Figure 1.
Tellurite-induction of β-galactosidase activity in E. coli reporter strains.
E. coli reporter strains ADA100 [AB734 λΦ(ibp::lacZ)], ADA310 [AB734λΦ (cspA::lacZ)], ADA410 [AB734 λΦ(p3RpoH::lacZ)] and ADA510 [AB734 λΦ(sulA::lacZ)] containing the stress-responsive promoters ibpA, cspA, p3RpoH and sulA fused to the lacZ 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.
Figure 2.
Generation of intracellular ROS by K2TeO3.
Cytoplasmic ROS content was evaluated measuring the H2DCFDA probe activation in E. coli cells treated with different sub lethal concentrations of tellurite [0 (▪), 0.2 (•), 0.5 (▴) and 1 µg/ml (◂)]. Fluorescence was measured 10 times at 2 min intervals. The inset shows probe activation at 28 min by tellurite at the indicated concentrations. See Methods for details.
Figure 3.
Tellurite increases the oxidation of cytoplasmic proteins and membrane lipids in E. coli.
Effects of K2TeO3 (0.5 µg/ml) and H2O2 (100 µM) on protein carbonyl (A) and TBARs content (B) of E. coli 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 E. coli BW25113 by the method described by Rice-Evans et al. [19].
Table 1.
Effect of potassium tellurite on E. coli malate dehydrogenase and aconitase.
Figure 4.
Effect of potassium tellurite on E. coli catalase and superoxide dismutase.
A, activity of catalases in crude protein extracts of E. coli BW25113 treated (•) or untreated (▪) with 0.5 µg/ml of K2TeO3. Cells were collected at 15 min intervals and catalase activity (µmol hydrogen peroxide/min/mg protein) was determined. B, activity of superoxide dismutases in crude protein extracts of E. coli BW25113 treated (•) and untreated (▪) with 0.5 µg/ml K2TeO3. Cells were harvested and SOD activity (U/mg protein) was determined.
Figure 5.
Tellurite induces katG and soxS mRNA synthesis in E. coli.
DNA fragments (300 bp) from E. coli sodA, sodB, katG, soxS and gapA 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 Material and Methods for details).
Table 2.
Minimal inhibitory concentrations (MIC) of K2TeO3 for E. coli BW25113 strains deficient in ROS-responsive genes.
Figure 6.
In vitro tellurite reduction generates superoxide in E. coli.
Superoxide generation was evaluated using an in vitro tellurite reduction assay previously developed in our laboratory [34]. 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).