Genetic evidence that the XylS regulator of the Pseudomonas TOL meta operon controls the Pm promoter through weak DNA-protein interactions

Abstract

The activation of the Pm promoter of the meta operon of the TOL plasmid of Pseudomonas putida by its cognate XylS activator protein in the presence and absence of benzoate inducers has been examined in specialized Escherichia coli strains carrying Pm-lacZ fusions and the xylS gene in different configurations in which all controlling elements are present in near native conditions and stoichometry. Expression of a chromosomal Pm-xylX::lacZ fusion was primarily dependent on the addition of an effector at a low xylS gene dosage, but such dependency decreased with increasing levels of the regulator, to the point that hyperproduced XylS could, in the absence of any aromatic effector, raise expression to a level 10(4)-fold higher than normal basal levels. Pm activity never reached a defined saturation level within the range of intracellular concentrations permitted by the intrinsic solubility of the protein, thus suggesting a low degree of occupancy of the OmR and OmL (Om right and left half-sites, respectively) operator sequences by XylS. This was confirmed by transcription interference experiments, which indicated that the frequency of occupation of Pm by active XylS is low. This property permits a fine tuning of Pm activity in vivo through changes in intracellular XylS concentrations, as is predicted in current models to account for the coordinated regulation of TOL operons.