Cpx Two-component System Research Articles

Mutation of the envelope stress-responsive protein CpxA capable of regulating the antimicrobial resistance and virulence of bacteria

CpxA is a key member of the envelope stress-responsive Cpx two-component system ubiquitous in Gram-negative bacteria. It is responsible for signal sensing and has dual activities of phosphatase and kinase. CpxA has been revealed to participate in the regulation of physiological processes such as virulence and antimicrobial resistance of bacteria. In recent years, the development of novel antimicrobials targeting CpxA has attracted much attention. Drugs developed based on inhibition of the phosphatase activity of CpxA have shown effectiveness in the treatment of urinary tract infections caused by Escherichia coli. This review introduces the structure and functional domains of CpxA and the activation of Cpx pathways by CpxA. Furthermore, it summarizes the roles of CpxA in the development of antimicrobial resistance and the regulation of bacterial virulence and reviews the latest progress in the development of new antimicrobials targeting this protein. It is expected to assist in the exploration of CpxA-targeting anti-infection strategies for severely antimicrobial-resistant bacteria whose clinical infections are of urgent need to be controlled.

The Cpx Stress Response Regulates Turnover of Respiratory Chain Proteins at the Inner Membrane of Escherichia coli.

The Cpx envelope stress response is a major signaling pathway monitoring bacterial envelope integrity, activated both internally by excessive synthesis of membrane proteins and externally by a variety of environmental cues. The Cpx regulon is enriched with genes coding for protein folding and degrading factors, virulence determinants, and large envelope-localized complexes. Transcriptional repression of the two electron transport chain complexes, NADH dehydrogenase I and cytochrome bo3, by the Cpx pathway has been demonstrated, however, there is evidence that additional regulatory mechanisms exist. In this study, we examine the interaction between Cpx-regulated protein folding and degrading factors and the respiratory complexes NADH dehydrogenase I and succinate dehydrogenase in Escherichia coli. Here we show that the cellular need for Cpx-mediated stress adaptation increases when respiratory complexes are more prevalent or active, which is demonstrated by the growth defect of Cpx-deficient strains on media that requires a functional electron transport chain. Interestingly, deletion of several Cpx-regulated proteolytic factors and chaperones results in similar growth-deficient phenotypes. Furthermore, we find that the stability of the NADH dehydrogenase I protein complex is lower in cells with a functional Cpx response, while in its absence, protein turnover is impaired. Finally, we demonstrated that the succinate dehydrogenase complex has reduced activity in E. coli lacking the Cpx pathway. Our results suggest that the Cpx two-component system serves as a sentry of inner membrane protein biogenesis, ensuring the function of large envelope protein complexes and maintaining the cellular energy status of the cell.

Contribution of the Locus of Heat Resistance to Growth and Survival of Escherichia coli at Alkaline pH and at Alkaline pH in the Presence of Chlorine.

The locus of heat resistance (LHR) confers resistance to extreme heat, chlorine and oxidative stress in Escherichia coli. This study aimed to determine the function of the LHR in maintaining bacterial cell envelope homeostasis, the regulation of the genes comprising the LHR and the contribution of the LHR to alkaline pH response. The presence of the LHR did not affect the activity of the Cpx two-component regulatory system in E. coli, which was measured to quantify cell envelope stress. The LHR did not alter E. coli MG1655 growth rate in the range of pH 6.9 to 9.2. However, RT-qPCR results indicated that the expression of the LHR was elevated at pH 8.0 when CpxR was absent. The LHR did not improve survival of E. coli MG1655 at extreme alkaline pH (pH = 11.0 to 11.2) but improved survival at pH 11.0 in the presence of chlorine. Therefore, we conclude that the LHR confers resistance to extreme alkaline pH in the presence of oxidizing agents. Resistance to alkaline pH is regulated by an endogenous mechanism, including the Cpx envelope stress response, whereas the LHR confers resistance to extreme alkaline pH only in the presence of additional stress such as chlorine.

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope.

