2-CHLOROMUCONATE AND CLCR-MEDIATED ACTIVATION OF THE CLCABD OPERON - IN-VITRO TRANSCRIPTIONAL AND DNASE-I FOOTPRINT ANALYSES

In Pseudomonas putida, the plasmid-borne clcABD operon encodes enzymes involved in 3-chlorocatechol degradation. Previous studies have demon strated that these enzymes are induced when P. putida is grown in the presence of 3-chlorobenzoate, which is converted to 3-chlorocatechol, and that ClcR, a LysR-type regulator, is required for this induction. The clcABD operon is believed to have evolved from the chromosomal cat BCA operon, which encodes enzymes that utilize catechol and is regulat ed by CatR. The inducer for the catBCA operon is an intermediate of th e catechol pathway, cis,cis-muconate. In this study, we demonstrate by the use of in vitro transcription assays and lacZ transcription fusio ns in vivo that the analogous intermediate of the 3-chlorocatechol pat hway, 2-chloromuconate, is the inducer of the clcABD operon. The DNase I footprints of ClcR with and without 2-chloromuconate were also dete rmined. An extended region of the promoter from -79 to -25 was occupie d in the absence of inducer, but the -35 region was unprotected. When 2-chloromuconate was added to the binding assays, the footprint contra cted similar to 4 bp at the proximal end of the promoter, and the -35 region was contacted. It is interesting to note that CatR actually ext ends its footprint 14 bp on the catBCA promoter in response to its ind ucer. Although CatR and ClcR change their nucleotide protection patter ns in different manners when exposed to their respective inducers, the ir final footprints resemble each other. Therefore, it is possible tha t their transcriptional activation mechanisms may be evolutionarily co nserved.