Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response

The Cpx envelope stress response of Escherichia coli is controlled by a two -component regulatory system that senses misfofded proteins in extracytopla smic compartments and responds by inducing the expression of envelope prote in folding and degrading factors. We have proposed that in the absence of e nvelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and th e sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tether itlg an MBP-CpxP fusion protein to the spheroprast inner membranes prevents full activation by this treatment. Spheroplast formation has previously be en demonstrated to induce the expression of a periplasmic protein of unknow n function, Spy. Analysis of spy expression in response to spheroplast form ation by Western blot analysis and by lacZ operon fusion in various cpx mut ant backgrounds demonstrated that spy is a member of the Cpx regulon. Inter estingly, although the only known spy homologue is cpxP, Spy does not appea r to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activati on of the sigma(E) stress response. Because the sigma(E) response is specif ically affected by alterations in outer membrane protein biogenesis, we thi nk it possible that Spy may be involved in this process.