Glutathione-Dependent Conversion of N-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

The electrophile N-ethylmaleimide (NEM) elicits rapid K⁺ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K⁺ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD⁺-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K⁺ efflux systems.     

Activation potassium efflux from Escherichia coli by glutathione metabolites

The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.     

Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems

KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.     

Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels.

The role of the KefB and KefC potassium efflux systems in protecting Escherichia coli cells against the toxic effects of the electrophile N-ethylmaleimide has been investigated. Activation of KefB and KefC aids the survival of cells exposed to high concentrations (> 100 microM) of NEM. High potassium concentrations reduce the protection afforded by activation of KefB and KefC, but the possession of these systems is still important under these conditions. The Kdp system, which confers sensitivity to the electrophile methylglyoxal, did not affect the survival of cells exposed to NEM. Survival is correlated with the reduction of the cytoplasmic pH upon activation of the channels. In particular, the kinetics of the intracellular pH (pHi) change are crucial to the retention of viability of cells exposed to NEM; slow acidification does not protect cells as effectively as rapid lowering of pHi. Cells treated with low levels of NEM (10 microM) recover faster if they activate KefB and KefC, and this correlates with changes in pHi. The pHi does not significantly alter the rate of NEM metabolism. The possible mechanisms by which protection against the electrophile is mediated are discussed.     

Activation of potassium channels during metabolite detoxification in Escherichia coli

In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram-negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation-translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram-negative bacteria, but not in the glutathione-deficient species of Gram-positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal-induced cell death. This establishes an essential physiological function for the KefB system.     

The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli

The glyoxalase I gene ( gloA ) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E . coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.     

Importance of RpoS and Dps in Survival of Exposure of Both Exponential- and Stationary-Phase Escherichia coli Cells to the Electrophile N-Ethylmaleimide

The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.     

Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.     

Potassium channel activation by glutathione-S-conjugates in Escherichia coli: protection against methylglyoxal is mediated by cytoplasmic acidification

Escherichia coli possesses two glutathione-gated potassium channels, KefB and KefC, that are activated by glutathione-S-conjugates formed with methylglyoxal. We demonstrate that activation of the channels leads to cytoplasmic acidification and that this protects cells during electrophilic attack. Further, we demonstrate that mutants lacking the channels can be protected against the lethal effects of methylglyoxal by acidification of the cytoplasm with a weak acid. The degree of protection is determined by the absolute value of the pH_i and the time at which acidification takes place. Alterations in the pH_i do not accelerate the rate of detoxification of methylglyoxal. The mechanism by which methylglyoxal causes cell death and the implications for pH_i-mediated resistance to methylglyoxal are discussed.     

Methylglyoxal production in bacteria: suicide or survival?

Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis. Received: 30 March 1998 / Accepted: 22 June 1998     

Survival of Escherichia coli cells exposed to iodoacetate and chlorodinitrobenzene is independent of the glutathione-gated K+ efflux systems KefB and KefC.

The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH. However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation. No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.     

Identification of an ancillary protein, YabF, required for activity of the KefC glutathione-gated potassium efflux system in Escherichia coli

A new subunit, YabF, for the KefC K(+) efflux system in Escherichia coli has been identified. The subunit is required for maximum activity of KefC. Deletion of yabF reduces KefC activity 10-fold, and supply of YabF in trans restores activity. IS2 and IS10R insertions in yabF can be isolated as suppressors of KefC activity consequent upon the V427A and D264A KefC mutations.     

Implicating the glutathione-gated potassium efflux system as a cause of electrophile-induced activated sludge deflocculation

The glutathione-gated K(+) efflux (GGKE) system represents a protective microbial stress response that is activated by electrophilic or thiol-reactive stressors. It was hypothesized that efflux of cytoplasmic K(+) occurs in activated sludge communities in response to shock loads of industrially relevant electrophilic chemicals and results in significant deflocculation. Novosphingobium capsulatum, a bacterium consistent with others found in activated sludge treatment systems, responded to electrophilic thiol reactants with rapid efflux of up to 80% of its cytoplasmic K(+) pool. Furthermore, N. capsulatum and activated sludge cultures exhibited dynamic efflux-uptake-efflux responses very similar to those observed by others in Escherichia coli K-12 exposed to the electrophilic stressors N-ethylmaleimide and 1-chloro-2,4-dinitrobenzene and the reducing agent dithiothreitol. Fluorescent LIVE/DEAD stains were used to show that cell lysis was not the cause of electrophile-induced K(+) efflux. Nigericin was used to artificially stimulate K(+) efflux from N. capsulatum and activated sludge cultures as a comparison to electrophile-induced K(+) efflux and showed that cytoplasmic K(+) efflux by both means corresponded with activated sludge deflocculation. These results parallel those of previous studies with pure cultures in which GGKE was shown to cause cytoplasmic K(+) efflux and implicate the GGKE system as a probable causal mechanism for electrophile-induced, activated sludge deflocculation. Calculations support the notion that shock loads of electrophilic chemicals result in very high K(+) concentrations within the activated sludge floc structure, and these K(+) levels are comparable to that which caused deflocculation by external (nonphysiological) KCl addition.     

