Identification of nucleotides critical for activity of the Pseudomonas putida catBC promoter.

Pseudomonas putida utilizes the catBC operon, which encodes cis,cis-muconate lactonizing enzyme I (MLEI; EC 5.5.1.1) and muconolactone isomerase (MI; EC 5.3.3.4), for growth on benzoate as a sole carbon source. This operon is positively regulated, and the promoter is located 64 bp upstream of the catB translational start site. Using site-specific mutagenesis, we identified nucleotides that influenced the induction of this promoter. Promoter activity was monitored with the promoter probe vector pKT240. Transcription of mRNA from mutant promoters was determined by primer extension mapping. Comparison of the initiation start site of mutant promoters with that of the wild-type promoter identified a single functional promoter.