Anaerobic RSH-dependent tellurite reduction contributes to Escherichia coli tolerance against tellurite

Strains and culture media

For all in vivo assays, MOPS buffered culture medium supplemented with 0.2% glucose (w/v) and 40 mM potassium nitrate (MOPS*n) was used. The in vitro assays were carried out with cell extracts obtained from cultures grown in LB medium supplemented with chloramphenicol. Escherichia coli BW25113 (E. coli WT, parental) was used as a wild strain. Additionally, the strains Δgor, ΔgshA, and ΔgshB from the Keio collection (NARA Institute, Japan) [7] were used in the in vivo reduction assay, RSH quantification, fitness determination, and ex vivo assay.

Growth conditions

The cell cultures were grown in the presence or absence of oxygen. For the condition of anaerobiosis, an anaerobiosis chamber was used keeping an atmosphere of 100% N2 (Coy chamber). All cultures were grown at 37 °C with agitation. To ensure the absence of oxygen during bacterial growth, all cultures were started using a pre-inoculum that was prepared from a plate culture in an aerobic medium and incubated for at least 16 h in the absence of oxygen. Likewise, the culture medium in which the pre-inoculum was seeded was purged of oxygen using the pre-chamber of the Coy chamber with the semi-open bottles and kept inside the chamber for 12 h before use.

Growth curves

Saturated bacterial cultures incubated overnight at 37 °C with agitation in aerobiosis or anaerobiosis, as appropriate, were used as pre-inoculum. Plates were used in whose wells was added 0 or 10 µg/ml of tellurite, 10 µL of saturated bacterial culture and completed at 1 mL volume with fresh MOPS*n medium. The curve was performed on the plate reader Tecan Infinite M200 Pro, at 37 °C with stirring at 150 rpm and measurements at 600 nm every 15 min for a total of 40 cycles. The curves were analyzed via R, obtaining the value of the area under the curve of each growth curve.

Quantification of the uptake of tellurite into the cell

The proportion of total tellurium present in the cells (sediment), in the medium (supernatant), and adsorbed on the membrane (washed) was determined by ICP-OES atomic absorption spectrophotometry. The cells were cultured in the presence or absence of oxygen to OD600 ~ 0.5 and then treated with tellurite 50 or 100 µg/ml for 30 or 60 min. After treatment, 1 mL of culture was taken for protein quantification and 5 mL for the assay. The 5 mL was centrifuged at 9000×g for 5 min recovering the supernatant. The sediment was immediately washed with 5 mL of 50 mM Tris–HCl pH 7.4 and centrifuged again recovering the 5 mL of wash. A volume of 65% HNO3 was added to both 5 mL of supernatant and wash so that the final acid concentration was 2%. The bacterial sediment was also treated with 65% HNO3 (final concentration, 2%) and incubated at 37 °C for 2 h with stirring. Then, the dissolved sediment was flushed to 5 mL with 50 mM Tris–HCl pH 7.4, so that the final acid concentration was 2%. Sediment, supernatant, and wash were filtered using 0.22 µm Nylon filters and stored at 4 °C until analysis. The standards were prepared using tellurite dissolved in 2% nitric acid; the standards used were 0.01; 0.1; 0.5; 1.0; 10.0; 100.0 and 250 µg/ml. The results obtained were normalized by protein concentration.

Determination of the viability percentage

The percentage of bacterial viability versus treatment with tellurite was performed in cultures by the micro drop method. Cultures of E. coli OD600 ~ 0.5 obtained as described above were plated and treated with increasing concentrations of tellurite in the range between 0 and 250 µg/ml. After the toxic addition, the cultures were incubated at 37 °C for 30 or 60 min. Of these, a 20 µL aliquot was taken that was diluted in 180 µL saline (0.9% NaCl w/v). From that dilution, serial dilutions were made 1/10 with saline solution until dilution 10–6. Then 4 µL of each dilution and the original culture were taken and deposited in drop form on LB agar plates 2% w/v. The plates were incubated at 37 °C for 16 h; subsequently, colonies were counted in the lowest dilution that contained countable colonies. The viability percentage was defined in relation to dilutions left untreated.

