles react with amino acid residues such as arginine, lysine and cysteine (Lo et al., 1994
). These compounds are known also to react with DNA and are highly mutagenic (Ferguson et al., 1998a
; Ferguson et al., 2000
). Electrophiles must be rapidly detoxified to prevent cellular and genetic damage. In bacteria, MG and NEM detoxification involves glutathione. MG is detoxified by glutathione-dependent glyoxalase systems (Ferguson et al., 1995
; MacLean et al., 1998
). In addition, both MG and NEM glutathione adducts activate the potassium transport system, KefB/KefC, resulting in acidification of the cytoplasm and protection against electrophile killing (Ferguson et al., 1995
).
Recently, we have shown that electrophiles such as NEM can modulate the oxidative stress responses in Xanthomonas spp. (Mongkolsuk et al., 1997b ; Vattanaviboon et al., 1999
). Exposure to low concentrations of NEM was shown to induce high levels of the peroxide-scavenging enzymes alkyl hydroperoxide reductase subunit C and catalase in an OxyR-dependent manner. This gives high-level protection against peroxide toxicity. Exposure of bacteria to low concentrations of some compounds can induce protection to subsequent exposure to lethal concentrations of the same compound (adaptive) or of other non-related compounds (cross-protection). These responses are important strategies for bacterial survival under stressful conditions. Thus, exposure to electrophiles will affect various physiological processes. Here, we determined the effects of pre-exposure to low concentrations of electrophiles on the response to subsequent exposure to lethal concentrations of electrophiles. A novel protective mechanism against electrophile toxicity was also investigated.
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METHODS |
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Quantitative determination of resistance levels to oxidants.
The induced adaptive and cross-protection response experiments were performed by adding either 0·01% (w/v) MG, 100 µM NEM or 100 µM iodoacetamide to exponential-phase X. c. pv. phaseoli cultures. The cultures were allowed to grow for 1 h before aliquots of cells were treated with lethal concentrations of MG (0·1% w/v) or NEM (1·0 mM). At the indicated times, samples were removed and washed twice with fresh SB medium before cells were plated on SB agar. Colonies were counted after 36 h incubation at 28 °C. Surviving fractions are defined as the number of c.f.u. recovered after the treatment divided by the number of c.f.u. prior to the treatment. To test the effect of sodium pyruvate on electrophile killing, experiments were performed as described, except that cells were plated on SB agar with and without 10 mM sodium pyruvate. The effects of hydroxyl radical scavengers were determined as previously described (Vattanaviboon & Mongkolsuk, 1998 ) by exposing X. c. pv. phaseoli cultures to protecting concentrations of DMSO (0·4 M) and glycerol (1·0 M) for 30 min prior to exposure to lethal concentrations of NEM (1·0 mM). All experiments were repeated at least four times and means and standard deviations are shown.
Qualitative determination of levels of resistance to lethal concentration of NEM.
The levels of resistance against a lethal concentration of NEM was qualitatively determined by using the inhibition zone method (Mongkolsuk et al., 2000 ). Exponential-phase cells (108 c.f.u. ml-1) were mixed with SB top agar and overlaid onto SB agar plates. Then 6 mm diameter filter paper discs soaked with 5 µl of 10 mM NEM were placed on top of the Xanthomonas cell lawn. The zone of growth inhibition was measured after 24 h incubation.
Catalase activity gel and assays.
Total catalase activity was assayed as previously described (Mongkolsuk et al., 1996 ). Preparation of cell lysates for enzyme assays and catalase activity gels were prepared according to Mongkolsuk et al. (1996)
. Essentially, cells were pelleted and washed once with 50 mM sodium phosphate buffer pH 7·0 and lysed by brief sonication followed by centrifugation at 10000 g for 10 min. Cleared lysates were used for enzyme assay and catalase activity gels. Catalase isozymes were visualized on native PAGE gels as described by Vattanaviboon & Mongkolsuk (2000)
. After electrophoresis gels were immersed at room temperature for 45 min in 50 mM sodium phosphate buffer pH 7·0 containing 50 µg ml-1 horseradish peroxidase (Sigma) and then in 50 mM sodium phosphate buffer pH 7·0 containing 5 mM H2O2 for 10 min. Subsequently, each gel was washed twice and finally treated with 0·5 mg ml-1 diamino benzidine. Catalase activity appeared as colourless bands against a dark brown background.
