TRANSDUCTION OF ENVELOPE STRESS IN ESCHERICHIA-COLI BY THE CPX 2-COMPONENT SYSTEM

Disruption of normal protein trafficking in the Escherichia call cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal tra nsduction pathways is a two-component regulatory system comprised of t he histidine kinase CpxA and the response regulator, CpxR. In this stu dy we characterized gain-of-function Cpx mutants in order to learn mo re about Cpx signal transduction. Sequencing demonstrated that the cpx mutations cluster in either the periplasmic, the transmembrane, or t he II-box domain of CpxA. Intriguingly, most of the periplasmic cpx g ain-of-function mutations cluster in the central region of this domain , and one encodes a deletion of 32 amino acids. Strains harboring thes e mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusio ns between the wild-type CpxA and a representative cpx mutant, CpxA10 1, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as auto kinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase ac tivity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx mutations suggest that interaction s with a periplasmic signaling molecule may normally dictate a decreas ed kinase/phosphatase ratio under nonstress conditions.