Envelope Stress Responses
DAWN M. MACRITCHIE AND TRACY L. RAIVIO*
[SECTION EDITOR: JOHN FOSTER]
Posted July 29, 2009
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
*Corresponding author. Mailing address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. Phone: (780) 492-3491, E-mail:
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The gram-negative bacterial envelope is a complex extracytoplasmic compartment responsible for numerous cellular processes. Among its most important functions is its service as the protective layer separating the cytoplasmic space from the ever-changing external environment. To adapt to the diverse conditions encountered both in the environment and within the mammalian host, Escherichia coli and Salmonella species have evolved six independent envelope stress response systems. Each stress response senses a unique activating signal that is indicative of a certain type of extracytoplasmic disturbance. By altering the gene expression profile of the respective organism, each stress response serves to alleviate the impending stress and return cellular homeostasis. In this chapter we will review the σE response, the CpxAR and BaeSR two-component systems, the phage shock protein response, and the Rcs phosphorelay system. These five signal transduction pathways represent the most studied of the six known stress responses. The final stress response, outer membrane vesicle formation, is reviewed in a separate chapter (see Chapter Outer Membrane Vesicles [121]) and as such will not be discussed in detail here. An overview of each pathway is provided at the beginning of the corresponding section.
The first and best characterized of the envelope stress responses is the σE signal transduction pathway. Because of its essentiality and important role in pathogenesis it has been studied extensively since its discovery in 1989 (61, 193). Here, we will discuss what is presently known about the physiological role of the σE pathway, placing emphasis on the mechanism underlying signal transduction.
The σE subunit of E. coli RNA polymerase (RNAP) was first identified as the transcription factor responsible for synthesis of the heat shock sigma, σ32 (RpoH), at elevated temperatures (61, 193). Characterization of the rpoH promoter region revealed that during extreme heat shock (50°C), the predominant form of rpoH mRNA is synthesized from the rpoHP3 promoter (62). Further investigation into rpoH regulation revealed that σE binds to the rpoHP3 promoter, thus indirectly driving expression of the heat shock genes. The same authors demonstrated that σE also binds to the promoter region of htrA (degP), a gene encoding a periplasmic protease required for survival at elevated temperatures (61). Based on this, σE was classified as an alternative sigma factor involved in thermotolerance (61).
In an effort to better understand the cellular function of σE, Gross and colleagues screened for positive regulators of σE activity (127). They found that overproduction of outer membrane proteins (OMPs) and mutations that caused OMP misfolding activated σE activity by way of a periplasmic signal (127). To investigate whether σE was specifically responding to the presence of misfolded envelope proteins the Georgopoulos group examined htrA and rpoHP3 lacZ fusions in mutants lacking important periplasmic protein folding factors (153). Activity from the reporters was elevated in strains lacking the disulfide oxidase DsbA or HtrA (DegP) alone, and the effect was even more pronounced in a dsbA htrA double mutant (153). Further characterization of rpoE revealed that σE regulates its own expression from one of two major promoters and that a functional rpoE gene is essential to survival at temperatures exceeding 40°C (153). It was proposed that envelope proteins that become misfolded at high temperatures generate a signal that activates a σE-dependent stress response (127, 153). It would later be shown that σE is essential at all temperatures suggesting that these early deletion mutants harbored uncharacterized suppressor mutations (44).
The proposal that σE was responding to disruptions in envelope protein biogenesis was soon corroborated. A transposon mutagenesis screen for regulators of σE activity uncovered a class of mutations that disrupted genes encoding envelope protein folding factors and resulted in the accumulation of misfolded periplasmic and outer membrane proteins (130). Interestingly, in addition to activating σE these mutants also induced expression of several periplasmic folding factors (SurA, Dsb, FkpA, and Skp) (130). Overproduction of these folding factors was sufficient to prevent σE activation in the mutant backgrounds (130). A separate study investigating protein folding in the periplasm discovered that mutants lacking the peptidyl prolyl isomerase SurA accumulated an unfolded form of the LamB OMP that was also found in cells overexpressing LamB (164). Rouviere and Gross (164) presented data suggesting that this unfolded OMP was part of the σE-activating signal. Interestingly, the surA- cells had defective outer membranes with altered protein composition (164). Together, these findings implied that σE monitors envelope protein biogenesis in an effort to maintain outer membrane integrity.
Another class of mutations found to activate σE consisted of previously uncharacterized genes. They were subsequently mapped to the same region as rpoE, and as such, were named RseA and RseB (for regulators of sigma E) (45, 131). Sequence analysis of the rpoE region revealed that rpoE is the first gene in a four-gene operon, also encoding RseA, RseB, and RseC (45, 131). Characterization of these regulatory proteins became a major focus of the research in this field during the next few years and enabled researchers to start piecing together the mechanism underlying σE regulation.
Characterization of the RseA, RseB, and RseC regulatory proteins was the first step to better understanding how the σE pathway senses and responds to extracytoplasmic stress. Biochemical and genetic analysis localized RseA to the inner membrane where it functions as an anti-sigma factor for σE (45, 131). RseB was found to reside in the periplasm, where it interacts with the C terminus of RseA and enhances inhibition of RseA (4, 33, 131). Finally, RseC was localized to the inner membrane and found to have no clear regulatory role (45, 131). Deletion of rseA resulted in a 25-fold increase in σE activity, while rseB mutants were still capable of wild-type σE activation (45, 131). This suggested to researchers that inactivation of RseA was essential for initial induction of the σE response (45). Further analysis of the regulatory role of RseA revealed that the cellular level of this protein is a major determinant of the amount of free σE available to counter extracytoplasmic stress (4). Because RseA becomes unstable under σE-activating conditions, researchers set out to identify the protease responsible for RseA degradation (4). By screening extracytoplasmic proteases for those involved in σE regulation, the Gross laboratory determined that the inner membrane protease DegS was essential for σE activation and, in turn, cell viability (4, 8). Based on the location of its active site, it was proposed that DegS degraded the periplasmic C-terminal end of RseA upon presentation of an inducing cue (4, 8). Interestingly, the N-terminal portion of RseA was found to be sufficient to inhibit σE, suggesting that additional proteases were also involved (4, 45, 131). It was soon discovered that another essential, inner membrane protease, RseP, formally called YaeL, degraded C-terminally truncated RseA (7, 42, 101, 102). Interestingly, RseP failed to degrade RseA in a degS null background (7, 8), which sent researchers on the hunt for the inhibitory mechanisms regulating the sequential cleavage of RseA under σE-activating conditions.
Both DegS and RseP possess PDZ domains. It is well documented that the PDZ domains of proteases are involved in protein-protein interactions that mediate substrate recognition and consequent cleavage (79). The Sauer group provided evidence that a specific sequence found in the exposed C terminus of unfolded OMPs binds to the PDZ domain of DegS (191). The transition between the inactive and active state of the DegS protease has been shown to be reversible due to the reorientation of residues in its activation domain (175, 200, 204). The interaction of the C terminus of misfolded OMPs with the PDZ domain of DegS was found to relieve PDZ-mediated proteolytic inhibition by stabilizing the active form of DegS (175, 191). The active form of this protease recognizes RseA as a substrate and initiates the proteolytic cascade that leads to activation of the σE response (191). The Ehrmann and Clausen groups also hypothesize that the PDZ domain of DegS serves as a scaffold, orienting OMP peptides correctly relative to the protease domain (200). Because the DegS switch between active and inactive conformations is mediated by allosteric ligands, the authors compare it with the phosphorylation/dephosphorylation steps of two-component signal transduction systems (80, 175).
