Response of the σE-dependent and the CpxA/R-regulated envelope stress pathways to inactivation and overexpression of ppiD. EσE (A) and Cpx (B) activities in the indicated strains carrying SurA (light gray bars), PpiD (dark gray bars), and Skp (black bars) encoding plasmids or an empty vector (pASK75; white bars) were assayed by monitoring the accumulation of β-galactosidase resulting from σE-dependent rpoH P3::lacZ and from Cpx-meditated cpxP-lacZ reporter expression, respectively. Cells were grown in LB (σE) or in LB buffered at pH 7.0 (Cpx) at 37°C and β-galactosidase activities were determined as described in Methods and compared to that of wild-type cells. Results represent the average of at least two independent experiments (*P ≤ 0.05; **P ≤ 0.01 Student's t-test). Qualitatively similar results were obtained from cells grown at 30°C (data not shown). (C) Western blot analysis of crude extracts derived from cells with (+) and without (-) pPpiD. A volume of sample equivalent to 4 × 107 cells was loaded onto each lane. The anti-PpiD antiserum showed a weak unspecific cross-reaction with a similar sized unknown protein. The intensity of the PpiD signal relative to that in the wild-type strain (rel. Int.) was calculated using MalE as the internal standard for each lane.