A TRICARBOXYLIC-ACID CYCLE INTERMEDIATE REGULATING TRANSCRIPTION OF ACHLOROAROMATIC BIODEGRADATIVE PATHWAY - FUMARATE-MEDIATED REPRESSION OF THE CLCABD OPERON

The ortho-cleavage pathways of catechol and 3-chlorocatechol are centr al catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA) cycle intermediat es; They, are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and de operons requires t he LysR-type transcriptional activators CatR and ClcR, respectively, a nd the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconat e, respectively. The regulation of the cat and de promoters has been w ell studied, but the extent to which these operons are repressed by gr owth in TCA cycle intermediates has not been explored. We demonstrate by transcriptional fusion studies that the expression from the de prom oter is repressed when the cells are grown on succinate, citrate, or f umarate and that this repression is ClcR dependent and occurs at the t ranscriptional level. The presence of these organic acids did not affe ct the expression from the cat promoter In vitro transcription assays demonstrate that the TCA cycle intermediate fumarate directly and spec ifically inhibits the formation of the clcA transcript. No such inhibi tion was observed when CatR was used as the activator on either the ca t or de template. Titration studies of fumarate and 2-Chloromuconate s how that the fumarate effect is concentration dependent and reversible , indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR. This is an interesting example of t he transcriptional regulation of a biodegradative pathway by the intra cellular sensing of the state of the TCA cycle.