The PDZ domain of RseP has also been shown to play an important role in RseA cleavage. Kanehara et al. (103) determined that a PDZ domain found in the periplasmic domain of RseP, as well as two glutamine (Gln)-rich regions in the C-terminal end of RseA, facilitate inhibition of RseA degradation by RseP. Deletion of either the RseP PDZ domain or the periplasmic domain of RseA enabled RseP to degrade full-length RseA in the absence of DegS (103). This led to the proposal that cleavage of the periplasmic domain of RseA by DegS removes the Gln-rich regions recognized by the PDZ domain of RseP, allowing RseP to take on an active form and target the membrane-spanning domain of RseA for cleavage (3, 103). Upon cleavage of RseA by RseP, a fragment of RseA still bound to σE is released into the cytoplasm (7, 20, 67, 102, 131). It is in the cytoplasm that a third proteolytic event takes place, effectively releasing σE from the inhibitory influence of RseA (67). This final step in the cascade is usually enacted by the AAA+ protease ClpXP working in concert with the adaptor protein SspB (67). If the cellular pool of ClpXP is overwhelmed by other substrates, alternate cytoplasmic proteases including Lon ensure that free σE is available to alter gene expression in response to extracytoplasmic stresses (29). See Fig. 1 for a model of the σE response.
Because of a strong correlation between the rate of RseA degradation and the level of σE activity it appears that RseA proteolysis transmits a signal to σE about the state of the envelope (5). Therefore, in the absence of an inducing cue, RseA should be a poor substrate for DegS and RseP. Additional factors have been identified that ensure the robustness of the system by inhibiting RseA cleavage by DegS and RseP. DegS-mediated cleavage is inhibited by the periplasmic regulatory protein, RseB (28). Its inhibitory affect is independent of the DegS PDZ domain and therefore independent of the signal generated by misfolded OMPs. The evidence suggests that RseB binds to the periplasmic domain of RseA where it interacts with the DegS recognition site making RseA a poor substrate (28). RseB also functions to inhibit RseP-directed cleavage of RseA (72). RseB has been shown to bind to the periplasmic domain of RseA between the two Gln-rich regions mentioned above (28, 103, 107). RseB was found to inhibit RseP but was impaired in its ability to inhibit RsePΔPDZ (72). Thus, it has been proposed that RseB forms a complex with the PDZ domain of RseP, effectively inhibiting cleavage of RseA (28, 107). It appears that this highly conserved protein functions to ensure that the initial cleavage event involving DegS remains the rate-limiting step in the process (29, 72). This guarantees that the σE response is governed by inducing signals and not the amount of protease present in the cell (29). Presently, it is unclear how RseB inhibition is relieved.
Characterization of one class of suppressors of an rpoE mutant in E. coli suggests that the rpoE gene is not essential per se, but that its absence results in cell death (19). This argues that a functional σE pathway is required for survival and demonstrates that this extracytoplasmic stress response carries out an important cellular function. It has long been thought that the key to understanding σE's essentiality lies in its regulon. Consequently, there has been extensive research aimed at characterizing the σE regulon in its entirety. The first major screen aimed at characterizing the entire σE regulon was performed by Dartigalongue and colleagues (42). Using two different genetic approaches, they identified 20 new σE-regulated promoters and provided further support for previously identified genes. The newly extended regulon included several periplasmic protein folding factors (skp, dsbC, fkpA, surA), the proteases degP and RseP, proteins involved in lipopolysaccharide (LPS) biogenesis (htrM, lpxD) and membrane-derived oligosaccharide synthesis (mdoS), regulatory genes (rseA, rseB, rseC) and several sigma factors (rpoE, rpoH, rpoD). Based on this, it appeared that the σE response had evolved a role in both envelope protein biogenesis and LPS biosynthesis (42). Two years later the Kormanec group used a two-plasmid system to identify promoters recognized by RNAP-σE (160, 161). This approach found 11 new promoters recognized by σE adding 15 genes to the list of σE regulon members. Among the newly identified regulon members were several genes involved in LPS and phospholipid synthesis (lpxP, psd), a sensory protein (sixA), four predicted inner and outer membrane proteins, and several proteins of unknown function. Interestingly, they also identified several genes linked to primary metabolism (fusA, tufA), which led the authors to propose yet another role for σE.
With its extensive regulon implicating the σE stress response in a wide range of cellular processes, researchers were left wondering what the true physiological role of the σE pathway really was. The Gross laboratory attempted to resolve this problem with the development and utilization of a σE promoter prediction model for E. coli coupled with extensive microarray analysis (162). This computational method was first validated in E. coli K12 and then used to predict σE regulon members in related species such as Salmonella enterica serovar Typhi, Salmonella enterica serovar Typhimurium, Shigella flexneri, and Yersinia pestis. This approach allowed the authors to generate an extended σE regulon comprising 89 unique transcriptional units identified across nine closely related genomes. Nineteen transcriptional units encoding 23 genes were highly conserved and designated the “core” σE regulon. The majority of the genes making up the “core” regulon facilitate the synthesis and assembly of LPS and OMPs or encode components of the σE signal transduction pathway itself (162). Therefore, it appears that the main role of the σE pathway is to ensure that there are adequate levels of LPS and OMPs available for integration into the outer membrane. This finding was corroborated by a separate study aimed at elucidating the σE regulon in serovar Typhimurium (171). Researchers identified 62 σE regulon members, 39 of which were previously found to be regulated in E. coli. A large portion of the regulon, including genes not regulated by E. coli σE, are thought to be involved in maintaining homeostasis in the outer membrane (171).
The extended σE regulon was found to encode numerous proteins that facilitate bacterial survival within the eukaryotic host. This portion of the regulon varied among pathogens, suggesting that the σE response has evolved a secondary function in pathogenesis that is species specific (162, 171). This finding is not surprising given the numerous published reports of degP, surA, and rpoE mutants being attenuated for virulence in animal models (89, 94, 154, 159, 165, 180, 182). In Salmonella, an organism for which rpoE is not an essential gene, rpoE mutants are unable to proliferate in macrophages and are highly attenuated in mice (89). This attenuation has been attributed to the inability of rpoE mutants to survive oxidative stress, because inactivation of the host phagocyte respiratory burst partially restores the pathogenic potential of the mutant (182). This is further supported by the increased sensitivity of rpoE mutants to oxidizing agents (89, 94). Because the rpoE mutant phenotype is more extreme than mutating any one regulon member, it appears that numerous functions of the pathway serve to facilitate pathogenesis (89). Similarly, in nonpathogenic E. coli, it seems unlikely that any one regulon member is the cause for this essentiality. It is more plausible that it is the cumulative functions of its regulon members that are important for cell vitality. In support of this the Ades group has provided strong evidence that the essentiality of σE in E. coli stems from its role in maintaining outer membrane integrity (81).
For a more in-depth discussion on the role of σE in pathogenesis, please refer to references 154, 165, and 171.
