ACTIVATION OF THE TRANSCRIPTIONAL REGULATOR XYLR OF PSEUDOMONAS-PUTIDA BY RELEASE OF REPRESSION BETWEEN FUNCTIONAL DOMAINS

In the presence of toluene, xylenes and other structural analogues, th e regulatory protein XylR, of the family of transcriptional regulators which act in concert with the sigma(54) factor, activate the promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putid a. Amino acid changes Val-219-Asp and Ala-220-Pro, introducing a proli ne kink at the hinge region between the N-terminal A domain and the ce ntral portion of XylR, resulted in a semi-constitutive phenotype which mimicked the activating effect of aromatic inducers. This phenotype w as further exacerbated by inserting extra amino acid residues within t he same inter-domain region. A truncated XylR protein devoid of the si gnal-receiving, aminoterminal portion of the protein stimulated the co gnate promoter Pu at high levels independently of inducer addition, bo th in Escherichia coil and in Pseudomonas putida. Replacement of the a mino-terminal domain by a heterologous peptide derived from the MS2 vi rus polymerase resulted in a hybrid protein still able to bind DNA to the same extent in vivo as XylR, but unable to stimulate transcription . These data indicate that a key event in the activation of XylR by to luene/xylenes is the release of the repression caused by the A domain of the protein on surfaces located at the central domain of the regula tor.