The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. The Cpx two-component system is an extensively studied ESRS in Escherichia coli that is active during exposure to a vast array of stresses and protects the envelope under those harmful circumstances. Overproduction of NlpE, a two-domain outer membrane lipoprotein of unclear function, has been used in numerous studies as a molecular trigger to turn on the system artificially. However, the mechanism of Cpx activation by NlpE, as well as its physiological relevance, awaited further investigation. In this paper, we provide novel insights into the role played by NlpE in the Cpx system. We found that, among all outer membrane lipoproteins in E. coli, NlpE is sufficient to induce Cpx when lipoprotein trafficking is perturbed. Under such conditions, fitness is increased by the presence of NlpE. Moreover, we show that NlpE, through its N-terminal domain, physically interacts with the Cpx sensor kinase CpxA. Our data suggest that NlpE also serves to activate the Cpx system during oxidative folding defects in the periplasm and that its C-terminal domain is involved in the sensing mechanism. Overall, our data demonstrate that NlpE acts as a sentinel for two important envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding, and they further establish NlpE as a bona fide member of the Cpx two-component system.IMPORTANCE Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gram-negative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli, since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.

Elucidating the contribution of the Pseudomonas aeruginosa CPX system in biofilm formation

An investigation of Pseudomonas aeruginosa attachment to a diverse range of polymers in a high-throughput microarray format resulted in the discovery of novel biofilm-resistant and biofilm-stimulating materials. These findings raised questions about the nature of the surface interactions involved and in particular the sensory mechanisms use by P. aeruginosa to distinguish between different surface chemistries. To further investigate this, Tn5 mutants of the P. aeruginosa PAO1 Washington sub-line were tested for biofilm formation on the polymer microarrays. This revealed that a Tn5::cpxR mutant had a significant difference in biofilm formation when compared with PAO1-W. In Escherichia coli cpxR forms an operon with cpxA and cpxP. Together they form the CPX two-component signalling system controlling biofilm formation in response to cell envelope stress. To further characterise the P. aeruginosa cpxsystem, ΔcpxR,ΔcpxP and ΔcpxA deletion mutants were constructed. No significant differences were observed in their growth or in the swimming, swarming or twitch motility phenotypes normally required for surface colonization. Unlike E. coli the PAO1-W ΔcpxAmutant was able to invade lung epithelial cells and did not display increased sensitivity to antibiotics. However, the ΔcpxRmutant showed increased biofilm formation on glass and eDNA secretion in both biofilm and liquid modes of growth. This work highlights the relationship between biofilm formation and the CPX system in P. aeruginosa. However, further assays need to be conducted in order to understand the sensory mechanism(s) involved in surface sensing via the CPX system.

Epigallocatechin Gallate-Mediated Cell Death Is Triggered by Accumulation of Reactive Oxygen Species Induced via the Cpx Two-Component System in Escherichia coli.

The high antimicrobial activity of epigallocatechin gallate (EGCG), the most bioactive component of tea polyphenol with a number of health benefits, is well-known. However, little is known about the mechanism involved. Here, we discovered the relationship between reactive oxygen species (ROS), the Cpx system, and EGCG-mediated cell death. We first found an increase in ampicillin resistance as well as the transcription level of a LD-transpeptidase (LD-TPase) involved in cell wall synthesis; ycbB transcription was upregulated whereas that of another LD-TPase, ynhG, appeared to be constant after a short exposure of Escherichia coli to sub-inhibitory doses of EGCG. Additionally, the transcription level of cpxP, a downstream gene belonging to the Cpx regulon, was positively correlated with the concentration of EGCG, and significant upregulation was detected when cells were treated with high doses of EGCG. Through analysis of a cpxR deletion strain (ΔcpxR), we identified a constant ROS level and a notable increase in the survival rate of ΔcpxR, while the ROS level increased and the survival rate decreased remarkably in the wild-type strain. Furthermore, thiourea, which is an antioxidant, reduced the ROS level and antimicrobial activity of EGCG. Taken together, these results suggest that EGCG induces ROS formation by activating the Cpx system and mediates cell death.