Upon presentation of stresses that interfere with OMP maturation, σE quickly downregulates OMP synthesis to prevent the buildup of unassembled protein in the envelope. Two separate research groups have reported a drastic decrease in OMP mRNA upon overexpression of rpoE (100, 162). This was surprising given that extensive characterization of the regulon failed to show direct regulation (except for ompX), or to identify a repressor of OMP gene expression (162). It was proposed that the decrease in OMP mRNA was due to mRNA decay caused by an unidentified auxiliary factor (162). This idea proved correct and researchers working in E. coli and Salmonella soon uncovered several small σE-regulated RNAs that target OMP mRNA for degradation (55, 65, 93, 144, 183, 186, 187).
The first small RNA shown to target OMP mRNA was MicF; its target was the osmoregulated porin protein, OmpF (6, 10). It was more than a decade later when another antisense regulator, MicA, was shown to inhibit OMP translation (186). Characterization of MicA revealed that its overexpression resulted in a decrease in OmpA (186). A separate group looking for suppressors that could compensate for the loss of the RseP protease identified another sRNA they named RseX (55). Overexpression of this sRNA led to a decrease in both OmpA and OmpC, which in turn allowed the cell to survive in the absence of a functional σE pathway. Using a more direct approach, Papenfort et al. (144) screened 50 sRNAs from Salmonella for the presence of a σE promoter. They identified two candidates, RybB and MicA, both found to be positively regulated by σE (144). Complementary work in E. coli also identified RybB and MicA as σE-regulated sRNAs (93, 183, 187). Overexpression of RybB was found to cause a decrease in OmpU and OmpW (93), along with several other σE-regulated promoters, effectively suppressing the σE response (183). The effect of RybB on OMP mRNA levels depended on Hfq (93, 183), a sRNA chaperone that accumulates on entry into stationary phase. It was proposed that Hfq-regulated sRNAs assisted in relieving envelope stress by directing sRNAs to OMP mRNA to facilitate decay (183). In support of this, a study in S. enterica found that the expression profile of a strain lacking hfq resembled cells exposed to a stress condition that induces the σE response (65). In this mutant background RseA is cleaved by DegS at an increased rate leading to elevated levels of σE activation. Interestingly, Hfq, σE, and the σE-regulated sRNA MicA accumulate upon entry into stationary phase (65). The inducing signal that causes σE activation during this stage of growth is not known at this time. Similar findings have been made for E. coli. A microarray experiment comparing wild-type E. coli with a Δhfq mutant found that σE expression increased, but that OMPs were not repressed in the absence of Hfq (74).
Researchers have successfully uncovered the mechanisms behind the rapid response of the σE signaling transduction pathway. The need for such quick action strongly supports the regulon studies, which suggest that the major role, and hence the essentiality, of this pathway is tied up in its ability to monitor and maintain outer membrane construction and inevitably its function.
The Cpx two-component system monitors surface organelle assembly and maintains envelope homeostasis by ridding the cell of potentially toxic misfolded proteins. In conjunction with this role, the Cpx pathway has been linked to bacterial pathogenesis. In the following section we will review the current understanding of the Cpx stress response system using what is known about its inducing cues and regulon to establish its physiological role.
McEwen and Silverman published a series of articles between 1980 and 1982 outlining the identification and characterization of two pleiotropic mutants, cpxA and cpxB (123, 124, 125, 126). These particular mutants were discovered during a mutagenesis screen aimed at identifying chromosomally encoded host proteins involved in the function of the conjugative F plasmid. The authors selected mutants for which conjugal functions (DNA donor activity and surface exclusion) were diminished. To distinguish these chromosomally encoded mutants from those encoded on the F plasmid itself, McEwen and Silverman (124) designated them “cpx” for “conjugative plasmid expression.” To understand how the cpx mutants were causing a decrease in surface exclusion, the authors examined the levels of an F-plasmid-encoded protein required for this process, TraT. They found that the outer membrane of cpx mutants had reduced levels of TraT. Further characterization of the cpx mutants revealed that they were temperature sensitive and isoleucine and valine auxotrophic and had characteristic inner and outer membrane protein profiles (123, 125, 126). While the surface of cpx mutant cells looked normal under the microscope, they had altered envelope protein composition that included a reduction in the level of a major outer membrane porin OmpF and the murein lipoprotein (126). McEwen and Silverman (126) concluded that the cpx mutations somehow affected the synthesis and/or translocation of select envelope proteins.
Sequence analysis revealed that the C terminus of CpxA was homologous to the C terminus of the inner membrane sensory protein, EnvZ (9). Topological predictions based on the primary structure of CpxA suggested that this inner membrane protein has a transmembrane domain flanking either side of an N-terminal periplasmic segment and a large C-terminal segment found in the cytoplasm (194). This information combined with fractionation experiments established CpxA as an inner membrane protein with a organizational structure similar to chemosensory transducers (110, 194). The sequence also exposed overlapping translation termination and initiation codons 5′ to cpxA, suggesting that cpxA is a 3′ gene in an operon (194). On examination of the upstream sequence researchers discovered a gene with homology to several transcription factors from the regulator class of two-component systems (52). The gene shared the most homology with members of the OmpR family of two-component regulatory proteins (52). Because CpxA shares homology with several cognate sensors of the OmpR subfamily, it appeared that the newly identified CpxR, together with CpxA, constituted a two-component signal transduction pathway (52).
Soon after its classification as a two-component system (TCS), the Silhavy group established a role for the Cpx pathway in envelope stress (36, 41, 174). While studying protein translocation, researchers isolated cpxA alleles capable of suppressing extracytoplasmic toxicity caused by high-level expression of a processing-defective outer membrane porin protein LamBA23D, and a tripartite fusion protein LamB-LacZ-PhoA (36, 174). Several cpxA alleles were shown to activate the expression of the periplasmic protease DegP (41). The activated cpxA alleles were collectively referred to as cpxA* mutants and shown to direct the degradation or stabilization of LamB-LacZ-PhoA and LamBA23D via DegP (36, 41). Because residual suppression of LamBA23D and LamB-LacZ-PhoA toxicities could be achieved by a cpxA* degP::Tn10 double mutant, it was suggested that the CpxAR two-component system directs the expression of additional folding factors in an effort to alleviate extracytoplasmic stress (36). Thus, the Cpx pathway was classified as an envelope stress response in the same class as σE.
After its classification as a stress-responsive two-component system, researchers began investigating the molecular mechanism by which CpxAR senses and responds to envelope stress. Early work suggested that CpxAR functions as a typical two-component system, with CpxA having autokinase, kinase and phosphatase activities and phosphorylated CpxR acting as a transcriptional regulator (40, 148, 157). In an effort to better understand Cpx-mediated signal transduction, Raivio and Silhavy (157) characterized cpxA gain-of-function mutations located in the periplasmic, transmembrane and cytoplasmic domains of CpxA. This approach confirmed the three enzymatic functions of CpxA and revealed that the ratio of CpxA kinase/phosphatase activity governs the expression of CpxR~P targets. Another interesting outcome of this study was the discovery that mutations to a central region of the periplasmic domain of CpxA were incapable of responding to known inducing cues. It was suggested that this particular region of CpxA was responsible for sensing and relaying the status of the extracytoplasmic environment to the cytoplasmic domain of CpxA (157). Because mutations to this putative sensing domain caused an increase in Cpx pathway activity under noninducing conditions, it was proposed that this domain interacts with an inhibitory signaling molecule in the absence of stress (157). Support for this came the next year with the identification of the Cpx-regulated protein CpxP (39). Overproduction of this small periplasmic protein was found to decrease Cpx pathway activity in a manner that depends on an intact CpxA sensing domain (156). Interestingly, CpxP is encoded immediately upstream of the cpxRA operon, which also belongs to the Cpx regulon (39, 156). It is thought that by autoregulating all three components of the signal transduction pathway, the Cpx response can quickly amplify a response to stress and rapidly shut off the pathway once the perceived stress has been removed (156) (Fig. 2).
