Functional characterisation of altered outer membrane proteins for tetracycline resistance in Escherichia coli

Abstract

Alterations in the outer membrane (OM) proteome in response to tetracycline have been reported in Escherichia coli K-12, but information regarding their functional profile is not available. In this study, upregulation of FimD, Tsx, OmpW, OmpC and TolC and downregulation of LamB in response to tetracycline were detected and confirmed by two-dimensional gel electrophoresis subproteomics and Western blotting, respectively. The ability of the six altered OM proteins was investigated using genetically modified strains with gene deletion or complementation. Of the six mutants tested, Delta tolC and Delta ompC showed decreased minimum inhibitory concentrations (MICs) and Delta lamB showed an increased MIC. These changes in the three mutants could be completely recovered by gene complementation. The survival capability of gene-deleted was significantly increased in Delta lamB mutants and significantly decreased in Delta tolC, Delta ompC, Delta ompW and Delta tsx with respect to the control; a more significant decrease was observed in Delta tolC and Delta ompC than Delta ompW and Delta tsx. These changes were also observed in their complemented strains. Our results indicate that LamB, OmpC and TolC are important OM proteins for tetracycline resistance in E. coli. Meanwhile, our data suggest that functional validation is required to investigate whether the altered proteins obtained from two-dimensional gel electrophoresis play a direct or indirect role in phenotypic changes. (C) 2008 Elsevier B. V. and the International Society of Chemotherapy. All rights reserved.