A-026. The YcgE Protein of Escherichia coli Is Involved in the Temperature Dependent Regulation of OmpF

V. Duval1, H. Nicoloff2, S. Levy1;
1Tufts Univ. Sch. of Med., Boston, MA, 2The Pennsylvania State Univ., University Park, PA.

Background: In Escherichia coli, an increased multidrug resistance (MDR) comes from the overproduction of the AcrAB-TolC efflux pump and/or from a decreased synthesis of the outer membrane porin F (OmpF) which allows the diffusion of many drugs into the periplasm. A strain that overproduces MarA produces a MDR by both mechanisms. MarA is a direct transcriptional activator of acrAB-tolC but also of micF which is a non-coding small RNA that inactivates the translation of ompF RNA. The rapid turnover of MarA is realized by the Lon protease which has many other specific substrates. Besides MarA, a network of transcriptional regulators control AcrAB and OmpF/micF expression. The purpose of this work is to identify new loci involved in MDR regulation. Methods: By transposome mutagenesis of an E. coli lon mutant strain, we isolated ycgE::Tn5 which expressed an increased multidrug resistance. We analysed by Northern blot the level of marA, ompF and micF expression. SDS-PAGE and Western blot were used to study the stability of MarA and YcgE and to quantify OmpF levels in outer membrane. Results: The increased resistance of the double lon and ycgE mutant comes from a decreased expression of OmpF especially at low temperature and without any effect on MarA RNA and protein levels. At 26°C, the Lon mutation leads to an increased transcription of micF and to a lower expression of ompF whereas ycgE::Tn5 inactivation causes a decreased ompF expression. Both mutations show cumulative effects on the OmpF protein level. In a lon mutant, complementation of the ycgE::Tn5 inactivation by overexpressing YcgE from a multicopy plasmid increases the production of OmpF in outer membrane by activating directly or indirectly the transcription of ompF without affecting micF RNA levels. By overproducing YcgE in wild type and lon mutant strains, we demonstrated that YcgE stability is not affected by Lon. Conclusion: YcgE and another protein affected by Lon are involved in the temperature dependent regulation of ompF.