The identification of a third component to an otherwise prototypical two-component system raised several questions as to the mechanism by which CpxP modulates CpxA activity. CpxP is not required for signal transfer between CpxA and CpxR and evidence suggests its role is to modulate CpxA autokinase activity (66, 156). It is commonly accepted that under noninducing conditions CpxP binds to the sensing domain of CpxA maintaining a low kinase/phosphatase ratio (156). Raivio et al. (156) provided indirect evidence for this model with the finding that tethering a MBP-CpxP fusion protein to the inner membrane can prevent full Cpx pathway induction by spheroplasting, a process leading to the release of the periplasmic contents. The first biochemical evidence for their direct interaction came with the reconstitution of the Cpx pathway in proteoliposomes (66). Researchers effectively demonstrated that a CpxP:CpxA ratio of 1:1 can inhibit autokinase activity by 50% (66). To learn more about the functional domains of CpxP, the Raivio group isolated and characterized CpxP loss of function mutations (18). They found that CpxP contains a highly conserved N-terminal domain that is important for its inhibitory function and overall stability. They also demonstrated that induction of the Cpx stress response by alkaline pH results in CpxP being degraded by the Cpx-regulated protease DegP. Around the same time the Silhavy group presented evidence suggesting that CpxP functions as an adaptor protein that delivers misfolded proteins to DegP and is subsequently degraded in concert with the misfolded protein (91). Presently it is not clear whether CpxP is titrated away from CpxA by misfolded proteins or if it undergoes a conformational change and simply dissociates from CpxA under inducing conditions (18, 91).
Recently researchers discovered that silent mutations in the CpxP signal sequence and replacement of the signal sequence with that of another periplasmic protein increased the periplasmic expression of CpxP (129). This suggests that the first few codons of the CpxP mRNA inhibit its periplasmic expression. Based on this, it appears that a posttranscriptional mechanism has evolved to influence a component of the Cpx signal transduction pathway. At this point it is not clear whether periplasmic expression of CpxP is simply inhibited by formation of a secondary structure in the mRNA of its signal sequence, or if it is the target of a small RNA (129).
Other important questions facing this field are how and what the sensing domain of CpxA senses. While researchers have strong evidence to suggest that misfolded protein is the major signaling molecule, solid biochemical evidence is still needed to confirm this. Also, researchers have found that not all inducing cues enter the Cpx pathway through the sensing domain of CpxA. The signal for adhesion to abiotic surfaces enters the pathway through the novel outer membrane lipoprotein NlpE (143), and activation on entry into the exponential phase of growth occurs independently of CpxA (48, 50). The topic of activating cues and what they tell us about signal sensing by the Cpx pathway is discussed in the next section.
One of the major challenges of working with an environmentally responsive signaling pathway is identifying the exact ligand responsible for initiating signal transfer. Most of what we know about signal sensing and the Cpx pathway comes from the inducing cues shown to activate the Cpx response. The first Cpx-activating signal to be identified was the overexpression of the novel outer membrane lipoprotein NlpE (41, 174). Overproduction of NlpE was found to suppress extracytoplasmic toxicity caused by aberrant envelope proteins by upregulating expression of degP in a CpxAR-dependent manner (41, 174). By mutating its signal sequence, researchers examined the ability of an inner membrane-associated NlpE to induce the Cpx response (132). Interestingly, the inner membrane-associated NlpE proved to be a more potent inducer than the wild-type form of the protein (132). This suggested that when overexpressed, some aberrant NlpE becomes mislocalized to the periplasmic face of the inner membrane leading to induction of the Cpx response (132). Researchers have shown that only one other, of the 90 lipoproteins in E. coli (YafY), can serve as an inducing cue for the Cpx response when overexpressed (132). This indicated that NlpE and YafY possess some unique structural characteristics that facilitate Cpx pathway activation.
Structural characterization of NlpE provided important clues as to how this lipoprotein might transmit a signal to CpxA. The crystal structure revealed an intrinsic structural instability in an N-terminal β-barrel, as well as a potentially active CXXC motif (86). The authors propose that correct folding and insertion of NlpE into the outer membrane by the Lol machinery could involve the formation of disulfide bonds at the CXXC motif. It is possible that overexpression of NlpE overwhelms the LolA chaperone pool leading to the accumulation of unfolded protein at the outer leaflet of the inner membrane (86). NlpE has also been implicated as an auxiliary factor in the Cpx signal transduction pathway (143) (Fig. 2). Otto and Silhavy (143) found that adherence of E. coli cells to an abiotic surface activates the Cpx response in a manner that requires NlpE. It is proposed that the lipoprotein is located upstream of the Cpx response and transmits the signal for adhesion to CpxA (143). A monomer model of the NlpE structure suggests that unfolded N-terminal residues can extend far enough through the periplasm to make contact with CpxA from its position in the outer membrane (86). Adhesion could disrupt NlpE causing unfolding of its unstable N-terminal domain, leading to activation of the Cpx response (86). The hypotheses derived from the biochemical analysis of NlpE await experimental testing. Elucidating the mechanism by which NlpE signals CpxA will undoubtedly shed light on how the CpxAR signaling pathway senses “stress.” It will also help determine whether NlpE is a true inducing cue or if its overexpression simply mimics the signal NlpE transduces to CpxA during stress.
Another informative inducing cue of the Cpx pathway was identified by researchers investigating the role of the PapD chaperone in P pilus biogenesis (95). Jones and colleagues observed that expression of P pilin subunits in the absence of their cognate chaperone allowed premature subunit-subunit interactions. This led to the formation of toxic aggregates of misfolded pilin proteins and the subsequent activation of degP expression (95). In the case of PapE, stimulation of degP transcription was found to be the result of Cpx pathway activation (95). It was later determined that, like NlpE, structural features and not instability or aggregation of off-pathway PapE subunits activate the Cpx pathway during P pilus assembly (115). An N-terminal extension of PapE is required for Cpx pathway activation (115). This domain is required for donor strand complementation, a process that mediates insertion of a new Pap subunit into a growing P pilus (115, 168). The unfolding of the N-terminal β-barrel of NlpE has been equated to the N-terminal extension of PapE (86, 115). It may be that CpxP and/or CpxA recognize a motif in this structural sequence that is normally hidden by a chaperone, or buried in the pilus structure (91, 115). It is easy to imagine a model similar to that of the σE stress response whereby CpxA recognizes a unique motif found in a specific group of periplasmic proteins.
Most recently, high concentrations of external copper were found to upregulate several members of the Cpx regulon (202). This finding, coupled with the fact that mutations in nlpE result in copper sensitivity (75), suggests that NlpE might function as a signaling molecule for more than just adhesion. It has been proposed that high concentrations of copper ions could lead to oxidation of a conserved CXXC motif in NlpE, which could cause unfolding of its N-terminal domain (86). It is also possible that the oxidative stress caused by high concentrations of external copper could cause other periplasmic proteins to become misfolded (201). More work needs to be done to elucidate how and why the Cpx response responds to external copper.
