Protective role of catalase in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide

The role of the two known catalases in Pseudomonas aeruginosa in protecting planktonic and biofilm cells against hydrogen peroxide (H2O2) was investig ated. Planktonic cultures and biofilms formed by the wild-type strain PAO1 and the katA and katB catalase mutants were compared for their susceptibili ty to H2O2. Over the course of 1 h, wild-type cell viability decreased stea dily in planktonic cells exposed to a single dose of 50 mM H2O2, whereas bi ofilm cell viability remained at approximately 90% when cells were exposed to a flowing stream of 50 mM H2O2. The katB mutant, lacking the H2O2-induci ble catalase KatB, was similar to the wild-type strain with respect to H2O2 resistance, The katA mutant possessed undetectable catalase activity. Plan ktonic katA mutant cultures were hypersusceptible to a single dose of 50 mM H2O2, while biofilms displayed a 10-fold reduction in the number of cultur able cells after a 1-h exposure to 50 mM H2O2. Catalase activity assays, ac tivity stains in nondenaturing polyacrylamide gels, and lacZ reporter genes were used to characterize the oxidative stress responses of planktonic cul tures and biofilms. Enzyme assays and catalase activity bands in nondenatur ing 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 ob tained with a katB::lacZ transcriptional reporter strain confirmed katB ind uction and that the increase in total cellular catalase activity was attrib utable to KatB. Biofilms upregulated the reporter in the constant presence of 50 mM H2O2, while planktonic cells were overwhelmed by a single 50 mM do se and were unable to make detectable levels of beta-galactosidase, The res ults of this study demonstrated the following: the constitutively expressed KatA catalase is important fur resistance of planktonic and biofilm P. aer uginosa 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 eit her planktonic cells or biofilm cells for acquired antioxidant resistance w hen initial levels of H2O2 are sublethal.