Faculty of Science, Department of Biochemistry, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, Thailand.
J Ind Microbiol Biotechnol. 2012 Dec;39(12):1801-10. doi: 10.1007/s10295-012-1180-3. Epub 2012 Aug 15.
Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P( todX ) promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP-pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9 h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose-response relationship, from which the K(½) value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57'sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.
苯胺和氯代苯胺（CA）被列为优先污染物；因此，需要一种有效的检测和监测方法。本研究在大肠杆菌 DH5α 中构建了一种基于绿色荧光蛋白的生物传感器，用于检测苯胺和 CA，并通过土壤和废水对其进行了特征和测试。该传感器的检测能力依赖于两个组件调控蛋白 TodS/TodT 和 Pseudomonas putida T-57（PpT57）的 P(todX)启动子之间的调控控制，因为基因表达的 todS、todT 和 todC2 会被 4-氯苯胺正向诱导。生物传感器系统（DH5α/pPXGFP-pTODST）的两个共存质粒具有明显的独特性。荧光响应的诱导性与培养基和时间有关。在 M9G 培养基中生长的细胞表现出低背景荧光水平，在暴露于 4CA 后 3 小时内很容易被诱导，在 9 小时达到最大诱导水平。在测试苯、甲苯、乙苯和二甲苯、苯胺和 CA 时，响应数据最符合 S 型剂量反应关系，从中确定了阳性效应物的 K(½)值。3CA 和 4CA 是相对较强的诱导剂，而一些多氯代苯胺也能诱导绿色荧光蛋白的表达。结果表明，PpT57 的 TodST 比以前报道的 P. putida 对其识别范围更广。环境样品的测试结果可靠，表明该生物传感器在受苯胺和/或 CA 污染地区的生态毒理学评估和生物修复方面具有潜在应用。
