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 beta-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 beta-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of beta-galactosidase in the bacteria. The concentration of beta-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.
Guan
X
X
Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
Ramanathan
S
S
Garris
J P
JP
Shetty
R S
RS
Ensor
M
M
Bachas
L G
LG
Daunert
S
S
D029681:Arabidopsis Proteins; D002396:Catechols / Q000032:analysis*; D018118:Chloride Channels / Q000235:genetics*; D017930:Genes, Reporter; D007763:Lac Operon; D008163:Luminescent Measurements; D009876:Operon; D010940:Plant Proteins; D016958:Pseudomonas putida / Q000737:chemistry / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C407524:ATCLC-A protein, Arabidopsis; D029681:Arabidopsis Proteins; D002396:Catechols; D018118:Chloride Channels; D010940:Plant Proteins; C031137:3-chlorocatechol; D001616:beta-Galactosidase; C038861:4-chlorocatechol