PROTEIN/DNA INTERACTION AT THE PHEBA PROMOTER

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Many microorganisms metabolize aromatic compounds via formation of catechol as an intermediate by a pathway called the ortho pathway. In Pseudomonas putida, the catechol degradative genes catB and catC are clustered together and organized as an operon. The catBC operon is regulated by a positive regulator CatR which binds upstream of the catB gene, and in presence of the inducer cis,cis-muconate (CCM), binds to two specific sites called repression binding site (RBS) and activation binding site (ABS). Binding at the RBS in absence of the inducer allows autoregulation by CatR of its own gene (catR) which is transcribed divergently from the catBC promoter, but does not lead to activation of the catBC operon. In presence of the inducer CcM, CatR undergoes a conformational change that allows it to bind the additional sequence ABS which leads to transcription initiation at the catBC promoter. Many microorganisms metabolize the toxic compound phenol via formation of catechol as an intermediate. However, in such cases, the catechol derived from phenol is metabolized by a different pathway called the meta pathway. In various well-studied systems, the meta pathway genes are also clustered to form an operon which is regulated by a positive regulator having homology to response regulators of the two-component system. Dr. Kivisaar's group has isolated a strain of P. putida that metabolizes phenol via catechol as an intermediate where the catechol, unlike other well-studied phenol pathways, undergoes mineralization via the chromosomally encoded ortho pathway. The evolved genes pheBA, encoding phenol hydroxylase (pheA gene product) and catechol l,2-dioxygenase (pheB gene product), have a promoter that resembles catBC promoter and allows binding by CatR for functional activation of the pheBA genes. Thus we propose to study comparative interactions of CatR with catBC and pheBA promoters to understand how and why evolution took a short-cut to simply evolve a couple of genes for phenol degradation, instead of a whole pathway that normally evolves in natural microorganisms.