Fig 2.

SslE-tetraCys is located on the outer surface of the outer membrane of EPEC strain E2348/69. (A) Titration of the inducer, anhydrotetracycline (ATc), to determine the amount required to induce expression of sslE-tetraCys to levels approximate to those of SslE in wild-type E2348/69. E2348/69ΔsslE(pJP169), where sslE-tetraCys was expressed from a tetA promoter, was grown in CAYE to an optical density at 600 nm (OD₆₀₀) of ∼2 and then induced for 2 h with 0, 30, 35, 40, or 50 ng/ml ATc (lanes 2 to 6, respectively). Proteins were separated by SDS-PAGE using 4–12% bis-Tris NuPAGE gels (Invitrogen) and stained with Coomassie brilliant blue R-250. (B) Fluorescent and phase-contrast images of E2348/69ΔsslE carrying plasmid pJP169 (pJP168::sslE-tetraCys) or the empty vector, pJP168, which was grown in CAYE to an OD₆₀₀ of ∼2 and then induced for 2 h with 35 ng/ml anhydrotetracycline. Bacteria were visualized by differential interference phase contrast on a Zeiss LSM700 inverted confocal microscope, and Lumio Green was excited using a 488-nm diode laser line. Bar, 2 μm. (C) E2348/69ΔsslE(pJP169) cells were grown as described above and incubated without (−) or with (+) polymyxin B and proteinase K, as indicated. Proteins, separated by SDS-PAGE using 4-12% bis-Tris NuPAGE gels (Invitrogen), were analyzed by Lumio Green detection for SslE and by immunoblotting for the controls, BamA (inner surface, outer membrane protein) and the cytoplasmic membrane protein F_oF₁ ATP synthase β-subunit (F₁β). The latter served as a control for sample loading, as it is protected from proteinase K by the inner membrane.