Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central ca tabolic pathways of Pseudomonas putida that convert aromatic and chloroarom atic compounds to tricarboxylic acid (TCA)-cycle intermediates. They are en coded by the evolutionarily related catBCA and clcABD operons, respectively . Expression of the cat and de operons requires the LysR-type transcription al activators CatR and ClcR, and the inducer molecules cis, cis-muconate an d 2-chloro-cis, cis-muconate. In addition to sequence similarities, CatR an d ClcR share functional similarities which allow catR to complement clcR mu tants. DNase-I footprinting, DNA bending and in vitro transcription analyse s with RNA polymerase mutants indicate that CatR and ClcR activate transcri ption via a similar mechanism which involves interaction with the C-termina l domain of the alpha-subunit (alpha-CTD) of RNA polymerase. In vitro trans cription assays with different regions of the clc promoter indicate that th e ClcR dimer bound to the promoter proximal site (the activation binding si te) interacts with the alpha-CTD. Gel shift assays and DNase-I footprinting have demonstrated that CatR occup ies two adjacent sites proximal to the catBCA promoter in the presence of i nducer and an additional binding site within the catB structural gene calle d the internal binding site (IBS). CatR binds the IBS with low intrinsic af finity that is increased by cooperativity in presence of the two promoter b inding sites. Site-directed mutations in the IBS indicate a probable cis-ac ting repressor function for the IBS. The location of the IBS within the cat B structural gene, the cooperativity observed in footprinting studies and p hasing studies suggest that the IBS participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA. Although the core transcriptional activation mechanisms of CatR and ClcR ha ve been conserved, nature has provided some flexibility to respond to diffe rent environmental signals in addition to the presence of inducer. Transcri ptional fusion studies demonstrate that the expression from the clc promote r is repressed when the cells are grown on succinate, citrate or fumarate a nd that this repression is ClcR-dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression f rom the cat promoter. In vitro transcription assays demonstrate that the TC A-cycle intermediate, fumarate, directly and specifically inhibits the form ation of the clcA transcript. No such inhibition was observed when CatR was used as activator on either the cat or clc template. Since both the catech ol and the chlorocatechol pathways feed into the TCA cycle, but only the ch lorocatechol pathway is inhibited by fumarate, there is a subtle difference in the regulation of these two pathways where intracellular sensing of a T CA-cycle intermediate leads to a reduction of chloroaromatic degradation. ( C) 1998 Elsevier Science B.V. All rights reserved.