Chlorocatechol detection based on a clc operon/reporter gene system

A sensitive and selective sensing system for chlorocatechols (3- chlorocatechol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteria harboring the plasmid pSMM50R-B'. In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme β-galactosidase. When bacteria containing components of the clc operon are grown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the β-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of β-galactosidase in the bacteria. The concentration of β-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal emitted with the use of a 1,2-dioxetane substrate. ClcR has a high specificity 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-chlorocatechol can be detected with this sensing system at concentrations as low as 8 x 10^-10 and 2 x 10^-9 M, respectively, using a 2-h induction period. 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 concentrations as high as 2 x 10^-4 M did not interfere with this system.