Activation of the toluene-responsive regulator XyIR causes a transcriptional switch between σ54 and σ70 promoters at the divergent Pr/Ps region of the TOL plasmid

The mechanism by which XylR, the toluene-responsive activator of the sigma(54)-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own sigma(70) promoter Pr has been examined. An in vitro transcription system was developed in order to reproduce the repression of Pr observed in cells of P. putida (pWW0) both in the presence and in the absence of the XylR inducer, benzyl alcohol. DNA templates bearing the two sigma(70)-RNA polymerase (RNAP) binding sites of Pr, which overlap the upstream activating sequences (UAS) for XylR in the divergent sigma(54) promoter Ps, were transcribed in the presence of a constitutively active XylR variant deleted of its N-terminal domain (XylR Delta A). The addition of ATP, known to trigger multimerization of the regulator at the UAS, enhanced the repression of Pr by XylR. Furthermore, we observed activation of the divergent sigma(54) promoter Ps during Pr downregulation by XylR Delta A. These results support the notion that activation of XylR by aromatic inducers in vivo triggers a transcriptional switch between Pr and Ps. Such a switch is apparently caused by the ATP-dependent multimerization and strong DNA binding of the protein required for activation of the sigma(54) promoter. This device could reset the level of XylR expression during activation of the sigma(54) Pu and Ps promoters of the TOL plasmid.