FIG 4.
NlpE senses oxidative folding defects in the periplasm. (A) The oxidoreductive state of NlpE modulates Cpx activity. β-Galactosidase activity from PcpxP-lacZ was measured in wild-type (GL43), nlpE::kanR (GL44), ΔdsbA (GL64), ΔdsbA nlpE::kanR (GL65), nlpE::nlpEC165A/C231A (GL375), and nlpE::nlpEC51A/C54A (GL252) cells. All values were normalized to the average activity obtained for GL43. Bars represent the averages of normalized values for at least three independent clones. Error bars indicate standard deviations. (B) Oxidative folding defects cause reduction of the disulfide bond in the C-terminal and N-terminal domains of NlpE. Western blotting using antibody raised against NlpE shows the NlpE migration profiles for wild-type (GL43), ΔdsbA (GL64), ΔdsbA nlpE::nlpEC51A/C54A (GL229), and ΔdsbA nlpE::nlpEC165A/C231A (GL372) cells. Samples were either not treated, treated with AMS (alkylated control), or treated with AMS and DTT (fully reduced alkylated control) as indicated. The potential redox states corresponding to the different bands are schematized at the bottom.