ACTIVATION OF THE TOLUENE-RESPONSIVE REGULATOR XYIR CAUSES A TRANSCRIPTIONAL SWITCH BETWEEN SIGMA-54 AND SIGMA-70 PROMOTERS AT THE DIVERGENT PR PS REGION OF THE TOL PLASMID/

The mechanism by which XylR, the toluene-responsive activator of the s igma(54)-dependent Pu and Ps promoters of the Pseudomonas TOL plasmid pWW0, downregulates its own sigma(70) promoter Pr has been examined. A n in vitro transcription system was developed in order to reproduce th e repression of Pr observed in cells of P. putida (pWW0) both in the p resence and in the absence of the XylR inducer, benzyl alcohol. DNA te mplates 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 presen ce of a constitutively active XylR variant deleted of its N-terminal d omain (XylR Delta A). The addition of ATP, known to trigger multimeriz ation of the regulator at the UAS, enhanced the repression of Pr by Xy lR. Furthermore, we observed activation of the divergent sigma(54) pro moter Ps during Pr downregulation by XylR Delta A. These results suppo rt the notion that activation of XylR by aromatic inducers in vivo tri ggers a transcriptional switch between Pr and Ps. Such a switch is app arently caused by the ATP-dependent multimerization and strong DNA bin ding of the protein required for activation of the sigma(54) promoter. This device could reset the level of XylR expression during activatio n of the sigma(54) Pu and Ps promoters of the TOL plasmid.