Interaction of two LysR-type regulatory proteins CatR and ClcR with heterologous promoters: functional and evolutionary implications.

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Abstract
The soil bacteria Pseudomonas putida can use benzoate or 3-chlorobenzoate as a sole carbon source. Benzoate and 3-chlorobenzoate are converted into catechol and 3-chlorocatechol, respectively, which are in turn converted into tricarboxylic acid cycle intermediates. The catabolic pathways of both compounds proceed through similar intermediates, have similar genetic organization, and have homologous enzymes responsible for different catabolic steps. This has led to suggestions that the plasmid-borne 3-chlorocatechol degradation genes evolved from the chromosomal catechol degradation genes. Both catechol and 3-chlorocatechol pathways are positively regulated by the homologous regulatory proteins CatR and ClcR, respectively. These proteins belong to the LysR family of DNA binding proteins and bind to highly conserved target sequences. We examined the ability of CatR and ClcR to cross-regulate the two pathways. CatR was shown in vitro by DNase I footprinting and gel-shift assays to interact with the clcABD promoter region. Likewise, ClcR was shown to interact in vitro with the catBC promoter region. In in vivo experiments, CatR complemented a ClcR- P. putida strain harboring the clcABD operon for growth on 3-chlorobenzoate. However, ClcR was not capable of complementing a CatR- P. putida strain for growth on benzoate. These observations were confirmed by lacZ-transcriptional fusion expression experiments. Differences in the CatR and ClcR binding sites and their in vitro binding characteristics may explain the ability of CatR and not ClcR to cross-activate. These differences may provide insight about the evolution of regulatory systems in P. putida.

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