P pilus assembly motif necessary for activation of the CpxRA pathway by PapE in Escherichia coli.

P pilus biogenesis occurs via the highly conserved chaperone-usher pathway, and assembly is monitored by the CpxRA two-component signal transduction pathway. Structural pilus subunits consist of an N-terminal extension followed by an incomplete immunoglobulin-like fold that is missing a C-terminal seventh beta strand. In the pilus fiber, the immunoglobulin-like fold of each pilin is completed by the N-terminal extension of its neighbor. Subunits that do not get incorporated into the pilus fiber are driven "OFF-pathway." In this study, we found that PapE was the only OFF-pathway nonadhesin P pilus subunit capable of activating Cpx. Manipulation of the PapE structure by removing, relocating within the protein, or swapping its N-terminal extension with that of other subunits altered the protein's self-associative and Cpx-activating properties. The self-association properties of the new subunits were dictated by the specific N-terminal extension provided and were consistent with the order of the subunits in the pilus fiber. However, these aggregation properties did not directly correlate with Cpx induction. Cpx activation instead correlated with the presence or absence of an N-terminal extension in the PapE pilin structure. Removal of the N-terminal extension of PapE was sufficient to abolish Cpx activation. Replacement of an N-terminal extension at either the amino or carboxyl terminus restored Cpx induction. Thus, the data presented in this study argue that PapE has features inherent in its structure or during its folding that act as specific inducers of Cpx signal transduction.

Signal detection and target gene induction by the CpxRA two-component system.

The Cpx pathway is a two-component signal transduction system that senses a variety of envelope stresses, including misfolded proteins, and responds by upregulating periplasmic folding and trafficking factors. CpxA resides in the inner membrane and has both kinase and phosphatase activities. CpxR, the response regulator, mediates a response by activating transcription of stress-combative genes. Signal transduction is subject to feedback inhibition via regulon member CpxP and autoamplification. Recently, it was shown that the Cpx pathway is also upregulated when cells adhere to hydrophobic surfaces and that this response is dependent on the outer membrane lipoprotein NlpE. Here we show that while NlpE is required for induction of the Cpx pathway by adhesion, induction by envelope stress and during growth is NlpE independent. We show that while all of the envelope stresses tested induce the Cpx pathway in a manner that is dependent on the periplasmic domain of CpxA, induction during growth is independent of CpxA. Therefore, we propose that the Cpx pathway can sense inducing cues that enter the signaling pathway at three distinct points. Although CpxP is not required for induction of the Cpx pathway, we show that its activity as a negative regulator of CpxA is inactivated by envelope stress. Moreover, the cpxP promoter is more inducible than any other regulon member tested. Consistent with these results, we suggest that CpxP performs a second function, most likely that of a chaperone. Finally, we show that two Cpx-regulated genes are differentially upregulated in response to different envelope stresses, suggesting the existence of three stress-responsive systems.