Potassium/proton antiport system of Escherichia coli

The intracellular level of potassium (K(+)) in Escherichia coli is regulated through multiple K(+) transport systems. Recent data indicate that not all K(+) extrusion system(s) have been identified (15). Here we report that the E. coli Na(+) (Ca(2+))/H(+) antiporter ChaA functions as a K(+) extrusion system. Cells expressing ChaA mediated K(+) efflux against a K(+) concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K(+) under conditions in which wild-type cells extruded K(+). The K(+)/H(+) antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K(+), which is toxic for these cells. These results suggest that ChaA K(+)/H(+) antiporter activity enables E. coli to adapt to K(+) salinity stress and to maintain K(+) homeostasis.     

pCMBS-induced swelling of dogfish (Squalus acanthias) rectal gland cells: role of the Na+,K(+)-ATPase and the cytoskeleton

(1) 0.1-1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark (Squalus acanthias) rectal glands, with an uptake of cell Na+ and a loss of K+. In contrast, 1 mM N-ethylmaleimide (NEM) does not swell rectal gland cells (RGC), while affecting cell cations. (2) The slow entry of [203Hg]pCMBS is linearly related to its external concentration (10 microM-1 mM) and a small accumulation of pCMBS (apparent gradient about 3) in the cells occurs in 2 h. Cell 203Hg rapidly washes out of the cells (fast rate constant 0.153.min-1; slow rate constant 0.0067.min-1), and this efflux is accelerated by 1mM dithiothreitol. Thus, a major portion of pCMBS inter-acts rather loosely with cell components. (3) pCMBS and NEM share: (a) a negligible effect on the efflux of 86Rb+ and of [14C]urea; (b) a gradual inhibition of the cell Na+,K(+)-ATPase activity. (4) NEM as well as agents lowering cell glutathione accelerate and increase the pCMBS-induced cell swelling. Conditions inhibiting the Na+,K(+)-ATPase (ouabain, absence of Na+) have the same effect. (5) pCMBS, but not NEM produce a disappearance of the F-actin-phalloidin fluorescence independent of cell volume changes, particularly at the basolateral RGC membrane. (6) The data are consistent with the following set of events: (a) pCMBS (but not NEM) affects the cell membrane by increasing the efflux of the cell osmolyte taurine (Ziyadeh et al. (1988) Biochim. Biophys. Acta 943, 43-52 and unpublished data); (b) on entry into the cells, pCMBS and NEM interact with cell -SH, including those of the Na+,K(+)-ATPase; this action produces the observed changes in cell cations. Also, pCMBS, but not NEM, decrease F-actin at the membrane; (c) the inhibition of the Na+,K(+)-ATPase activity together with the decreased resistance of the cell membrane to stretch (absence of F-actin) produces the observed pCMBS-induced cell swelling by osmotic forces (intracellular non-diffusible anions).     

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na(+) and K(+) concentrations is important for bacterial cells, which, in the absence of Na(+) extrusion systems, cannot grow in the presence of high external Na(+). Likewise, bacteria require K(+) uptake systems when the external K(+) concentration becomes too low to support growth. At present, we have little knowledge of K(+) toxicity and bacterial outward-directed K(+) transport systems. We report here that high external concentrations of K(+) at alkaline pH are toxic and that bacteria require K(+) efflux and/or extrusion systems to avoid excessive K(+) accumulation. We have identified the first example of a bacterial K(+)(specific)/H(+) antiporter, Vp-NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cation : proton antiporter-1 (CPA1) family, was able to mediate K(+) extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V. parahaemolyticus Na(+)/H(+) antiporters, Vp-NhaA and Vp-NhaB, which also exhibit a novel ion specificity toward K(+), implying that they work as Na(+)(K(+))/H(+) exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K(+) extrusion against a K(+) chemical gradient, indicating that E. coli also possesses an unidentified K(+) extrusion system(s).     

Evidence for multiple K+ export systems in Escherichia coli.

