FIG. 6.
Protease accessibility of PvdN in spheroplasts. Wild-type PAO1 and PAOΔtatABC mutant strains expressing pvdNV5H6 were grown in phosphate-limited medium. pvdNV5H6 expression was induced in the exponential phase by incubation with 0.1 mM IPTG for 2 h. Cells were then collected and treated for spheroplast preparation. Wild-type (WT) and tatABC (tat) spheroplast samples equivalent to 0.1 OD600 unit of the original cultures were further treated with increasing concentrations of trypsin and analyzed by 11% SDS-PAGE, followed by Western blotting using anti-XcpQ (B) and anti-V5 (C) antibodies. Panel A is a red Ponceau-stained nitrocellulose membrane used for Western blotting. This shows that for each trypsin concentration, the same amount of protein is loaded for the wild-type strain and the tat mutant, indicating that the smaller amount of PvdN observed in the wild-type strain compared to the tat mutant is not due to overloading. This experiment was repeated twice.