Genetic evidence of separate repressor and activator activities of the XylR regulator of the TOL plasmid, pWWO, of <i>Pseudomonas putida</i>

<jats:title>Summary</jats:title><jats:p>The XylR protein encoded by pWWO, the TOL (toluene biodegradation) plasmid of <jats:italic>Pseudomonas putida</jats:italic>, activates at a distance the transcription of <jats:italic>Pu</jats:italic> and <jats:italic>Ps</jats:italic>, which are the two σ<jats:sup>54</jats:sup>‐dependent promoters of the plasmid, but it also downregulates its own σ<jats:sup>70</jats:sup>‐promoter, <jats:italic>Pr</jats:italic>, which divergently overlaps the upstream activating sites of <jats:italic>Ps.</jats:italic> All regulatory elements that control <jats:italic>Pr</jats:italic> activity have been faithfully reproduced in <jats:italic>Escherichia coli</jats:italic>, and the basis of the autoregulation of XylR transcription has been examined by monitoring the activity <jats:italic>in vivo</jats:italic> of different combinations of mutant proteins and promoters in <jats:italic>rpoN<jats:sup>+</jats:sup></jats:italic> and <jats:italic>rpoN<jats:sup>‐</jats:sup></jats:italic> genetic backgrounds. By using <jats:italic>PsIPr</jats:italic> regions bearing deleted or offset binding sites for XylR and the σ<jats:sup>54</jats:sup>‐containing RNA polymerase, we could show that formation of a nucleoprotein complex involving the polymerase bound to the divergent promoter <jats:italic>Ps</jats:italic> is not required for downregulation of <jats:italic>Pr.</jats:italic> Mutant XylR proteins, G268N and A311V (mutated within the NTP‐binding region of XylR) or R453H (affected in multi‐merization), which are unable to activate (‐dependent transcription from <jats:italic>Ps</jats:italic>, were indistinguishable from the wild‐type XylR in their ability to repress a reporter <jats:italic>Pr‐lacZ</jats:italic> fusion. Autoregulation of XylR is therefore due exclusively to the binding of the protein to its target sites at the <jats:italic>Pr</jats:italic> promoter. This allows one to define <jats:italic>sensu stricto</jats:italic> XylR as a transcriptional repressor, independently of its activator role in other promoters.</jats:p>