Additional inducers of the Cpx response include alkaline pH, spheroplasting, and mutations to several genes linked to membrane biogenesis and growth (37, 39, 112, 128). Known activating signals enter the Cpx pathway at three points: NlpE (adhesion), CpxR (growth), and CpxA (almost all others) (50). Activation by each of these inducers can be explained in terms of envelope protein misfolding, and provide further evidence that the Cpx pathway responds to environmental stress that disrupts protein biogenesis in the periplasm. As is evident by the signals feeding into the Cpx response, this pathway has evolved to sense a diverse group of inducing cues that, in turn, lead to significant changes in gene expression. The myriad of inducing cues that upregulate the Cpx response has enabled researchers to identify genes that are specifically regulated by CpxR. The Cpx regulon further implies that the main physiological role of the pathway is to sense and respond to envelope stress and also implicates the pathway in several unexpected cellular processes.
Because of its general role in periplasmic protein biogenesis, the Cpx stress response is involved in an ever-growing list of envelope-associated functions. Early work showing that the Cpx response upregulates expression of the periplasmic protease DegP led the Silhavy and Beckwith groups (40, 148) to examine whether the pathway regulates other prominent folding factors. They found that activation of the Cpx pathway led to an increase in expression of the disulfide oxidoreductase dsbA as well as degP. By analyzing the promoters of dsbA and degP, a putative CpxR consensus sequence [GTAAN(6-7)GTAAA] was generated and used to screen the E. coli genome for potential regulon members (148). This approach led to the identification of an additional folding factor, the peptidyl prolyl isomerase, PpiA. Using DNase I footprinting, it was confirmed that CpxR~P binds upstream of the promoter regions of degP, dsbA, and ppiA (148). Two years later the CpxR consensus sequence was again used to screen promoters in E. coli. This time researchers identified two unexpected and negatively regulated members of the Cpx regulon (motABcheAW and tsr), implicating the Cpx pathway in the processes of chemotaxis and motility (48). That same year, researchers found evidence that activation of the Cpx pathway also represses expression of the curlin adhesin protein (CsgA) important for biofilm formation (54). Using sequence information from the putative CpxR binding sites of the aforementioned regulon members, De Wulf and colleagues (49) generated a weighted CpxR recognition matrix. This approach led to changes to the putative CpxR binding sequence and added eight more candidates to the growing list of Cpx-regulated genes (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) (49). The newly identified genes encoded proteins with sensory function, those involved in membrane biogenesis, protein transport, and, most surprisingly, the regulatory components of the σE envelope stress response (49).
Researchers became aware of the functional overlap between the Cpx and σE signaling pathways early on, but the finding that the Cpx pathway directly inhibited expression of σE came as a surprise (49). It seems counterintuitive for one envelope stress response to repress expression of another, especially given that they both upregulate expression of DegP in response to adverse environmental conditions (34, 37). This is not the only example of the Cpx response influencing the activity of another signaling pathway. In addition to regulating all three components of its own signaling pathway (cpxAR, cpxP), CpxR has also been shown to act at promoters targeted by the BaeSR and EnvZ/OmpR two-component systems. In the case of the Bae envelope stress response, both CpxR and BaeR activate transcription of spy, a small periplasmic protein of unknown function (152). Additionally, CpxR modulates BaeR activity at the promoters of two multidrug efflux genes, mdtA and acrD (83). CpxR binding sites have also been identified at the promoters of the genes encoding the classical porin proteins OmpC and OmpF (12). Porin regulation is coordinated by OmpR~P levels in response to osmolarity. OmpR is involved in both activation and repression of ompC and ompF. Similarly, CpxR~P has been shown to repress expression of the larger OmpF porin and activate expression of OmpC, binding at sites overlapping those of OmpR (12). Further, CpxR binds to multiple sites upstream of the curlin regulatory gene csgD, including at the single site recognized by OmpR, leading to repression of csgD transcription (99). Perhaps, by reducing the expression of large outer membrane porins and surface appendages, the Cpx pathway can reduce protein traffic in the periplasm while attempting to return cellular homeostasis.
The most recent class of genes added to the Cpx regulon includes those involved in copper homeostasis (202). Two separate microarray experiments revealed that exposure of E. coli cells to high levels of external copper leads to upregulation of several Cpx regulon members (106, 202). These studies led to the identification of nine copper-associated genes that are regulated by CpxR (aroG, ydeH, ftnB, ybaJ, yccA, yebE, yqjA, and ycdN) (106, 201, 202). Because no function has been assigned to the majority of the Cpx-regulated copper-inducible genes it is difficult to elucidate the exact role of the Cpx pathway in copper regulation. As mentioned in the previous section, one model shows external copper causing oxidative stress that triggers protein misfolding and, in turn, activation of the Cpx stress response. Interestingly, ycdN was recently found to encode part of a Cpx-regulated Fe2+ transporter (EfeUOB) in enterohemorrhagic E. coli (22). This connection to copper and iron regulation suggests the Cpx pathway has evolved a role in metal homeostasis in conjunction with its role as an envelope stress response system. More work is needed to determine the molecular mechanism behind these processes.
While the Cpx stress response has been studied predominantly in E. coli, it is important to mention that all three components of the Cpx signaling pathway are present in S. flexneri, Y. pestis, serovar Typhi and serovar Typhimurium (47, 154). In the case of serovar Typhimurium and S. flexneri, CpxA, CpxR, and CpxP are >95% identical to those of E. coli, and are predicted to function in a similar manner (154). In support of this, the Cpx pathway of serovar Typhimurium is autoregulated, induced by overexpression of NlpE, and regulates dsbA, the gene immediately upstream of dsbA, yihE/rdoA, and rpoE, as reported in E. coli (88, 179). Studies investigating the effect of cpx mutants on the virulence potential of Salmonella and E. coli species suggest a conserved function for the Cpx pathway in gram-negative bacterial pathogenesis. This topic will be explored further in the next section.
The first report implicating the Cpx response in pathogenesis came from an investigation into P pilus assembly in uropathogenic E. coli (UPEC). P pili of UPEC are encoded by the pap operon and elaborated via a chaperone-usher pathway. Researchers discovered that expression of the entire pap operon, in the absence of the PapD chaperone, led to activation of the Cpx stress response (95). The authors proposed that pilus subunits failing to interact with chaperone become misfolded and serve as an inducing signal for CpxA (95). It was thought that the Cpx pathway facilitated P pilus assembly by upregulating periplasmic folding factors important for elaboration (90, 95). Further characterization found a more direct role for the pathway, as CpxR~P binds at several sites within the pap regulatory region serving as a negative regulator for pap gene expression (82, 90). This suggests that when pilus assembly does not proceed properly, the misfolded subunits activate the Cpx response. The Cpx pathway would in turn upregulate protein folding and degrading factors that either fold or degrade potentially toxic misfolded envelope proteins. In addition, the Cpx pathway would downregulate pap gene expression in an effort to avoid making the situation worse by continuing to make pilus subunits. The Cpx pathway has also been shown to influence the assembly of the bundle forming pili (BFP) of enteropathogenic E. coli (EPEC). BFP are rope-like pili composed of a single repeating subunit and are assembled at the inner membrane. EPEC cpxR mutants fail to produce full-length BFP and have a reduced capacity to adhere to epithelial cells (139). Cpx-regulated DsbA is required for proper folding of BFP subunits suggesting that the Cpx pathway influences BFP elaboration at the posttranscriptional level (53, 139). Ongoing work in our laboratory is addressing the role of the Cpx pathway in BFP synthesis and assembly.
