Phoenix P, Keane A, Patel A, Bergeron H, Ghoshal S, Lau P C K
Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2.
Environ Microbiol. 2003 Dec;5(12):1309-27. doi: 10.1111/j.1462-2920.2003.00426.x.
A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260-residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.
在恶臭假单胞菌F1（PpF1）中，一个调控甲苯降解（tod途径）的双组分todST磷酸化信号转导系统下游发现了一个新的基因簇，命名为sepABC以及一个反向转录的sepR。sepABC编码的推导氨基酸序列与已知参与溶剂外排或多药泵的细菌蛋白具有高度同源性。SepA、SepB和SepC分别被认为是周质、内膜和外膜外排蛋白。与野生型相比，各种PpF1突变体在甲苯存在下的生长情况表明，溶剂外排系统在溶剂胁迫环境中可能具有保护作用。对互补突变体的生长测试证实了Sep蛋白参与赋予溶剂耐受性。sepR基因编码一个260个残基的多肽，它是大肠杆菌IclR阻遏蛋白家族的成员。通过在大肠杆菌和PpF1中进行sep-lacZ转录融合测试、在DNA结合试验中以麦芽糖结合融合蛋白形式表达SepR以及mRNA分析，确定了SepR的阻遏作用。Southern杂交实验和恶臭假单胞菌KT2440基因组序列分析表明，与sepABC基因的同源物相比，sepR相对较少见。我们开发了一种全细胞生物发光生物传感器PpF1G4，它包含一个基于染色体的sep-lux转录融合。该生物传感器显示，多种芳香分子，包括苯、甲苯、乙苯和二甲苯的所有三种异构体（BTEX）、萘以及脂肪族和芳香族烃的复杂混合物，均可显著诱导sepABC基因。PpF1G4代表了第二代生物传感器，它不是基于分解代谢启动子，但仍可被芳香污染物诱导，而且在营养丰富的条件下也能发挥作用。
