Genetic evidence of separate repressor and activator activities of the XylR regulator of the TOL plasmid, pWW0, of Pseudomonas putida

The XylR protein encoded by pWW0, the TOL (toluene biodegradation) plasmid of Pseudomonas putida, activates at a distance the transcription of Pu and Ps, which are the two σ54-dependent promoters of the plasmid, but it also downregulates its own σ70-promoter, Pr, which divergently overlaps the upstream activating sites of Ps. All regulatory elements that control Pr activity have been faithfully reproduced in Escherichia coli, and the basis of the autoregulation of XylR transcription has been examined by monitoring the activity in vivo of different combinations of mutant proteins and promoters in rpoN+ and rpoN- genetic backgrounds. By using Ps/Pr regions bearing deleted or offset binding sites for XylR and the σ54-containing RNA polymerase, we could show that formation of a nucleoprotein complex involving the polymerase bound to the divergent promoter Ps is not required for downregulation of Pr. Mutant XylR proteins, G268N and A311V (mutated within the NTP-binding region of XylR) or R453H (affected in multimerization), which are unable to activate σ54-dependent transcription from Ps, were indistinguishable from the wild-type XylR in their ability to repress a reporter Pr-lacZ fusion. Autoregulation of XylR is therefore due exclusively to the binding of the protein to its target sites at the Pr promoter. This allows one to define sensu stricto XylR as a transcriptional repressor, independently of its activator role in other promoters.