Figure 4.

Signal sequences that further increase the periplasmic level of CpxP. (A) Schematic representation of genetic constructs using the CpxP signal sequence (pTCP), the mutated CpxP signal sequence (pTC*P), the MalE signal sequence (pTMP), and the DsbA signal sequence (pTDP) fused to the mature coding sequence of CpxP. (B) MM2 strain harboring pTCP (1), pTC*P (2), pTMP (3), and pTDP (4) was grown at 37°C with IPTG induction, then fractionated by spheroplast preparation. Whole-cell extracts and periplasmic fractions, normalized to the same amount of cells, were separated by SDS-PAGE and stained by Coomassie Blue. The position of CpxP is indicated by an arrow and the relative amount of this protein is indicated under the corresponding lane. (C) Effect of cpxP overexpression on the activity of its own promoter. SR4046 strain, carrying a chromosomal cpxP-lacZ fusion and transformed by the indicated plasmids, was grown at 37°C with IPTG induction. Miller units of lacZ encoded β-galactosidase were calculated using the average of four independent experiments. Inhibition of the cpxP promoter activity is indicated at the top of the bars.