Metabolomic Investigation of the Bacterial Response to a Metal Challenge

Pseudomonas pseudoalcaligenes KF707 is naturally resistant to the toxic metalloid tellurite, but the mechanisms of resistance are not known. In this study we report the isolation of a KF707 mutant (T5) with hyperresistance to tellurite. In order to characterize the bacterial response and the pathways leading to tolerance, we utilized Phenotype MicroArray technology (Biolog) and a metabolomic technique based on nuclear magnetic resonance spectroscopy. The physiological states of KF707 wild-type and T5 cells exposed to tellurite were also compared in terms of viability and reduced thiol content. Our analyses showed an extensive change in metabolism upon the addition of tellurite to KF707 cultures as well as different responses when the wild-type and T5 strains were compared. Even in the absence of tellurite, T5 cells displayed a “poised” physiological status, primed for tellurite exposure and characterized by altered intracellular levels of glutathione, branched-chain amino acids, and betaine, along with increased resistance to other toxic metals and metabolic inhibitors. We conclude that hyperresistance to tellurite in P. pseudoalcaligenes KF707 is correlated with the induction of the oxidative stress response, resistance to membrane perturbation, and reconfiguration of cellular metabolism.     

In Vivo 31P Nuclear Magnetic Resonance Investigation of Tellurite Toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by ³¹P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by ³¹P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli

Potassium tellurite (K₂TeO₃) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.     

Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages

Background

Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections.

Principal Findings

In vitro treatment of Y. pestis culture with sodium tellurite (Na₂TeO₃) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype.

Conclusions

These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.     

Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli

Tellurite (TeO3(2-)) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.     

Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.     

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k_cat and K_m, selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H₂O₂ production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.     

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K₂TeO₃ to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.     

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

This work examines the effects of potassium tellurite (K₂TeO₃) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO₃²⁻) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te⁰ crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.     

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD₅₀ = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF‐Ts, and PPIase) displayed increased abundance in tellurite‐stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3‐hydroxy acyl‐CoA dehydrogenase, acyl‐CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.     

The highly toxic oxyanion tellurite (TeO<Stack><Subscript>3</Subscript><Superscript>2−</Superscript></Stack>) enters the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> via an as yet uncharacterized monocarboxylate transport system

The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 μM acetate. Conversely, tellurite at 100 μM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.     

Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant E.coli

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level.In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins.Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control.The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.     

Treatment of human cancer cells with selenite or tellurite in combination with auranofin enhances cell death due to redox shift

Selenium is an essential trace element incorporated as selenocysteine in 25 human selenoproteins. Among them are thioredoxin reductases (TrxR) and glutathione peroxidases, all central proteins in the regulation of the cellular thiol redox state. In this paper the effects of selenite and tellurite treatment in human cancer cells are reported and compared. Our results show that both selenite and tellurite, at relatively low concentrations, are able to increase the expression of mitochondrial and cytosolic TrxR in cisplatin-sensitive (2008) and -resistant (C13) phenotypes. We further investigated the cellular effects induced by selenite or tellurite in combination with the specific TrxR inhibitor auranofin. Selenite pretreatment induced a dramatic increase in auranofin cytotoxicity in both resistant and sensitive cells. Investigation of TrxR activity and expression levels as well as the cellular redox state demonstrated the involvement of TrxR inhibition and redox changes in selenite and auranofin combined action.     

Protein–protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster “ter” from pathogenic Escherichia coli

Gene cluster “ter” conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein–protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein–protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.     

A CYTOCHEMICAL LOCALIZATION OF REDUCTIVE SITES IN A GRAM-NEGATIVE BACTERIUM : Tellurite Reduction in Proteus vulgaris

In order to obtain information on the exact location of the respiratory enzyme chain in Gram-negative bacteria, an electron microscopic study was made of the sites of reducing activity of cells that had, in the living state, incorporated tellurite. In the test object Proteus vulgaris, the reduced tellurite was found to be deposited in bodies contiguous with the plasma membrane but different in structure from those described in the Gram-positive Bacillus subtilis (2). In fact, the bodies proved to consist of a conglomerate of elements which contained the strongly electron-scattering reduced tellurite and a delicately granular "matrix." A limiting membrane was not observed around these complexes. In serial sections details of the complexes are illustrated. Reduced tellurite was not deposited in the plasma membrane to any important degree. Since no other sites of deposition of the reduced product were revealed, it is assumed that the complexes represent the mitochondrial equivalents in the investigated organism. In addition, the bodies might function as the basal granules of the flagella.     

Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V_max and K_m were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min⁻¹ · mg⁻¹ and 0.12 mM were obtained for V_max and K_m, respectively, whereas the V_max values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.     

Acetate Permease (ActP) Is Responsible for Tellurite (TeO32?) Uptake and Resistance in Cells of the Facultative Phototroph Rhodobacter capsulatus

The highly toxic oxyanion tellurite has to enter the cytoplasm of microbial cells in order to fully express its toxicity. Here we show that in the phototroph Rhodobacter capsulatus, tellurite exploits acetate permease (ActP) to get into the cytoplasm and that the levels of resistance and uptake are linked.Tellurite exerts its toxic action in the cytoplasmic compartment mostly by triggering an increase in the generation of reactive oxygen species (ROS) (4, 5, 10). This implies that the acquisition of the oxyanion by the cell is a prerequisite for the full development of its toxicity and suggests, as a consequence, that diminished uptake would result in increased resistance.The link between tellurite uptake and toxicity in bacterial cells has been previously discussed in a limited number of reports (2, 3, 7). In Escherichia coli, it was shown that mutations in a phosphate transport system confer a high level of resistance to tellurite and that the oxyanion competes very efficiently for ³²P_i transport (7). In the Gram-positive bacterium Lactococcus lactis, mutations associated with proteins PstA and PstD (phosphate transport), but also those associated with protein ChoQ (proline/glycine-betaine/carnitine/choline transport), were shown to confer a higher level of resistance relative to the wild type (8). Finally, in the phototrophic species Rhodobacter capsulatus it was suggested that a phosphate transporter might also be responsible for tellurite uptake (3). This hypothesis has recently been questioned because of the extremely high concentration of P_i needed to significantly inhibit tellurite uptake (2). Conversely, acetate was found to decrease the uptake of tellurite, when present at concentrations as low as 1 μM (2). This finding, along with the fact that lactate and pyruvate, but not malate and succinate, act similarly as competitors of tellurite uptake, led to the proposal that, in R. capsulatus, the oxyanion enters the cell via a monocarboxylate transport system (2).In this work, we show that an acetate transport system is utilized by tellurite to enter R. capsulatus cells and that limitation of this uptake drastically increases the resistance to the oxyanion.