The Vibrio cholerae VexGH RND Efflux System Maintains Cellular Homeostasis by Effluxing Vibriobactin.

ABSTRACTResistance-nodulation-division (RND) superfamily efflux systems have been widely studied for their role in antibiotic resistance, but their native biological functions remain poorly understood. We previously showed that loss of RND-mediated efflux in Vibrio cholerae resulted in activation of the Cpx two-component regulatory system, which mediates adaptation to stress resulting from misfolded membrane proteins. Here, we investigated the mechanism linking RND-mediated efflux to the Cpx response. We performed transposon mutagenesis screening of RND-deficient V. cholerae to identify Cpx suppressors. Suppressor mutations mapped to genes involved in the biosynthesis of the catechol siderophore vibriobactin. We subsequently demonstrated that vibriobactin secretion is impaired in mutants lacking the VexGH RND efflux system and that impaired vibriobactin secretion is responsible for Cpx system activation, suggesting that VexGH secretes vibriobactin. This conclusion was bolstered by results showing that vexGH expression is induced by iron limitation and that vexH-deficient cells exhibit reduced fitness during growth under iron-limiting conditions. Our results support a model where VexGH contributes to cellular homeostasis by effluxing vibriobactin. In the absence of vexGH, retained vibriobactin appears to chelate iron from iron-rich components of the respiratory chain, with the deferrated proteins functioning to activate the Cpx response. Our collective results demonstrate that a native function of the V. cholerae VexGH RND efflux system is in vibriobactin secretion and that vibriobactin efflux is critical for maintenance of cellular homeostasis.

Dynamic interaction between the CpxA sensor kinase and the periplasmic accessory protein CpxP mediates signal recognition in E. coli.

Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.

Reciprocal regulation of resistance-nodulation-division efflux systems and the Cpx two-component system in Vibrio cholerae.

The Cpx two-component regulatory system has been shown in Escherichia coli to alleviate stress caused by misfolded cell envelope proteins. The Vibrio cholerae Cpx system was previously found to respond to cues distinct from those in the E. coli system, suggesting that this system fulfills a different physiological role in the cholera pathogen. Here, we used microarrays to identify genes that were regulated by the V. cholerae Cpx system. Our observations suggest that the activation of the V. cholerae Cpx system does not induce expression of genes involved in the mitigation of stress generated by misfolded cell envelope proteins but promotes expression of genes involved in antimicrobial resistance. In particular, activation of the Cpx system induced expression of the genes encoding the VexAB and VexGH resistance-nodulation-division (RND) efflux systems and their cognate outer membrane pore protein TolC. The promoters for these loci contained putative CpxR consensus binding sites, and ectopic cpxR expression activated transcription from the promoters for the RND efflux systems. CpxR was not required for intrinsic antimicrobial resistance, but CpxR activation enhanced resistance to antimicrobial substrates of VexAB and VexGH. Mutations that inactivated VexAB or VexGH efflux activity resulted in the activation of the Cpx response, suggesting that vexAB and vexGH and the cpxP-cpxRA system are reciprocally regulated. We speculate that the reciprocal regulation of the V. cholerae RND efflux systems and the Cpx two-component system is mediated by the intracellular accumulation of an endogenously produced metabolic by-product that is normally extruded from the cell by the RND efflux systems.

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram-negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ(E) envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two-component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram-negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ(E) envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ(E) and Cpx responses in non-pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ(E) response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K-12. Cpx pathway activation resulted in part from overexpression of the bundle-forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.

The Cpx Stress Response System Potentiates the Fitness and Virulence of Uropathogenic Escherichia coli

Strains of uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of the cpxRA operon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with the cpxP deletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.

The crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA.