NAD+/NADH ratio determination

The quantification was performed by the MTT method as described previously [24]. Briefly, sediments obtained from 10 ml of treated or untreated cultures in the presence or absence of oxygen were suspended in 2 mL of PBS. The volume was separated into 2 tubes that were centrifuged at 14,000×g for 5 min, discarding the supernatant. The sediment was suspended in 125 µL of 0.2 M HCl (NAD+ extraction) or 0.2 M NaOH of (NADH extraction). Then, the suspensions were heated at 100 °C for 10 min and immediately centrifuged as before. A calibration curve was prepared using commercial NADH or NAD+ of concentration 1.5 to 0.02 mM. With the samples and calibration curve prepared, the following solutions were prepared, which were applied in each reaction well in the indicated volumes; (1) Mix1, containing 20 µL of 1 M Bicin pH 8.0; 50 µL neutralization buffer (0.1 M HCl for NADH, 0.1 M NaOH for NAD+); 20 µL of 40 mM EDTA pH 8.0; 20 µL of 100% ethanol; (2) Mix2, containing 20 µL of PES (phenazine ethosulfate) 16 mM; 20 µL MTT (3-[4,5-dimethylthiazol-2-y] 2,5-diphenyltetrazolium bromide). From the tube for extraction of NAD+ 50 µL was taken and mixed with 4 µL of ADHII (yeast alcohol dehydrogenase II). The plate was incubated in the dark at 30 °C with stirring for 25 m. Once the incubation period elapsed, the reaction wells containing 50 µL of extract (NADH or NAD+), 110 µL of Mix1, and 40 µL of Mix2 were added. The absorbance measurement was started immediately at 570 nm every 30 s with stirring intervals using the Tecan Infinite M200 PRO reader. The calibration curve was constructed and the concentration in the samples was determined. The NADH and NAD+ concentrations obtained were normalized by protein concentration.

Quantification of total reduced thiols (RSH).

E. coli cultures were incubated to OD600 ~ 0.5 and separated in identical volume samples, one was treated with tellurite 100 µg/mL and one was left untreated (control); both groups were incubated for 1 h at 37 °C with shaking at 150 rpm in the initial growth condition. At the end of the treatment, 500 µL aliquots were taken and centrifuged at 14,000×g for 1 min, discarding the supernatant. The sediments were stored at − 20 °C until the time of measurement. Each sample was suspended in 1 mL of RSH buffer, containing 50 mM Tris–HCl pH 8.0, 5 mM EDTA, 0.1% SDS and 0.1 mM DTNB (5,5'-dithiobis (2-nitrobenzoic acid)). The suspension was incubated at 37 °C for 30 min, homogenized in a vortex for 10 s and centrifuged for 10 min at 14,000×g. Subsequently, the absorbance of the supernatant of each fraction obtained was evaluated spectrophotometrically at 412 nm. Using the extinction coefficient molar of oxidized DTNB (1.36 × 104 M−1 cm−1), the concentration of reduced thiols was determined. The determined RSH concentrations were normalized by protein concentration.

Protein quantification

For this purpose, the Bradford method was used [9], repeating the calibration curve protocol, that is, in 150 µL wells 150 µL of Bradford reagent, 1–10 µL of protein sample and water miliQ completing the volume of the well. The determination was made on the Tecan Infinite M200 Pro plate reader by recording the absorbance of the sample at 595 nm.

Determination of specific tellurite reducing activity in vitro

This was determined using crude protein extracts and/or purified proteins. In both cases tellurite reductase buffer was used [12, 37], changing the pH of the reaction (20 mM sodium phosphate buffer (pH 7.0 and 8.0) or Tris–HCl buffer (pH 9.0), 0.1 mM NADH and/or NADPH, 1 mM β-mercaptoethanol and 0.5 µg/mL potassium tellurite) and 40 µg of protein. The reaction was performed on the Tecan Infinite M200 pro reader, evaluating the absorbance at 500 nm at 37 °C for 15 min for purified protein or 60 min for crude extracts. An enzyme unit (U) was defined as the amount of enzyme needed to increase the absorbance at 500 nm in 0.001 units in 1 min under the test conditions; the specific activity (SA) was determined normalized by the protein concentration (U/mg protein).

Determination of tellurite reduction in vivo

Cultures initiated from saturated pre-inoculums prepared at least 16 h in advance, were incubated in the measurement condition (aerobiosis or anaerobiosis, 37 °C, and agitation at 150 rpm) up to OD600 ~ 0.5 (the acceptable range it was considered between 0.45 and 0.55). At that point, the cultures were treated with 100 µg/ml tellurite, and the initial absorbance at 600 nm and 500 nm was immediately evaluated. The cultures were incubated at 37 °C for 1 h with shaking at 150 rpm, at the end of this period the absorbance at 500 nm was evaluated again. An enzyme unit (U) was defined as the amount of enzyme needed to increase absorbance at 500 nm in 0.001 units in 1 min under the test conditions; the specific activity (SA) was determined by dividing U by the concentration of proteins (U/mg/ml proteins).

Data analysis

Statistical analysis and graphs were carried out using GraphPad Prism 6.0 (GraphPad Software, Inc.). The confidence interval in the analysis of variance (ANOVA) was set at p < 0.05 with post hoc analysis false discovery rate (FDR) correction.

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