Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription

Abstract

The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P. putida PaW85, which is involved in phenol catabolism. We have used an in vitro transcription system to study the roles of CatR, cis,cis-muconate, Escherichia coli RNA polymerase, and promoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transcript formation. We also examined the in vitro transcription of three site-directed mutants of the catBCA promoter; the results obtained compared favorably with previous in vivo data. The requirement of the alpha subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha subunit of RNA polymerase has been implicated in direct protein-protein contact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons because RNA polymerases with truncated alpha subunits were deficient in activation. Further experiments demonstrated the arginine at position 265 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previous results, we propose a model to explain the interaction of the different regulatory components leading to CatR-dependent activation of the catBCA operon.