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

Envelope stress responses play important physiological roles in a variety of processes, including protein folding, cell wall biosynthesis, and pathogenesis. Many of these responses are controlled by extracytoplasmic function (ECF) sigma factors that respond to external signals by means of a membrane-localized anti-sigma factor. One of the best-characterized, ECF-regulated responses is the sigma(E) envelope stress response of Escherichia coli. The sigma(E) pathway ensures proper assembly of outer-membrane proteins (OMP) by controlling expression of genes involved in OMP folding and degradation in response to envelope stresses that disrupt these processes. Prevailing evidence suggests that, in E. coli, a second envelope stress response controlled by the Cpx two-component system ensures proper pilus assembly. The sensor kinase CpxA recognizes misfolded periplasmic proteins, such as those generated during pilus assembly, and transduces this signal to the response regulator CpxR through conserved phosphotransfer reactions. Phosphorylated CpxR activates transcription of periplasmic factors necessary for pilus assembly.

The Cpx two-component signal transduction pathway of Escherichia coli mediates adaptation to envelope protein misfolding. However, there is experimental evidence that at least 50 genes in 34 operons are part of the Cpx regulon and many have functions that are undefined or unrelated to envelope protein maintenance. No comprehensive analysis of the Cpx regulon has been presented to date. In order to identify strongly Cpxregulated genes that might play an important role(s) in envelope protein folding and/or to further define the role of the Cpx response and to gain insight into what makes a gene subject to strong Cpx regulation, we have carried out a uniform characterization of a Cpx-regulated lux reporter library in a single-strain background. Strongly Cpx-regulated genes encode proteins that are directly linked to envelope protein folding, localized to the envelope but uncharacterized, or involved in limiting the cellular concentration of noxious molecules. Moderately Cpx-regulated gene clusters encode products implicated in biofilm formation. An analysis of CpxR binding sites in strongly regulated genes indicates that while neither a consensus match nor their orientation predicts the strength of Cpx regulation, most genes contain a CpxR binding site within 100 bp of the transcriptional start site. Strikingly, we found that while there appears to be little overlap between the Cpx and Bae envelope stress responses, the E and Cpx responses reciprocally regulate a large group of strongly Cpx-regulated genes, most of which are uncharacterized.

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The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain-and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance.

The Cpx envelope stress response (ESR) has been linked to proteins that are integrated into and secreted across the inner membrane for several decades. Initial studies of the cpx locus linked it to alterations in the protein content of both the inner and outer membrane, together with changes in proton motive driven transport and conjugation. Since the mid 1990s, the predominant view of the Cpx envelope stress response has been that it serves to detect and respond to secreted, misfolded proteins in the periplasm. Recent studies in Escherichia coli and other Gram negative organisms highlight a role for the Cpx ESR in specifically responding to perturbations that occur at the inner membrane (IM). It is clear that Cpx adaptation involves a broad suite of changes that encompass many functions in addition to protein folding. Interestingly, recent studies have refocused attention on Cpx-regulated phenotypes that were initially published over 30years ago, including antibiotic resistance and transport across the IM. In this review I will focus on the insights and models that have arisen from recent studies and that may help explain some of the originally published Cpx phenotypes. Although the molecular nature of the inducing signal for the Cpx ESR remains enigmatic, recently solved structures of signaling proteins are yielding testable models concerning the molecular mechanisms behind signaling. The identification of connections between the Cpx ESR and other stress responses in the cell reveals a complex web of interactions that involves Cpx-regulated expression of other regulators as well as small proteins and sRNAs. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

To detect and effectively respond to damage to the cell envelope, Gram‐negative bacteria possess multiple envelope stress responses. Among these, the CpxAR two‐component system has been shown to sense the presence of misfolded periplasmic proteins and increase the production of envelope‐localized protein folding and degrading factors in response. However, recent studies have revealed that additional parameters, such as adhesion and central metabolism, can also be sensed by the Cpx signalling system. The discovery that the Cpx regulon contains dozens to hundreds of genes indicates that the cellular functions of the Cpx response are also likely much broader than previously realized. These newly recognized functions include other aspects of envelope maintenance, communication with other regulatory pathways, and pathogenesis. A new model is emerging in which the Cpx response integrates diverse signals and promotes cell survival by protecting the envelope in multiple ways.

SummaryEscherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The σ σ σ σ E and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The σ σ σ σ E pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.