In contrast to the positive effect of the Cpx response on pilus assembly in UPEC and EPEC, our laboratory has demonstrated a negative role for the Cpx response in the assembly and function of the EPEC-type three-secretion system (T3SS). We found that a functional Cpx signaling pathway is not required for efficient secretion of EPEC effectors, but that activation of the pathway leads to a drastic reduction in secretion due in part to a decrease in expression of several key T3SS components and substrates (117). This finding has been corroborated by work in Yersinia pseudotuberculosis (25). The involvement of the Cpx response in E. coli and Y. pseudotuberculosis pathogenesis is mirrored in the intracellular pathogen, serovar Typhimurium. While cpxR mutants resemble wild-type cells in an in vivo infection model, cpxA mutants fail to grow in mice, and cpxA* gain-of-function mutants are impaired in their ability to attach to and invade eukaryotic cells (88). It is thought that by knocking out cpxA, CpxR~P accumulates in the cell because of phosphorylation by small-molecular phosphodonors, mimicking Cpx pathway-activating conditions (88, 154). Therefore, it appears that induction of the Cpx response inhibits virulence-associated structures in serovar Typhimurium as it does in E. coli and Yersinia species (25, 26, 82, 117). In contrast to the negative effects of the Cpx response on virulence determinant expression described above, it has been shown that CpxA is required for activation of HilA, a key regulator of invasion genes in serovar Typhimurium , at low pH (138). Activation of HilA is not directly mediated by CpxR, suggesting that the change in hilA transcription may be a secondary effect of activating the Cpx response (138). Further research is required to better understand the mechanism by which the Cpx pathway inhibits Salmonella pathogenesis.
Cumulatively, these studies suggest that the Cpx response has evolved to downregulate virulence determinant expression. This may serve to reduce protein traffic in the periplasm and conserve energy until the perceived stress has been alleviated. This appears to be a conserved function of the Cpx pathway in gram-negative pathogenesis.
The BaeSR two-component system upregulates the expression of several efflux pumps that serve to expel toxic compounds from the cell. It is presently thought that BaeS senses membrane-damaging compounds from its position in the inner membrane. Little is known about the specific signal received by BaeS, or the small group of genes regulated by BaeR. The pathway seems to function under very specific environmental conditions suggesting that it is not a general extracytoplasmic stress response like the Cpx or σE pathways.
The bacterial adaptive response regulated by the products of the baeSR operon was identified in a screen for sensor/regulator pairs capable of restoring ompC-lacZ or phoA expression to envZ and phoRcreC mutants, respectively (137). Characterization of BaeS and BaeR revealed the conserved features of the EnvZ and OmpR families of histidine kinases and response regulators, respectively (137). In vitro phosphotransfer experiments provided further evidence that BaeSR functions as a prototypical two-component system (137). A putative role for the BaeSR signaling pathway was uncovered almost a decade later by researchers looking for a regulator responsible for spheroplast-induced synthesis of the periplasmic protein Spy (76, 152). It had previously been shown that spy belongs to the Cpx regulon, and that deletion of the spy gene lead to induction of σE-regulated genes (155). Because spy expression could be induced in the absence of a functional Cpx pathway, researchers were interested in finding additional regulators of this uncharacterized gene (152). Raffa and Raivio (152) isolated a baeS1:Tn10 gain-of-function mutant capable of activating expression of a spy::lacZ fusion independent of CpxR. Further characterization of the BaeSR two-component system found that a baeR cpxR double mutant was more sensitive to envelope stress than either single mutant (152). This, combined with evidence that the Bae pathway is induced by known envelope stresses, such as indole, spheroplast formation, and PapG overexpression, led to BaeSR being classified as the third extracytoplasmic stress response in E. coli (152) (Fig. 3).
In the same year that the BaeSR TCS was classified as an envelope stress response, it was demonstrated that overexpressing BaeR increased resistance of hypersensitive E. coli cells to novobiocin, deoxycholate, and bile salts (11, 136). Activation of the Bae pathway was found to prevent cellular accumulation of novobiocin, implicating it in the process of active efflux (11). Examination of the sequence immediately upstream of baeSR revealed the yegMNOB (mdtABCD) multidrug resistance locus (11). Disruption of the mdt locus in cells overexpressing BaeR abolished Bae-mediated novobiocin resistance. Further study revealed that BaeR binds to the mdtA promoter region, and is capable of activating its expression (11). The presence of overlapping start-and-stop codons between each of the six genes suggests that mdtABCDbaeSR constitutes a single operon that is positively regulated by BaeR (11). Two studies looking at response regulator overexpression and drug resistance found that, in addition to novobiocin and deoxycholate, BaeR confers resistance to low levels of sodium dodecyl sulfate and β-lactam antibiotics (84, 85). The studies showed that BaeR-mediated resistance was due to upregulation of mdtABC and an additional multidrug efflux gene, acrD (84). As mentioned earlier in this chapter, both BaeR and CpxR bind to the promoters of mdtA and acrD (83). While both genes are upregulated by BaeSR in the absence of CpxAR, Cpx-mediated induction requires baeS and baeR (83). It is proposed that CpxR binding to the mdtA and acrD promoters enhances BaeR binding, making CpxR a modulator of BaeR activity (83). It appears that the CpxAR and BaeSR TCSs of E. coli cooperatively combat drug-induced envelope stress thru active efflux.
The BaeSR TCS has also been shown to mediate drug resistance in Salmonella (87, 141). A baeR null mutant in serovar Typhimurium was found to be more sensitive to ceftriaxone (87). Examination of the outer membrane protein profile of a baeR null strain exposed to this drug revealed that the outer membrane porin, OmpW, is upregulated in the absence of BaeR (87). OmpW of E. coli encodes a colicin S4 receptor protein (147). It has been implicated in methyl viologen (MV) resistance in serovar Typhimurium (70). OmpW was shown to export small amounts of MV from the cell, suggesting it is not the main exporter, but functions when high levels of MV are present (70). It is possible that OmpW is a backup efflux system used when other drug transporters are inactivated or overwhelmed. As BaeR regulates mdtA and acrD in S. enterica, a baeR null strain would fail to upregulate these efflux systems in the presence of ceftriaxone, explaining the need for increased levels of OmpW (87, 141). Similar to E. coli, the Bae pathway of S. enterica mediates resistance to oxacillin, novobiocin, deoxycholate, β-lactams, and indole (141). Based on a report that the Bae pathway of E. coli is induced by zinc (114), Nishino and colleagues (141) tested whether copper or zinc serve as activating cues for the Bae pathway in Salmonella. They found that exposure to zinc and copper lead to increases in mdtA and acrD expression (141). This finding suggests that the Bae pathway is not only involved in drug efflux, but also metal resistance. The Cpx pathway has also been implicated in metal homeostasis, demonstrating another incidence of functional overlap between these two stress-responsive TCSs. Interestingly, research in E. coli recently identified copper-responsive CpxR binding sites, as well as zinc-responsive BaeR binding sites, in the promoter region of spy (203). It should be noted that, unlike Salmonella, the Bae pathway of E. coli is not induced by copper (203). Based on this, it appears that the Cpx and Bae envelope stress response systems of E. coli have developed independent roles in metal homeostasis that somehow involve Spy. Further study is needed to determine the function of Spy during envelope stress.
