Signal Detection and Target Gene Induction by the CpxRA Two-Component System

In gram-negative bacteria, the cell envelope, which consists of the outer membrane, the inner membrane, and the periplasm, comes into direct contact with a variety of environments and is consequently involved in a number of adaptive responses. In order to preserve envelope integrity, at least two stress response pathways in Escherichia coli sense and respond to environmental stress. The σE and Cpx signal transduction pathways both control the synthesis of proteins involved in repairing damaged proteins in the envelope; however, each pathway has unique inputs and outputs (6, 24, 30, 33). It is thought that the σE pathway monitors assembly of outer membrane β-barrel proteins, whereas the primary functions of the Cpx pathway are to maintain surface structures such as pili (19) and to detect the attachment of the bacterial cell to surfaces (19, 29). The Cpx pathway is a prototypical two-component signal transduction system. The input signal is transduced across the inner membrane by the sensor kinase CpxA. CpxA consists of a periplasmic domain and a conserved cytoplasmic signaling domain separated by two transmembrane α-helices (44). It is thought that upon detection of envelope stress, CpxA autophosphorylates by using ATP at a conserved histidine and then transfers this phosphate to a conserved aspartate in the amino-terminal domain of the response regulator CpxR. CpxR-phosphate (CpxR∼P) then activates transcription of genes whose products are involved in protein physiology in the envelope (33). Known Cpx regulon members include the disulfide oxidase DsbA, the protease and chaperone DegP, the peptidyl-prolyl isomerase PpiA, and Spy, a periplasmic protein of unknown function (8, 30, 31). Recent work revealed that the Cpx regulon may contain >100 members (14). The Cpx regulon is positively autoregulated and is feedback inhibited by regulon member CpxP, a small periplasmic protein (13, 32). It is thought that CpxP negatively regulates by interacting with the periplasmic domain of CpxA, keeping the pathway off. During envelope stress, CpxP is thought to be titrated from CpxA, perhaps by misfolded proteins, allowing for activation of the pathway (32, 34). NlpE is an outer membrane lipoprotein that upregulates the Cpx pathway when overproduced (40). Adhesion to hydrophobic surfaces activates the Cpx pathway in an NlpE-dependent manner, suggesting that NlpE is an upstream component of the Cpx pathway (7, 29). The role of NlpE in the transduction of other stresses is not known. A large number of environmental conditions upregulate the Cpx pathway (4, 5, 20, 21, 25, 29, 40). How, then, does the Cpx pathway appropriately sense and respond to diverse stresses? Here we show that induction of Cpx by known envelope stresses requires the periplasmic domain of CpxA, but not NlpE; we argue that all of these stresses present a single periplasmic signal to the Cpx pathway. We also show that CpxP is not required for signal transduction, but its activity as a negative regulator is inactivated under conditions of envelope stress. Moreover, we report that several Cpx-regulated target genes are coregulated by other stress responses.