MODULATION OF THE FUNCTION OF THE SIGNAL RECEPTOR DOMAIN OF XYLR, A MEMBER OF A FAMILY OF PROKARYOTIC ENHANCER-LIKE POSITIVE REGULATORS

The XylR protein controls expression from the Pseudomonas putida TOL p lasmid upper pathway operon promoter (Pu) in response to aromatic effe ctors. XylR-dependent stimulation of transcription from a Pu::lacZ fus ion shows different induction kinetics with different effectors, With toluene. activation followed a hyperbolic curve with an apparent It of 0.95 mM and a maximum beta-galactosidase activity of 2,550 Miller uni ts, With o-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent K of 0.55 mM and a Hill coefficient of 2.65, m- Nitrotoluene kept the XylR regulator in an inactive transcriptional fo rm, Therefore, upon binding of an effector, the substituent on the aro matic ring leads Co productive or unproductive XylR forms, The differe nt transcriptional stairs of the XylR regulator are substantiated by X ylR mutants, XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of m-nitrotoluene: however, its response to m-aminotoluene was negligible, in contrast w ith the wild-type regulator, These results illustrate the importance o f the electrostatic interactions in effector recognition and in the st abilization of productive and unproductive forms by the regulator upon aromatic binding, XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcriptinn from Po in the absence of effecter s, whereas substitution of Glu for Asp135 in XylRD135E resulted in a m utant whose ability to recognize effecters was severely impair ed. The refore, the conformation of mutant XylRD135Q as well as XylRD135N: see med to mimic that of the wild-type regulator when effector binding occ urred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an in hibitor molecule such as m-nitrotoluene or m-aminotoluene.