All the information gathered about the Bae pathway thus far suggests that its primary physiological role is to regulate active efflux in response to a diverse group of antimicrobials. In addition to the inducers described above, sensitivity of bae mutants to myricetin, gallic acid, nickel chloride, sodium tungstate, and, most recently, condensed tannins suggests that the Bae pathway also mediates expulsion of these toxic agents (205, 206). Examination of all the compounds known to activate the Bae pathway has failed to reveal a common structural feature (T. L. Raivio, unpublished observations). It is predicted that each of the aforementioned compounds somehow disrupts the outer and/or inner membrane generating a common signal that is sensed by BaeS in the inner membrane. Alternatively, there may be additional components of the Bae pathway involved in signal relay. Researchers have employed several strategies in an effort to characterize the Bae regulon in its entirety (11, 140). Along with those previously mentioned (spy, mdtABCD, acrD), BaeR is thought to positively regulate a putative cysteine hydrolase encoded by ycaA, and two uncharacterized genes yicO and ygcL (11, 140). As no one approach has yielded every regulon member, it seems plausible that there are additional BaeR targets. The identification of new BaeR targets, characterization of existing regulon members, as well as elucidation of the mechanism for signal sensing by BaeS requires further investigation. Ongoing work in our laboratory is addressing some of these important questions.
Regulation of the phage shock protein (Psp) signal transduction system is mediated by protein-protein interactions and is thought to respond to dissipation of the proton motive force. Activation of the Psp response leads to upregulation of a limited Psp regulon, which encodes the components of the Psp signaling pathway and several Psp-specific effector proteins.
Researchers observed that E. coli cells infected with filamentous phage (f1) overproduced an uncharacterized protein that was not detected in uninfected E. coli (16). The polypeptide was appropriately named the phage shock protein, or Psp, and was found to be specifically induced by phage-encoded gene IV (pIV) (16). pIV encodes an integral outer membrane protein involved in phage assembly (15, 166). It is synthesized as a precursor and assembled into the outer membrane independent of other phage-encoded proteins (15, 16). The induction of Psp required continuous synthesis of periplasmic-targeted pIV, suggesting that a transient form of the phage protein generates the Psp inducing signal (16). Because several heat shock proteins are also upregulated during a phage infection, Brissette and colleagues (16) examined whether Psp levels were also affected by classic heat shock-inducing conditions. The authors found that extremely high temperatures, osmotic shock, and ethanol exposure all led to accumulation of the phage shock protein, in a manner that was independent of the heat shock σ factor, σ32. It was proposed that Psp represented a new stress-responsive protein with a function that paralleled that of the heat shock response (16). Examination of the sequence adjacent to the psp locus uncovered the pspABCDE operon, which is transcribed as a single polycistronic mRNA under Psp inducing conditions (17). Psp, renamed PspA, turned out to be the major component of the phage shock protein response, later classified as an extracytoplasmic stress response (Fig. 4).
The identification and subsequent characterization of the PspABCDE operon enabled researchers to start piecing together the mechanisms governing Psp expression under inducing and noninducing conditions. Deletion analysis revealed that PspC alone, or in conjunction with PspB, is required for full activation of the Psp response (17, 196). The requirement for PspB seems to depend on the activating signal (17, 196). The original phage shock protein, PspA, was found to be a negative regulator of Psp expression (196). Removal of pspA led to constitutive activation of the pathway, while its overexpression repressed the pathway even in the presence of an inducer (196). Fractionation experiments placed PspB and PspC in the inner membrane, while PspA was shown to associate with the inner membrane under inducing conditions (17, 108). Based on the available information, it was proposed that PspB and PspC work together to relieve PspA-mediated inhibition of the Psp response, by sequestering PspA at the cytoplasmic face of the inner membrane (108, 196). Tommassen and colleagues (1) provided support for this model by successfully cross-linking PspB to PspC, and PspA to PspC. An interaction between PspA and PspB was only achieved when the entire pspABCDE operon was overexpressed (1). The remaining members of the Psp operon, PspD and PspE, have not been shown to influence Psp expression. The pspE gene is transcribed from its own promoter under normal conditions and as part of the psp operon during stress (17, 108). It encodes a periplasmic rhodanese and, as such, is proposed to function in energy metabolism (2). PspE is found in E. coli and serovar Typhimurium but is absent from the Psp response of Yersinia, suggesting that it is not an essential component of the pathway (2). In the case of pspD, early experiments failed to detect a PspD protein, leading to its classification as a hypothetical gene (17, 108, 196). Researchers recently detected PspD for the first time using antibody raised against synthetic peptides (1). Like PspA, PspD localizes with the inner membrane and the cytoplasm, suggesting it is a peripheral inner membrane protein (1). To date, no function has been assigned to PspD.
In addition to encoding its own positive and negative regulators, mapping of the Psp promoter region revealed a single σ54-consensus sequence required for activation (196). An rpoN mutant failed to accumulate PspA under inducing conditions confirming that σ54-RNAP drives transcription from the psp promoter (196). Further promoter analysis identified two integration host factor (IHF) binding sites located between the σ54-recognition site and an upstream activator sequence (UAS) (196). IHF-mediated DNA bending is typically required to facilitate contact between the σ54-RNAP and a UAS-bound activator protein (158, 197). The transcriptional activator is needed to catalyze ATP hydrolysis, which powers formation of an open complex (158, 197). Researchers used transposon mutagenesis to identify the σ54-activator protein required for transcription of the psp operon, PspF (98). PspF is encoded directly upstream of the pspABCDE operon in the reverse orientation (98). It is a cytoplasmic protein with homology to the enhancer binding protein family (98). Early work suggested that DNA-bound PspF was not the target of PspA, but rather that PspA sequesters free PspF in the cytoplasm (56, 98). In an effort to better understand the mechanism behind PspA-mediated inhibition, Elderkin et al. (60) performed binding assays in which they preincubated PspF and σ54 with PspA. This effectively prevented formation of a PspF:σ54 complex. The N-terminal part of PspA was later shown to bind a surface-exposed region near the ATPase active site within the PspF AAA+ domain (59, 60). PspA binding inhibits the ATPase activity of wild-type PspF, preventing formation of a transcriptionally competent open complex at the psp promoter (60). Therefore, the phage shock protein response represents a stress-responsive signal transduction pathway regulated through protein-protein interactions (Fig. 4).
As with the other stress response pathways discussed thus far, the Psp response is upregulated by multiple activating cues. A commonality among several of the Psp-activating signals is a reduction in membrane electrochemical potential, which drives the proton motive force (PMF). PMF dissipation disrupts numerous functions associated with the inner membrane and in turn the entire envelope. The first evidence that the Psp response was somehow involved with inner membrane homeostasis came from work on protein translocation in E. coli. Kleerebezem and Tommassen (109) observed that overexpression of PhoE mutants, defective for normal biogenesis, caused an increase in PspA levels. Since induction of PspA expression depended on prePhoE entering the Sec export machinery, the authors proposed that overloading or blocking the translocation apparatus generated a Psp inducing signal. In addition, the translocation rate of mutant prePhoE was reduced in psp mutants (108, 109). This phenotype could be complemented by adding back PspA on a high-copy plasmid, suggesting that PspA facilitates clearing of the translocation channel, thereby increasing the rate of translocation (108). In support of this, mutations to secD, secF, and yidC, all of which are involved in the later stages of Sec-mediated translocation, led to upregulation of PspA (96, 109). Similarly, mutations to genes encoding structural components of the Tat export system induce PspA expression (46). In addition to PhoE, overexpression of several other mutant and native inner membrane-associated envelope proteins leads to upregulation of the Psp response (24, 119, 163).
