<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 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

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

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

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