Construction and comparison of Escherichia coli whole-cell biosensors capable of detecting aromatic compounds.
The XylR regulatory protein is a transcription factor involved in the BTEX (benzene, toluene, ethylbenzene, and xylene) degradation pathway in Pseudomonas species. When XylR-dependent stimulation of transcription from a plasmid containing XylR and its cognate promoters Pr and Pu was monitored as firefly luciferase activities in Escherichia coli, a notably high level of basal activity was observed in the absence of inducers. To improve the response specificity of XylR in this system, two related but different promoters were tested for their activities; the XylS activator promoter Ps and the DmpR activator promoter Po. Po with the deletion of its own upstream activating sequences (UASs; Po') showed a very low level of basal activity compared to Pu and Ps. The maximum level with the addition of inducers was increased 3151-fold by o-xylene with Po', while it was 31.5 and 74.1 fold by m-xylene with Pu and Ps, respectively. Gel mobility shift assay showed that the purified XylR without inducers can bind to Pr/Pu but not to Pr/Po', implying that XylR multimerization with Pr/Pu could be formed for initiation of transcription in this system. The data suggest that Po' can be an excellent alternative in constructing a signal-intensified, whole-cell biosensor in response to the xenobiotics.
Kim
Mi Na
MN
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea.
Park
Hoo Hwi
HH
Lim
Woon Ki
WK
Shin
Hae Ja
HJ
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D015152:Blotting, Northern; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006841:Hydrocarbons, Aromatic / Q000032:analysis*; D049409:Luciferases, Firefly / Q000032:analysis; D008163:Luminescent Measurements; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006841:Hydrocarbons, Aromatic; D014157:Transcription Factors; C074322:XylR protein, Pseudomonas; D012313:RNA; D049409:Luciferases, Firefly