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Applied and Environmental Microbiology, September 2000, p. 4119-4123, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Hydrogen Peroxide Sensitivity of Catechol-2,3-Dioxygenase: a Cautionary Note on Use of xylE Reporter Fusions under Aerobic Conditions

Daniel J. Hassett,1,* Urs A. Ochsner,2 Stephanie L. Groce,3 Kislay Parvatiyar,1 Ju-Fang Ma,1 and John D. Lipscomb3

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05241; Department of Microbiology and Immunology, University of Colorado Health Sciences, Denver, Colorado 802622; and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455-03263

Received 16 March 2000/Accepted 15 June 2000

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H2O2) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta -galactosidase) or -xylE fusion plasmids to H2O2 stimulated beta -galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H2O2, while a 10,000-fold excess was required for similar inhibition of beta -galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H2O2 nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.

* Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0524. Phone: (513) 558-1154. E-mail: Daniel.Hassett{at}UC.Edu.

Applied and Environmental Microbiology, September 2000, p. 4119-4123, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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