GENETIC-EVIDENCE OF SEPARATE REPRESSOR AND ACTIVATOR ACTIVITIES OF THE XYIR REGULATOR OF THE TOL PLASMID, PWW0, OF PSEUDOMONAS-PUTIDA

The XylR protein encoded by pWW0, the TOL (toluene biodegradation) pla smid of Pseudomonas putida, activates at a distance the transcription of Pu and Ps, which are the two sigma(54)-dependent promoters of the p lasmid, but it also downregulates its own sigma(70)-promoter, Pr, whic h divergently overlaps the upstream activating sites of Ps. All regula tory elements that control Pr activity have been faithfully reproduced in Escherichia coli, and the basis of the autoregulation of XylR tran scription has been examined by monitoring the activity in vivo of diff erent combinations of mutant proteins and promoters in rpoN(+) and rpo N(-) genetic backgrounds. By using Ps/Pr regions bearing deleted or of fset binding sites for XylR and the sigma(54) containing RNA polymeras e, we could show that formation of a nucleoprotein complex involving t he polymerase bound to the divergent promoter Ps is not required for d ownregulation of Pr. Mutant XylR proteins, G268N and A311V (mutated wi thin the NTP-binding region of XylR) or R453H (affected in multimeriza tion), which are unable to activate sigma(54)-dependent transcription from Ps, were indistinguishable from the wild-type XylR in their abili ty to repress a reporter Pr-lacZ fusion. Autoregulation of XylR is the refore due exclusively to the binding of the protein to its target sit es at the Pr promoter. This allows one to define sensu stricto XylR as a transcriptional repressor, independently of its activator role in o ther promoters.