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Applied and Environmental Microbiology, July 2005, p. 3512-3518, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3512-3518.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Philipp De Spiegeleer, Jan Sermon, Kristof Vanoirbeek, Abram Aertsen, and Chris W. Michiels^*

Department of Food and Microbial Technology, Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium

Received 4 October 2004/ Accepted 24 January 2005

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN^–) from thiocyanate (SCN^–), using H₂O₂ as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN^–.

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* Corresponding author. Mailing address: K.U. Leuven, Laboratory of Food Microbiology, Kasteelpark Arenberg 22, B-3001 Leuven, Belgium. Phone: 32-16-32 15 78. Fax: 32-16-32 19 60. E-mail: chris.michiels{at}agr.kuleuven.ac.be.

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Applied and Environmental Microbiology, July 2005, p. 3512-3518, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3512-3518.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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