Bacterial Toxicity of Potassium Tellurite: Unveiling an Ancient Enigma

Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2−)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2−) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2′7′-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite

Catalases Are NAD(P)H-Dependent Tellurite Reductases

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3) (2−)) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical

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

The Geobacillus stearothermophilus V iscS Gene, Encoding Cysteine Desulfurase, Confers Resistance to Potassium Tellurite in Escherichia coli K-12

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters

Tellurite increases the oxidation of cytoplasmic proteins and membrane lipids in <i>E. coli.</i>

<p>Effects of K₂TeO₃ (0.5 µg/ml) and H₂O₂ (100 µM) on protein carbonyl (A) and TBARs content (B) of <i>E. coli</i> BW25113 cells exposed to these compounds for 30 min. A, protein oxidation was determined by a chemical protein carbonyl assay by derivatizing total cellular proteins with DNPH and reading specific carbonyls absorbance at 370 nm. B, membrane peroxidation products were determined as thiobarbituric acid-reactive substances present in crude extracts of <i>E. coli</i> BW25113 by the method described by Rice-Evans et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000211#pone.0000211-RiceEvans1" target="_blank">[19]</a>.</p

Minimal inhibitory concentrations (MIC) of K₂TeO₃for <i>E. coli</i> BW25113 strains deficient in ROS-responsive genes.

<p>Numbers are the mean of 4 independent determinations.</p

<i>In vitro</i> tellurite reduction generates superoxide in <i>E. coli.</i>

<p>Superoxide generation was evaluated using an <i>in vitro</i> tellurite reduction assay previously developed in our laboratory <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000211#pone.0000211-Caldern1" target="_blank">[34]</a>. The system makes use of the O₂⁻ specific reactive compound WST-1. WST-1 reduction was determined in the presence of catalase and NADPH (Cat); catalase, tellurite and NADPH (Cat/Te); catalase, tellurite, NADPH and purified superoxide dismutase (Cat/Te/SOD); catalase, tellurite, NADPH and β-amylase (Cat/Te/amylase).</p

Tellurite-induction of β-galactosidase activity in <i>E. coli</i> reporter strains.

<p> <i>E. coli</i> reporter strains ADA100 [AB734 λΦ(<i>ibp</i>::<i>lacZ</i>)], ADA310 [AB734λΦ (<i>cspA</i>::<i>lacZ</i>)], ADA410 [AB734 λΦ(p3<i>RpoH</i>::<i>lacZ</i>)] and ADA510 [AB734 λΦ(<i>sulA</i>::<i>lacZ</i>)] containing the stress-responsive promoters <i>ibpA, cspA, p3RpoH</i> and <i>sulA</i> fused to the <i>lacZ</i> gene respectively, were used to study transcription induction in cells treated or untreated with K₂TeO₃ (0.5 µg/ml). β-galactosidase activity was evaluated at time 0 and after 3 h with or without tellurite treatment. The fold induction was calculated dividing the value obtained at 3 h by the value at time 0. Results are the average of at least 4 determinations.</p

Tellurite induces <i>katG</i> and <i>soxS</i> mRNA synthesis in <i>E. coli.</i>

<p>DNA fragments (300 bp) from <i>E. coli sodA, sodB, katG, soxS</i> and <i>gapA</i> genes were amplified by RT-PCR and fractionated by electrophoresis on agarose gels (1.5%). Total RNA from cells grown with (K₂TeO₃) or without (control) 0.5 µg/ml potassium tellurite was used as template for the RT-PCR. The estimated DNA (ng) content determined for each band is shown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000211#s4" target="_blank">Material and Methods</a> for details).</p