FIG. 3.

Effects of inducing NlpE on the rdoA promoter and DsbA protein levels. (A) A comparison of promoter activity of rdoA with or without induction of the Cpx response via expression of NlpE by comparing rdoA::lacZ (pPrdoA) in cells containing an arabinose-inducible nlpE (pND18) or control (pBAD18) vector. The cpxR::Ω strain is included to demonstrate that the effect on the rdoA promoter is mediated by the Cpx pathway. Cells were grown to an OD₆₀₀ of 0.3 in the presence of 0.2% glucose. The cultures were then split, and 0.4% arabinose was added to one. Cultures were assayed for β-galactosidase activity at the time of adding arabinose (t = 0) and several times later. (B) Upper panel, protein gel of periplasmic extracts from cells grown under NlpE-uninduced (−) and induced (+) conditions at two loading concentrations (5 and 10 μl) stained with Coomassie blue to indicate total protein levels (DsbA is not visible on this gel); lower panel, Western immunoblot developed with anti-DsbA antiserum. DsbA is more abundant when NlpE is overexpressed. (C) Upper panel, protein gel of periplasmic extracts from the wild-type strain (NLM2247), dsbA null strain (NLM267), rdoA null strain (NLM2255), and rdoA null strain containing a copy of rdoA in trans (NLM2269) grown under NlpE-inducing conditions (all strains contain pDN18 and were grown in the presence of arabinose) stained with Coomassie blue to indicate total protein levels (DsbA is not visible); lower panel, Western immunoblot developed with anti-DsbA antiserum. Inducing NlpE does not increase the DsbA levels to that of the wild type (NLM2247) in the rdoA null (NLM2255) strain. The wild-type phenotype is regained when rdoA is provided in trans (NLM2269).