Department of Molecular Biology, Faculty of Natural Science, Comenius University, Bratislava, Slovakia.
PLoS One. 2013 Nov 11;8(11):e78010. doi: 10.1371/journal.pone.0078010. eCollection 2013.
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 E. coli at the protein level. 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. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter 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 E. coli 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. 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. 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.
碲酸盐化合物被用于工业过程，其排放量的增加会导致特定的污染，很可能导致环境污染，并对公众健康产生潜在的不利影响。本研究的目的是在蛋白质水平上揭示碲酸盐敏感和碲酸盐抗性大肠杆菌的毒性机制。在这项工作中，我们使用基于凝胶的质谱分析方法来识别与碲酸盐毒性相关的差异蛋白质谱，并解决 ter 操纵子介导的碲酸盐抗性机制。通过将抗性基因 ter 引入 pLK18 质粒，对 BL21 大肠杆菌进行基因操作以获得碲酸盐抗性。向培养物中添加亚碲酸钾，以获得最终 3.9 微米的浓度。将碲酸盐敏感和碲酸盐抗性大肠杆菌的蛋白质在 2-D 凝胶电泳上运行，挑取感兴趣的斑点，进行胶内消化，然后通过纳升 LC-MS/MS（离子阱）进行分析。此外，进行 Western 印迹和酶活性测量以验证某些候选蛋白质的表达。与碲酸盐抗性细菌相比，在暴露于碲酸盐后，敏感细胞表现出抗氧化酶超氧化物歧化酶、过氧化氢酶和氧化还原酶 YqhD 的水平增加。胱氨酸脱硫酶与碲酸盐毒性有关，以及参与蛋白质折叠的蛋白质：GroEL、DnaK 和 EF-Tu 在敏感细胞中上调。在抗性细菌中，观察到四个必需 Ter 蛋白的几种同工型，并且在处理碲酸盐后，与敏感对照相比，上述蛋白质水平没有显示任何明显的蛋白质组变化。抗性细菌中缺乏针对碲酸盐毒性的一般防御机制，这进一步证明了 ter 操纵子的四个蛋白质通过特定的作用模式在碲酸盐抗性机制中起作用，可能涉及抗氧化和蛋白质折叠途径的蛋白质级联。
