Genetic evidence that the XylS regulator of the Pseudomonas TOL meta operon controls the Pm promoter through weak DNA-protein interactions.
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.
Kessler
B
B
Gesellschaft für Biotechnologische Forschung, Bereich Mikrobiologie, Braunschweig, Germany.
Herrero
M
M
Timmis
K N
KN
de Lorenzo
V
V
D051777:AraC Transcription Factor; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014050:Toluene / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D001565:Benzoates; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C078720:XylS protein, Pseudomonas putida; D014050:Toluene; D019817:Benzoic Acid