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

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol d egradation resembles the catBCA promoter and is activated by CatR, the regu lator of the chromosomally encoded catechol-degradative catBCA genes in Pse udomonas putida. In this study, site-directed mutagenesis of the pheBA prom oter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homolog ous to that of the catBCA promoter. However, the RES was shown not to be th e sole important feature for high-affinity binding of CatR to this site. Mu tagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleo tides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the Ass negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA an d catBCA promoters between the -10 and -35 hexamers is 19 bp, which is long er than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal bi nding site (IBS) for CatR is located downstream of the transcriptional star t site of the catBCA genes and it negatively regulates the operon. A simila r IBS was identified in the case of the pheSA operon and tested for its fun ctionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This univer sal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substra te range via horizontal transfer of the phenol degradative genes.