The role of the K+ transport systems encoded by the kefB (formerly trkB) and kefC (formerly trkC) genes of Escherichia coli in K+ efflux has been investigated. The rate of efflux produced by N-ethylmaleimide (NEM), increased turgor pressure, alkalinization of the cytoplasm, or 2,4-dinitrophenol in a mutant with null mutations in both kef genes was compared with the rate of efflux in a wild-type strain for kef. The results show that these two genes encode the major paths for NEM-stimulated efflux. However, neither efflux system appears to be a significant path of K+ efflux produced by high turgor pressure, by alkalinization of the cytoplasm, or by addition of high concentrations of 2,4-dinitrophenol. Therefore, this species must have at least one other system, besides those encoded by kefB and kefC, capable of mediating a high rate of K+ efflux. The high, spontaneous rate of K+ efflux characteristic of the kefC121 mutation increases further when the strain is treated with NEM. Therefore, the mutational defect that leads to spontaneous efflux in this strain does not abolish the site(s) responsible for the action of NEM.     

Antibiotic effects on SO4(2-) uptake in human erythrocytes

The erythrocyte is a cell highly exposed to oxygen pressure that, in turn, provokes oxidative stress involving loss of SH-groups, cell shrinkage by activation of K(+)-Cl(-) cotransport (KCC) and membrane destabilization which plays an important role in the premature haemolysis of red blood cells (RBCs). Oxidative stress provoked by chemicals frequently occurs in human erythrocytes. The aim of this study was to test whether the antibiotics alter the redox state and investigate their influences on band 3 protein that is involved in the facilitated electro neutral exchange of Cl(-) for HCO(3)(-) across the membrane of mammalian erythrocytes. Normal erythrocytes were treated with some antibiotics and thiol oxidizing agent N-ethylmaleimide (NEM) and tested for sulphate uptake, K(+) efflux and for glutathione (GSH) concentration as an index of oxidative stress. The rate constant of SO(4)(=) uptake measured in erythrocytes treated with antibiotics as well as NEM was decreased with respect to control cells as a result of band 3 SH-groups oxidation or the stress-induced K(+)-Cl(-) symport-mediated cell shrinkage. In fact, this hypothesis was verified by increased K(+) efflux and decreased GSH values measured in treated erythrocytes compared to controls.     

Metabolic control of the potassium permeability in pancreatic islet cells

The K(+) permeability of pancreatic islet cells was studied by monitoring the efflux of (86)Rb(+) (used as tracer for K(+)) from perifused rat islets and measuring the uptake of (42)K(+). Glucose markedly and reversibly decreased (86)Rb(+) efflux from islet cells and this effect was antagonized by inhibitors of the metabolic degradation of the sugar, i.e. mannoheptulose, iodoacetate, glucosamine and 2-deoxyglucose. Among glucose metabolites, glyceraldehyde reduced the K(+) permeability even more potently than did glucose itself; pyruvate and lactate alone exhibited only a small effect, but potentiated that of glucose. Other metabolized sugars, like mannose, glucosamine and N-acetylglucosamine, also decreased (86)Rb(+) efflux from islet cells. Fructose was effective only in the presence of glucose. Non-metabolized sugars like galactose, 2-deoxyglucose and 3-O-methylglucose had no effect. The changes in K(+) permeability by agents known to modify the concentrations of nicotinamide nucleotides, glutathione or ATP in islet cells were also studied. Increasing NAD(P)H concentrations in islet cells by pentobarbital rapidly and reversibly reduced (86)Rb(+) efflux; exogenous reduced glutathione produced a similar though weaker effect. By contrast, oxidizing nicotinamide nucleotides with phenazine methosulphate or Methylene Blue, or oxidizing glutathione by t-butyl hydroperoxide increased the K(+) permeability of islet cells. Uncoupling the oxidative phosphorylations with dicumarol also augmented (86)Rb(+) efflux markedly. In the absence of glucose, but not in its presence, methylxanthines reduced (86)Rb(+) efflux from the islets; such was not the case for cholera toxin or dibutyryl cyclic AMP. Glucose and glyceraldehyde had no effect on (42)K(+) uptake after a short incubation (10min), but augmented it after 60min; the effect of glucose was suppressed by mannoheptulose and not mimicked by 3-O-methylglucose. The results clearly establish the importance of the metabolic degradation of glucose and other substrates for the control of the K(+) permeability in pancreatic islet cells and support the concept that a decrease in the K(+) permeability represents a major step of the B-cell response to physiological stimulation.     

Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Mutations at the trkB and trkC loci of Escherichia coli produce an abnormal efflux of K+. The mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. The mutations can be reverted by insertion of Tn10 into the mutated gene, and spontaneous revertants are fully recessive to the mutant allele in diploids. K+ efflux produced by NEM* and by DNP* persists in strains with presumed null mutations at either locus, indicating neither gene product is the primary target for the effect of these inhibitors on K+ efflux. The results are consistent with the view that trkB and trkC encode independent systems for K+ efflux. Mutations at these loci alter regulation of the process so that K+ efflux occurs inappropriately. A second mutation to the null state abolishes this abnormal K+ efflux. These genes may encode K+/H+ antiporters, an activity postulated to mediate K+ efflux and demonstrated to exist in E. coli and other bacteria.