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Applied and Environmental Microbiology, October 1999, p. 4594-4600, Vol. 65, No. 10
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
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
-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.
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