Figure 3

Increased PpiD levels reduce σ^E and Cpx activity in surA skp cells. (A) SurA-depletion strains carrying either the chromosomal σ^E-dependent rpoH P3::lacZ or the Cpx-regulated cpxP-lacZ reporter fusions were cultivated at 37°C in LB buffered at pH 7.0 ± IPTG as described in Methods. Once growth of P _Llac-O1 -surA Δskp cells ceased in the absence of IPTG, samples were taken and assayed for σ^E and Cpx activities, respectively, by determining β-galactosidase activity. The strains contained either an empty vector (pASK75) or a plasmid encoding wild-type PpiD, PpiD^I350A, PpiDΔParv, and PpiDΔTM (soluble His₆-PpiD), respectively. The data shown are representative of at least two independent experiments. (B) Western blot detection of SurA and of DegP in crude extracts of cells after 240-minute growth at 37°C in LB ± IPTG. A volume of extracts equivalent to 4 × 10⁷ cells was loaded onto each lane. Signal intensities were calculated using Hsc66 as the internal standard for each lane and are shown relative to those in the wild-type strain (rel. Int.). Western analysis was performed a minimum of two times for each σ^E and Cpx reporter assay, and data for one representative experiment are shown.