Chlorocatechol detection based on a clc operon/reporter gene system

A sensitive and selective sensing system for chlorocatechols (3-chlorocatec hol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteri a harboring the plasmid pSMM50R-B'. In this plasmid, the regulatory protein of the de operon, ClcR, controls the expression of the reporter enzyme bet a-galactosidase, When bacteria containing components of the cb operon are g rown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the beta-galactosidase gene. Thus, the conce ntration of chlorocatechols can be related to the production of beta-galact osidase in the bacteria. The concentration of beta-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal em itted with the use of a 1,2-dioxetane substrate. ClcR has a high specificit y for chlorocatechols and provides the sensing system with high selectivity . This was demonstrated by evaluating several structurally related organic compounds as potential interfering agents. Both 3-chlorocatechol and 4-chlo rocatechol can be detected with this sensing system at concentrations as lo w as 8 x 10(-10) and 2 x 10(-9) M, respectively, using a 2-h induction peri od. In the case of 3-chlorocatechol, a highly selective sensing system was developed that can detect this species at concentrations as low as 6 x 10(- 8) M after a 5-min induction period; the presence of 4-chlorocatechol at co ncentrations as high as 2 x 10(-4) M did not interfere with this system.