The Cpx two-component system of Gram-negative bacteria senses extracytoplasmic stresses using the histidine kinase CpxA, a membrane-bound sensor, and controls the transcription of the genes involved in stress response by the cytosolic response regulator CpxR, which is activated by the phosphorelay from CpxA. CpxP, a CpxA-associated protein, also plays an important role in the regulation of the Cpx system by inhibiting the autophosphorylation of CpxA. Although the stress signals and physiological roles of the Cpx system have been extensively studied, the lack of structural information has limited the understanding of the detailed mechanism of ligand binding and regulation of CpxA. In this study, we solved the crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA (VpCpxA-peri) to a resolution of 2.1 Å and investigated its interaction with CpxP. VpCpxA-peri has a globular Per-ARNT-SIM (PAS) domain and a protruded C-terminal tail, which may be required for ligand sensing and CpxP binding, respectively. The direct interaction of the PAS core of VpCpxA-peri with VpCpxP was not detected by NMR, suggesting that the C-terminal tail or other factors, such as the membrane environment, are necessary for the binding of CpxA to CpxP.

Structural Basis for Two-component System Inhibition and Pilus Sensing by the Auxiliary CpxP Protein

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like "left hands" forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.

A Periplasmic LolA Derivative with a Lethal Disulfide Bond Activates the Cpx Stress Response System

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.

Genetic Analysis of Activation of the Vibrio cholerae Cpx Pathway

The Cpx two-component system is thought to mediate envelope stress responses in many gram-negative bacteria and has been implicated in the pathogenicity of several enteric pathogens. While cues that activate the Escherichia coli Cpx system have been identified, the nature of the molecular signals that stimulate this pathway is not well understood. Here, we investigated stimuli that trigger this system in Vibrio cholerae, a facultative pathogen that adapts to various niches during its life cycle. In contrast to E. coli, there was no basal activity of the V. cholerae Cpx pathway under standard laboratory conditions. Furthermore, several known stimuli of the E. coli pathway did not induce expression of this system in V. cholerae. There were no defects in intestinal growth in V. cholerae cpx mutants, arguing against the idea that this pathway promotes V. cholerae adaptation to conditions in the mammalian host. We discovered that chloride ions activate the V. cholerae Cpx pathway, raising the possibility that this signal transduction system provides a means for V. cholerae to sense and respond to alterations in salinity. We used a genetic approach to screen for mutants in which the Cpx pathway is activated. We found that mutations in genes whose products are required for periplasmic disulfide bond isomerization result in activation of the Cpx pathway, suggesting that periplasmic accumulation of proteins with aberrant disulfide bonds triggers the V. cholerae Cpx pathway.

Activation of the Cpx Envelope Stress Response Down-Regulates Expression of Several Locus of Enterocyte Effacement-Encoded Genes in Enteropathogenic Escherichia coli

The Cpx two-component system regulates an extracytoplasmic stress response that functions to rid the envelope of misfolded and mislocalized proteins that may interfere with normal cellular processes. The Cpx pathway is also involved in pathogenesis. This study investigated the role of the Cpx response in enteropathogenic Escherichia coli (EPEC) type III secretion (T3S). It was determined that a functional Cpx pathway is not required for T3S but that pathway activation inhibits secretion by reducing the cellular pools of T3S substrates. The EPEC T3S system structural components, as well as a number of its substrates, are encoded on the locus of enterocyte effacement (LEE) pathogenicity island. Transcriptional fusions to the five major operons of the LEE were constructed and examined under Cpx pathway-activating conditions. Induction of the Cpx response caused a decrease in the transcription of several LEE operons, with the most pronounced effect on LEE4 and LEE5. Collectively, these two operons encode components of the T3S translocation apparatus, the bacterial adhesin intimin, and the translocated bacterial receptor Tir. These data show for the first time that activation of the Cpx envelope stress response in EPEC inhibits T3S of both translocators and effectors, likely through down regulation of LEE transcription. Coupled with recent findings, our results suggest that Cpx-mediated down regulation of virulence is a conserved theme in a number of bacterial pathogens.