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RESULTS AND DISCUSSION |
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The different electrophile-induced adaptive responses were unexpected. They suggested that X. c. pv. phaseoli has evolved the ability to mount an adaptive response against a man-made electrophile NEM but not against an endogenously produced one, MG. The ability of NEM to induce high-level protection against both MG and itself implied that it is a potent inducer of a pathway which can detoxify both NEM and MG. In contrast, MG is not an inducer of this detoxification pathway(s) and is unable to confer protection against itself. Since MG-induced protection against NEM was found to be independent of new protein synthesis, MG probably effects some modification of existing cellular components that results in an increase in resistance to NEM killing. A likely mechanism for MG-induced resistance is via the KefB/KefC system induced by MG/glutathione conjugates, resulting in cytoplasmic acidification and protection against NEM killing (Ferguson et al., 1995 ; Ferguson, 1999
). This process is independent of new protein synthesis (Ferguson et al., 1995
). In Xanthomonas, unlike E. coli, cytoplasm acidification is not sufficient to confer protection against MG toxicity, as shown by lack of MG-induced adaptation. In addition, the inability of MG to induce high-level protection against electrophile killing could be due to the ability of the bacteria to metabolize MG effectively. This could prevent the intracellular concentration of MG from reaching the critical level required to activate a protective pathway(s), while NEM is more slowly metabolized; so its concentration could increase rapidly to the level needed to induce the protective pathways. The data show that in Xanthomonas, NEM and MG toxicity arise from several routes and there are protective pathways against these compounds. Variation in the ability of each electrophile to induce a protective response(s) is likely to have significant physiological effects.
Catalase has important protective roles against electrophile killing
We were interested to determine the mechanisms responsible for induced adaptive and cross-protection responses to MG and NEM killing. We have shown that exposure of Xanthomonas spp. to low concentrations of NEM induces cross-protection to H2O2 killing (Vattanaviboon et al., 1999 ). NEM induces a more than 10-fold increase in each of the peroxide-scavenging enzymes, catalase and alkyl hydroperoxide reductase subunit C (AhpC) in an OxyR (a peroxide sensor and transcription regulator)-dependent manner (Vattanaviboon et al., 1999
). This mechanism is likely to be responsible for NEM-induced cross-resistance to peroxide killing. We have observed that a X. c. pv. phaseoli H2O2-resistant mutant has a more than 200-fold increase in both catalase and alkyl hydroperoxide reductase activities and is more resistant to NEM killing (Fuangthong & Mongkolsuk, 1997
). E. coli strains with suppressors of oxyR mutants which have high levels of catalase and AhpC show reduced susceptibility to NEM killing (Greenberg & Demple, 1988
). We suspect that peroxide-scavenging enzymes might help protect against electrophile killing. We tested this idea by determining the resistance levels to MG and NEM killing in X. c. pv. phaseoli harbouring recombinant plasmids containing genes for peroxide-scavenging enzymes. X. c. pv. phaseoli harbouring pUFR047 (vector alone), pahpCF [containing genes for alkyl hydroperoxide reductase C and F subunits, ahpC and ahpF (Loprasert et al., 1997
; Mongkolsuk et al., 1997a
)], pkat [containing the monofunctional catalase gene, katX (Mongkolsuk et al., 1996
)], pohr [containing a gene conferring organic peroxide resistance (Mongkolsuk et al., 1998a
)] were treated with lethal concentrations of MG and NEM. The results (Fig. 2a
, b
) clearly showed that only cells expressing high levels of catalase were protected from MG and NEM killing. High-level expression of genes for all other peroxide-scavenging enzymes tested had no protective effect. The ability of catalase to protect bacterial cells against MG and NEM killing is a novel finding and could be generally important in other bacteria.
