INTERACTION OF 2 LYSR-TYPE REGULATORY PROTEINS CATR AND CLCR WITH HETEROLOGOUS PROMOTERS - FUNCTIONAL AND EVOLUTIONARY IMPLICATIONS

The soil bacteria Pseudomonas putida can use benzoate or 3-chlorobenzo ate as a sole carbon source. Benzoate and 3-chlorobenzoate are convert ed 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 p lasmid-borne 3-chlorocatechol degradation genes evolved from the chrom osomal catechol degradation genes. Both catechol and 3-chlorocatechol pathways are positively regulated by the homologous regulatory protein s CatR and ClcR, respectively. These proteins belong to the LysR famil y of DNA binding proteins and bind to highly conserved target sequence s. 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-shif t assays to interact with the clcABD promoter region. Likewise, ClcR w as shown to interact in vitro with the catBC promoter region. In in vi vo experiments, CatR complemented a ClcR(-) P. putida strain harboring the clcABD operon for growth on 3-chlorobenzoate. However, ClcR was n ot capable of complementing a CatR(-) P. putida strain for growth on b enzoate. These observations were confirmed by lacZ-transcriptional fus ion expression experiments. Differences in the CatR and ClcR binding s ites and their in vitro binding characteristics may explain the abilit y of CatR and not ClcR to cross-activate. These differences may provid e insight about the evolution of regulatory systems in P. putida.