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Journal of Bacteriology, May 2008, p. 3658-3669, Vol. 190, No. 10
0021-9193/08/$08.00+0     doi:10.1128/JB.00022-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Peptidoglycan-Associated Lipoprotein OprL Helps Protect a Pseudomonas aeruginosa Mutant Devoid of the Transactivator OxyR from Hydrogen Peroxide-Mediated Killing during Planktonic and Biofilm Culture ^,

Departments of Molecular Genetics, Biochemistry, and Microbiology,¹ Internal Medicine,² Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524,⁴ Veterans Administration Medical Center, Cincinnati, Ohio 45220,³ Laboratory of Bacterial Pathogenesis, Department of Pathology, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210-1214⁵

Received 4 January 2008/ Accepted 14 February 2008

OxyR controls H₂O₂-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10⁷/ml) organisms are easily killed by micromolar H₂O₂. The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H₂O₂. We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H₂O₂ than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H₂O₂. Given the sensitivity of oxyR oprL bacteria to planktonic H₂O₂, we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H₂O₂-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H₂O₂. Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H₂O₂ in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.

Published ahead of print on 29 February 2008.

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