Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting

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Abstract
CatR, a LysR family protein, positively regulates the Pseudomonas putida catBC operon, which is required for growth on benzoate as a sole carbon source. Transcriptional studies show that the catR and catBC promoters are divergent and overlapping by 2 bp. A beta-galactosidase promoter probe vector was constructed to analyze expression from the catR and catBC promoters under induced and uninduced conditions. As predicted, the catBC promoter is expressed only under induced conditions, while the catR promoter is constitutive. CatR has been shown to specifically bind the catRBC promoter region, and this property was used to devise a purification protocol for CatR. Linear M13 DNA containing the catRBC control region was covalently bound to cyanogen bromide-activated Sepharose in order to construct a DNA affinity column. Crude extracts containing hyperproduced CatR protein were then incubated with the affinity resin under binding conditions, and the CatR protein was eluted with 1 M NaCl. CatR was also purified by heparin-agarose chromatography. This highly purified protein was used for gel retardation and hydroxyl-radical footprinting studies. From this analysis, it was shown that CatR binds upstream of the catBC promoter within the transcribed region of catR.