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Applied and Environmental Microbiology, October 1999, p. 4594-4600, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Protective Role of Catalase in Pseudomonas aeruginosa Biofilm Resistance to Hydrogen Peroxide

James G. Elkins,1,2 Daniel J. Hassett,3 Philip S. Stewart,2,4 Herbert P. Schweizer,5 and Timothy R. McDermott1,2,*

Department of Land Resources and Environmental Sciences,1 Center for Biofilm Engineering,2 and Department of Chemical Engineering,4 Montana State University, Bozeman, Montana 59717; Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-05243; and Department of Microbiology, Colorado State University, Fort Collins, Colorado 805235

Received 21 June 1999/Accepted 9 August 1999

The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investigated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibility to H2O2. Over the course of 1 h, wild-type cell viability decreased steadily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas biofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-inducible catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance. The katA mutant possessed undetectable catalase activity. Planktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of culturable cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, activity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cultures and biofilms. Enzyme assays and catalase activity bands in nondenaturing polyacrylamide gels showed significant KatB catalase induction occurred in biofilms after a 20-min exposure to H2O2, suggesting that biofilms were capable of a rapid adaptive response to the oxidant. Reporter gene data obtained with a katB::lacZ transcriptional reporter strain confirmed katB induction and that the increase in total cellular catalase activity was attributable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM dose and were unable to make detectable levels of beta -galactosidase. The results of this study demonstrated the following: the constitutively expressed KatA catalase is important for resistance of planktonic and biofilm P. aeruginosa to H2O2, particularly at high H2O2 concentrations; KatB is induced in both planktonic and biofilm cells in response to H2O2 insult, but plays a relatively small role in biofilm resistance; and KatB is important to either planktonic cells or biofilm cells for acquired antioxidant resistance when initial levels of H2O2 are sublethal.

* Corresponding author. Mailing address: Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2190. Fax: (406) 994-3933. E-mail: timmcder{at}montana.edu.

Applied and Environmental Microbiology, October 1999, p. 4594-4600, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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