A Sensor of the Two-Component System CpxA Affects Expression of the Type III Secretion System through Posttranscriptional Processing of InvE

The chief function of the Cpx two-component system is perceiving various cell envelope stresses, but CpxR is also known to regulate the expression of the type III secretion system (TTSS) of Shigella sonnei through transcription of the primary regulator virF. Here, we have isolated novel cpxA mutants that exhibited decreased TTSS expression from Escherichia coli HW1273, which carries the virulence plasmid of S. sonnei. The cpxA deletion strain of HW1273 expressed beta-galactosidase activity levels from the virF-lacZ fusion similar to those of HW1273. However, the second regulator InvE (VirB) and the TTSS component IpaB proteins were apparently expressed at a low level. In the cpxA strain, beta-galactosidase activity levels from the invE-lacZ transcriptional fusion remained similar to those of HW1273, whereas the beta-galactosidase activity level from the translational fusion of invE-lacZ was reduced to 21% of that of HW1273. Therefore, the deletion of the cpxA gene influenced TTSS expression chiefly at the posttranscriptional processing of InvE. In addition, the cpxA deletion strain of S. sonnei showed the same phenotype. These results indicate that the Cpx two-component system is involved in virulence expression through posttranscriptional processing of the regulatory protein InvE, a novel feature of the Cpx two-component system in posttranscriptional processing and virulence expression of Shigella.

Effects of Lipoprotein Overproduction on the Induction of DegP (HtrA) Involved in Quality Control in the Escherichia coli Periplasm

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli. Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high temperatures. However, it is not completely clear why NlpE overproduction causes degP expression. Moreover, among newly confirmed lipoproteins, there may be others that also induce degP expression. Therefore, we overproduced each of the 90 lipoproteins and examined the level of degP expression as beta-galactosidase activity by using a degP promoter-lacZ fusion. The extent of degP expression caused by NlpE overproduction was dependent on the mode of degP-lacZ fusion. On the other hand, new inner membrane lipoprotein YafY strongly induced degP expression irrespective of the mode of fusion even though the level of overproduced YafY was lower than that of NlpE. The induction of degP expression by YafY overproduction was dependent on the Cpx two-component system. Alteration of the lipoprotein-sorting signals of NlpE and YafY did not abolish the degP induction. However, a YafY derivative possessing the outer membrane signal remained on inner membranes. The non-lipidated derivative of NlpE did not induce degP expression, indicating that membrane anchoring is essential for degP induction. The amino acid sequences of YafY and YfjS, another inner membrane lipoprotein, are highly identical, but overproduction of the latter did not induce degP expression. Construction of various YafY-YfjS chimeric lipoproteins revealed that only a few residues located in the N- and C-terminal regions were important for the induction of DegP.

Molecular components of physiological stress responses in Escherichia coli.

In order to survive under and adapt to different conditions Escherichia coli has evolved elaborate systems that are able to sense and respond to environmental stimuli. Very often, different stresses act on a bacterium simultaneously and a variety of stresses have similar effects on cellular molecules and processes. Therefore, the various stress response systems have to interact (cross talk) with each other. A complex network of global regulatory systems with a multitude of molecular components ensures a coordinated and effective answer. Such regulatory components include DNA, mRNAs, sRNAs, proteins, such as DNA-and RNA binding proteins, alternative sigma factors and two-component systems, as well as small molecular weight molecules, as for example (p)ppGpp. These regulatory systems govern the expression of a plethora of further effectors that aim at maintaining stability of the cellular equilibrium under the various conditions. Using five of the most important stress response systems, we will discuss the roles and mechanisms of such regulatory and effector molecules in more detail. The heat shock response, controlled by the sigma factor sigma32, and the envelope stress response, controlled by the sigma factor sigmaE and the Cpx two-component system, both result in an increased expression of chaperones and proteases in response to misfolded proteins. The cold shock response governs expression of RNA chaperones and ribosomal factors, ensuring accurate translation at low temperatures. The general stress response depends on the sigma factor sigmaS, which controls the expression of more than 50 genes conferring resistance to many different stresses. The (p)ppGpp-dependent stringent response reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift. A lot has been learned in recent years about the mechanisms of action of single components. However, the main challenge for the future is to achieve an understanding of the interactions of these components under different physiological conditions.

Periplasmic stress and ECF sigma factors.

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.