Construction and comparison of Escherichia coli whole-cell biosensors capable of detecting aromatic compounds

The XyIR regulatory protein is a transcription factor involved in the BTEX (benzene. toluene. ethylbertzene, and xylene) degradation pathway in Pseudomonas species. When XyIR-dependent stimulation of transcription from a plasmid containing XyIR and its cognate promoters Pr and Pit 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 XyIR in this system. two related but different promoters were tested for their activities; the XyIS 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 Pit 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 Pit and Ps, respectively. Gel mobility Shift assay showed that the purified XyIR without inducers can bind to Pr/Ptt but not to Pr/Po', implying that XyIR 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. (C) 2004 Elsevier B.V. All rights reserved.