Transduction of envelope stress in Escherichia coli by the Cpx two-component system

Abstract

Disruption of normal protein trafficking in the Escherichia coli 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 transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity 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 interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.A distinct set of protein folding factors and proteases are located in the cell envelope (inner membrane, periplasm, outer membrane) of the gram-negative bacterium Escherichia coli. These include the periplasmic protease DegP (37, 65), a number of enzymes involved in the catalysis of disulfide bond formation, DsbA to D (3-5, 29, 45, 48, 58), and three different peptidyl-prolyl isomerases, FkpA (21), PpiA (38), and SurA (32, 54). Several of these are directly involved in protein folding or degradation within the bacterial envelope. For example, DegP is required for survival above 40°C (35-37, 64, 65) and functions to degrade some abnormal or misassembled envelope proteins (12,31,63,64

The extracytoplasmic adaptor protein CpxP is degraded with substrate by DegP

“…It encodes a small, stress-combative periplasmic protein capable of negatively regulating the Cpx response (12)(13)(14). In this study, we provide evidence that CpxP is degraded by the periplasmic protease DegP, together with misfolded PapE or PapG.…”

“…All strains were grown in Luria broth (LB) at 30°C unless otherwise indicated. Strains containing the cpxA24 allele were maintained on LB agar containing amikacin (Sigma) at 1.5 g͞ml (13). Plasmid-containing strains were grown in either ampicillin (Sigma) or chloramphenicol (Sigma) at concentrations of 125 g͞ml and 20 g͞ml, respectively.…”

“…In contrast, the Cpx stress response is a prototypical two-component regulatory system consisting of a sensory inner membrane kinase (CpxA) and a cytoplasmic response regulator (CpxR). When a stress is sensed, CpxA autophosphorylates on a conserved histidine in the cytoplasmic kinase domain and then activates CpxR via phosphorylation of a conserved aspartate residue (3). CpxRphosphate is a transcriptional activator that both autoregulates the cpxRA operon and up-regulates the expression of various periplasmic stress-combative activities, some of which overlap with those of E and some that are unique to Cpx (4).…”

The DNA-binding domain of the Escherichia coli CpxR two-component response regulator is constitutively active and cannot be fully attenuated by fused adjacent heterologous regulatory domains

“…The proteins were either directly stained with Coomassie brilliant blue or transferred to nitrocellulose, stained with acidic Ponceau S and subjected to Western blot analysis. Rabbit polyclonal antibody raised against a maltosebinding protein (MBP)-CpxR hybrid (Raivio & Silhavy, 1997) was used at a 1 : 30 000 dilution and revealed with horseradish-peroxidaseconjugated goat anti-rabbit IgG (Bio-Rad). Enhanced chemiluminescence reagents were purchased from Pierce and used according to the manufacturer's instructions.…”

ADP reduces the oxygen-binding affinity of a sensory histidine kinase, FixL: The possibility of an enhanced reciprocating kinase reaction

“…A positive feedback, for example, can account for the stabilization, amplification, or prolongation of the outputs in the course of a signaling cascade. In fact, the feedback loops have been reported in two-component systems with some transcriptional factors looping downstream of the signaling pathways (13)(14)(15). However, the issue of whether ligand recognition or the ligandbinding affinity of sensory histidine kinases is directly modulated by such a feedback loop remains unclear, because few ligandbinding studies for other two-component sensory histidine kinases have been reported because of the experimental difficulties of measuring their binding affinities.…”