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These observations raised the question of how catalase protects cells from electrophile toxicity. H2O2 is the only substrate for monofunctional catalase. It is unlikely that the enzyme can react directly with either MG or NEM, suggesting that MG and NEM toxicity might reflect accumulation and/or increased production of H2O2. Studies on MG and NEM toxicity in other bacteria have not conclusively shown that H2O2 is involved in electrophile-induced toxicity (Ferguson et al., 1995 , 1998a
; Ferguson, 1999
). Nevertheless, there is evidence that oxidative stress might be involved in electrophile toxicity. For example, expression of an oxidative stress protective gene (dps) has been shown to increase resistance to electrophile killing (Ferguson et al., 1998a
). Support for the role of H2O2 in electrophile toxicity came from two observations. First, addition of 10 mM sodium pyruvate [a compound that chemically inactivates peroxide (Nath et al., 1995
)] to the growth medium increased X. c. pv. phaseoli resistance levels more than 100-fold to MG and NEM killing (Fig. 4
). Secondly, H2O2 killing of bacteria involves production of highly reactive hydroxyl radicals. In Xanthomonas, compounds which absorb hydroxyl radicals protect cells from H2O2 killing (Vattanaviboon & Mongkolsuk, 1998
). The effects of hydroxyl radical scavengers (DMSO and glycerol) on NEM killing was investigated. The results in Fig. 5(a)
show that both DMSO and glycerol produced around 10-fold protection against NEM killing. The question as to how exposure to NEM or MG leads to an increase in the level of H2O2 remains obscure. Electrophiles are known to react with amino acids in proteins, especially with cysteine residues (Lo et al., 1994
). Many peroxide-scavenging enzymes, such as alkyl hydroperoxide reductase or the regulator of the peroxide stress response OxyR, have cysteine residues at their active sites (Loprasert et al., 1997
; Mongkolsuk et al., 1997a
). Thus, electrophiles could react directly with cysteine residues at active sites of these proteins and inactivate their biological functions. This, in turn, would reduce the cells ability to detoxify toxic peroxides, leading to intracellular accumulation of these compounds. Glutathione is an important component of electrophile-detoxification systems. High levels of glutathione have protective effects against electrophile toxicity (Ferguson et al., 1995
). Electrophiles could lower the ratio of reduced versus oxidized glutathione. Depletion of glutathione would directly affect the rate of electrophile metabolism, electrophile resistance levels and the redox balance of the cell. The condition not only reduces the rate of electrophile metabolism and the associated resistance level but also the inducing oxidative stress condition(s). These factors might be expected to combine to increase intracellular concentrations of H2O2. Thus, a thiol reagent such as iodoacetamide that causes depletion of glutathione (Kondo et al., 1987
) is expected to affect electrophile killing of the bacteria. Xanthomonas cultures were pretreated with 100 µM iodoacetamide prior to exposure to lethal concentrations of NEM. The results clearly showed that iodoacetamide induced high-level (1000-fold) protection against NEM killing (Fig. 5b
). Iodoacetamide caused over 10-fold induction of total catalase activity (data not shown). In addition, we observed that a reducing agent such as DTT partially reversed the effects of NEM-induced protection against itself (Fig. 1a
). Thus, in cells that produce high levels of catalase, or when grown in media with high levels of pyruvate or hydroxyl radical scavengers (DMSO or glycerol), H2O2 accumulation is prevented. This results in the observed increase in resistance to electrophile killing.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 June 2000;
revised 15 August 2000;
accepted 26 September 2000.
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