Figure 4

Overproduction of PpiD in surA skp cells stimulates synthesis and folding of OmpA. The SurA- depletion strains P _Llac-O1 -surA (SB44454) and P _Llac-O1 -surA Δskp (SB44452; Δskp) were grown at 37°C in LB buffered at pH 7.0 supplemented with 0.2% maltose ±of IPTG. Cells contained either pPpiD (+) or the empty vector pASK75 (-). The data shown are representative for a minimum of two independent experiments. (A) Total cellular levels of SurA and of OmpA in SurA-depletion strains grown for 240 min as described above. Extracts corresponding to 8 × 10⁷ cells were loaded onto each lane and analyzed by western blotting. Signal intensities were calculated using cytoplasmic Hsc66 as the internal standard for each lane and are shown relative to those in the SurA-depleted P _Llac-O1 -surA strain (rel. Int.). (B) Levels of unfolded OmpA (u-OmpA) and folded OmpA (f-OmpA) species in SurA-depletion strains grown as described above. Culture samples corresponding to an equal number of cells were taken at the indicated time points and cell extracts prepared by gentle lysis. Samples of cell extracts corresponding to 1.3 × 10⁸ cells were loaded onto each lane and analyzed by western blotting. Relative signal intensities (rel. Int.) for u-OmpA (u) and f-OmpA (f) were calculated as in A.