Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons

Abstract

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.