Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant <i>E.coli</i>

<div><p>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 <i>E. coli</i> at the protein level.</p><p>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.</p><p><i>E. coli BL21</i> was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring <i>ter</i> 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 <i>E. coli</i> 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.</p><p>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.</p><p>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.</p></div

Images of two 2D electrophoresis gels.

<p>Crude protein extracts (600 µg) were separated on strips pH 4–7 and SDS-PAGE on 15% polyacrylamide gradient gels. Spots with significant differences in expression level are marked on the gel: <i>E. coli BL21/pACYC184</i> cells (A) untreated and (B) treated with K₂TeO₃, and <i>E. coli BL21/pLK18</i> cells (C) untreated and (D) treated with K₂TeO₃.</p

Determination of the “sub-lethal” concentration for the sensitive strain <i>E. coli</i> BL21(pACYC184).

<p>Cells were incubated for a defined time interval of 10°C in the presence of potassium tellurite (at 0.3–4.5 µM).</p

The immunoblotting patterns of proteins verifying results from two-dimensional gel electrophoresis are shown.

<p>Quantification results expressed as arbitrary units of optical density are shown (<b>A</b>) GroEL, (<b>B</b>) DnaK. Experiments were carried out in triplicates; ns - not significant, *P<0.05, ***P<0.001.</p

Proteins with at least a two-fold difference in expression (P<0.05) identified by nano-LC-ESI-CID/ETD-MS/MS for <i>E. coli BL21</i>(pACYC184) and <i>E. coli BL21</i>(pLK18).

<p><b>S</b> - <i>E. coli BL21</i>(pACYC184) untreated, <b>S+Te</b><i>E. coli BL21</i>(pACYC184) treated with K₂TeO₃, <b>R</b> - <i>E. coli</i> BL21(pLK18) untreated, <b>R+Te</b><i>E. coli BL21</i>(pLK18) treated with K₂TeO₃, <b>+</b> - up-regulation of a protein in a specific strain and condition.</p

Effect of potassium tellurite on <i>E. coli</i> catalase, superoxide dismutase and aldehyde reductase activity.

<p>(<b>A</b>) Activity of catalases, (<b>B</b>) superoxide dismutases and (<b>C</b>) YqhD aldehyde-reductase activity in crude protein extracts of tellurite-sensitive and tellurite resistant <i>E. coli</i> BL21 untreated or treated with 0.5 µM and 3.9 µM of K2TeO3. Cells were collected after 10 min and catalase activity (µmol hydrogen peroxide/min/mg protein), SOD activity (U/mg protein) and YqhD activity (µmol/min/mg) were determined. Error values represent the standard deviation of triplicate experiments, ns - not significant, *P<0.05, ***P<0.001.</p