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J Bacteriol, February 1998, p. 600-604, Vol. 180, No. 3
Department of Biochemistry, Molecular and
Cellular Biology of Plants, Estación Experimental del
Zaidín, Consejo Superior de Investigaciones
Científicas, E-18008 Granada, Spain
Received 12 June 1997/Accepted 20 November 1997
The XylR protein controls expression from the Pseudomonas
putida TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from
a Pu::lacZ fusion shows different induction
kinetics with different effectors. With toluene, activation followed a
hyperbolic curve with an apparent K of 0.95 mM and a
maximum
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Modulation of the Function of the Signal Receptor
Domain of XylR, a Member of a Family of Prokaryotic Enhancer-Like
Positive Regulators


-galactosidase activity of 2,550 Miller units. 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 form. Therefore, upon binding of an
effector, the substituent on the aromatic ring leads to productive or
unproductive XylR forms. The different transcriptional states of the
XylR regulator are substantiated by XylR 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 with the
wild-type regulator. These results illustrate the importance of the
electrostatic interactions in effector recognition and in the
stabilization of productive and unproductive forms by the regulator
upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators
that are able to stimulate transcription from Pu in the absence of
effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted
in a mutant whose ability to recognize effectors was severely impaired.
Therefore, the conformation of mutant XylRD135Q as well as XylRD135N
seemed to mimic that of the wild-type regulator when effector binding
occurred, whereas mutant XylRD135E seemed to be blocked in a
conformation similar to that of wild-type XylR and XylRE172K upon
binding to an inhibitor molecule such as m-nitrotoluene or
m-aminotoluene.
*
Corresponding author. Present address: Department of
Biochemistry and Molecular Biology, School of Pharmacy, University of Granada, E-18071 Granada, Spain. Phone: 34-58-246363. Fax:
34-58-248960. E-mail: rsalto{at}goliat.ugr.es.
Present address: Department of Microbiology and Molecular Genetics,
University of California, Los Angeles, CA 90095-1489.
Present address: Department of Biochemistry, School of Veterinary
Sciences, University of Córdoba, 14071 Córdoba, Spain.
§
Present address: Department of Genetics, School of Biology,
University of Seville, 41012 Seville, Spain.
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