FIG. 5.

(A) Western blot analysis of inner and outer membranes prepared from the ΔpmrC1.1 strain containing either the pBAC108L vector (EG14656) or the ppmrCFLAG plasmid (EG14592), which carries a pmrC gene directed by its own promoter and expresses a PmrC protein tagged with a FLAG epitope at its C terminus. Bacteria were grown to the logarithmic phase in N-minimal medium, pH 7.7, with 10 μM MgCl₂. Inner and outer membranes were prepared by sucrose density gradient centrifugation. Twenty micrograms of protein from the inner and outer membranes was boiled for 10 min, run in an SDS-10% polyacrylamide gel, transferred onto a nitrocellulose membrane, and developed by using anti-FLAG antibodies. To examine the purity of the membrane preparations, we determined the NADH oxidase activity by measuring the oxidation of NADH at 340 nm, and these values are expressed as follows: 100 × μmol of substrate oxidized/min/mg of protein. The analysis demonstrates that the PmrC protein localizes to the inner membrane. (B) Kyte-Doolittle hydropathy plot (25) of the PmrC protein generated by DNA Strider 1.3 software. (C) The left panel shows the predicted topology of the PmrC protein. The numbers correspond to the positions in the PmrC protein at which in-frame fusions were generated to the PhoA and LacZ proteins. The right panel shows alkaline phosphatase and β-galactosidase activities displayed by the phoN strain (EG14286) harboring plasmids pPmrC₁₅₀-lacZ′, pPmrC₁₅₀-phoA′, pPmrC₁₈₁-lacZ′, pPmrC₁₈₁-phoA′, pPmrC₂₉₅-lacZ′, and pPmrC₂₉₅-phoA′ when streaked onto LB agar plates containing either XP (40 μg/ml) or X-Gal (40 μg/ml). These data suggest that the C-terminal region (amino acids 177 to 547) of PmrC localizes to the periplasm.