Around the same time researchers connected the Psp response to protein translocation, the Model group determined that PspA was strongly upregulated by CCCP, a proton ionophore that diminishes the PMF (198). This led researchers to test whether translocation of mutant prePhoE had an effect on the PMF, measured as ΔμH+ (108). Interestingly, overexpression of prePhoE in a pspA mutant background led to a marked decrease in the ΔμH+, while cellular ΔμH+ was unaffected in a pspA mutant under normal conditions (108). This led to the proposal that the Psp response senses a signal associated with changes in the PMF (108). Further evidence for this came out of elegant work by the Pugsley group (73). The authors showed that synthesis of the PulD secretin in the absence of its pilot protein PulS led to insertion of PulD in the inner membrane (73). This caused a minor decrease in membrane electrochemical potential and induction of the Psp response. Expression of PulD in the absence of PspA and PulS resulted in a significant decrease in the PMF and the eventual death of the cells (73). Work in serovar Typhimurium implicated not only the Psp response, but also the σE stress response in PMF maintenance (13). This stems from microarray data showing that PspA is elevated in an rpoE mutant grown to stationary phase (13). Both stationary phase pspA and rpoE mutant cells exhibited lower ΔμH+ compared with wild-type cells, suggesting that both pathways help maintain the PMF late in the growth cycle (13). At this time there are insufficient data to claim that all Psp inducing cues influence the PMF. Researchers have yet to determine the exact signal generated by changes in PMF, the mechanism behind signal sensing by PspB-PspC and recruitment of PspA, or how the Psp effectors alleviate inner membrane stress.
Microarray data from E. coli, Salmonella, and Yersinia all suggest that the Psp response is limited to regulating a small group of Psp-specific genes (116, 169). Lloyd et al. (116) performed transcriptional profiling of E. coli and Salmonella cells overproducing pIV. They discovered that the only genes affected by activation of the Psp response were the members of the pspABCDE operon and yjbO, renamed pspG. PspG is an inner membrane protein whose gene is physically unlinked to the psp operon (116). It has been implicated in the process of motility because its overexpression leads to a decrease in this PMF consuming process (116). In an effort to address the biological function of Psp proteins, Jovanovic et al. (97) performed transcriptional profiling on E. coli cells either lacking or overexpressing key members of the Psp response. This approach yielded evidence to suggest that PspA and PspG function to switch the cell to anaerobic fermentation/respiration and to downregulate motility. The authors argued that the Psp response maintains the PMF by reducing the cells dependence on PMF costly processes. In line with this model, the same authors reported that the redox sensor, ArcB, was required for the induction of the Psp response (97). These findings were later called into question when the Darwin group working with Yersinia enterocolitica failed to obtain the same transcriptional profile using a natural inducer of the pathway (169). Darwin and colleagues argue that ArcB is not required for Psp pathway induction and that the Psp response is a self-limited pathway in which the effectors PspG and PspA work in concert to directly mediate a physiological response (169). Since these studies were performed in different bacteria, it is possible that this accounts for the observed discrepancy regarding ArcB.
The Rcs phosphorelay is a complicated signaling pathway involved in the regulation of surface-associated structures in response to membrane perturbation and peptidoglycan stress. The Rcs response has been implicated in the pathogenesis of both E. coli and Salmonella species by direct regulation of virulence genes as well as by regulating virulence-associated structures involved in motility and host recognition.
Researchers began investigating the complexities of the Rcs phosphorylation cascade more than two decades ago. The first members of the pathway were uncovered in a search for regulators of capsular polysaccharide (CPS) synthesis in E. coli. The production of CPS depends on the expression of the cps operon and leads to the formation of a mucoid layer over the surface of the bacteria (199). A search for genes that influence the expression of a cps::lac fusion led to the identification of the regulators of capsule synthesis encoded by rcsA, rcsB, and rcsC (71). Sequence analysis of rcsA and rcsB showed that both proteins possess homology to the LuxR family of transcriptional activators (176, 177). It had previously been shown that removal of the ATP-dependent protease Lon leads to upregulation of cps expression (185). As such, researchers tested whether RcsA or RcsB served as substrates for the Lon protease (184). Increased stability of RcsA in the absence of the protease proved responsible for elevated cps expression in the mutant (184). RcsB was not targeted by Lon and was actually found to stabilize RcsA in the presence of the protease (177, 184). Interestingly, RcsA-mediated activation of capsule genes required RcsB, while RcsB could function independent of RcsA (14). These findings led to our present understanding that RcsA is an auxiliary factor in RcsB-mediated capsule regulation. Further studies into the regulation of RcsA have shown that, in addition to being limited by the Lon protease, the rcsA gene is subject to HNS silencing (172) and autoregulation (57). It appears that E. coli has evolved several mechanisms to modulate Rcs pathway activity.
The third regulatory gene, rcsC, was found to contain an N-terminal end that resembles the histidine kinases of two-component systems, and a C-terminal end that shares limited homology with the N-terminal receiver domain of response regulators (176). It was proposed that RcsC and RcsB constituted an unorthodox two-component regulatory system involved in capsular regulation (176). Because it is unusual for a hybrid kinase to have His/Asp domains with no histidine-containing phosphotransfer (Hpt) domain, researchers were interested in characterizing RcsC (His/Asp) to RcsB (Asp) phosphotransfer. This led to the discovery of a new component of the Rcs regulatory pathway, YojN, later renamed RcsD (181). RcsD possesses a unique Hpt domain shown to mediate phosphoryl transfer between RcsC and RcsB (181). Deletion of rcsD, or mutations to its conserved His domain (H2), abolish cps induction (181). The same was found for mutations to the His (H1) and Asp (D1) domains of RcsC (31). It appeared that the Rcs pathway was a complex phosphorelay system involving an H1-D1-H2-D2 phosphotransfer reaction that ends with phosphorylated RcsB (31). There is evidence to suggest that this reaction is reversible, with RcsC acting as a phosphatase as well as a kinase (31, 68). Clarke and colleagues (31) showed that expression of rcsC from an inducible promoter represses the activity from a cpsB::lacZ fusion in a manner dependent upon the RcsC D1 domain (31). The RcsC phosphatase activity responsible for this repression explains earlier work that suggested RcsC was a negative regulator of capsule gene expression (14, 176).
Another component of the Rcs pathway, RcsF, was identified for its ability to induce the Rcs phosphorelay when overexpressed (69, 118). Epistasis experiments place RcsF upstream of RcsC in the signaling cascade, implicating it as a sensory protein (27). As with NlpE of the Cpx pathway, RcsF is an outer membrane lipoprotein orientated toward the periplasm (27). While several inducing cues require RcsF to effectively activate the Rcs phosphorelay, others have been shown to bypass RcsF, presumably entering the pathway at RcsC (27, 77, 118, 170).
From these findings a model for the Rcs phosphorelay has been assembled (Fig. 5). In brief, under noninducing conditions, RcsC serves as a phosphatase for RcsB, effectively maintaining the pathway in an off, or downregulated, state. Upon presentation of an inducing cue, RcsC autophosphorylates at a conserved histidine residue (H1) and then transfers the phosphoryl group to its conserved aspartate residue (D1). The Hpt domain of RcsD (H2) then mediates the phosphotransfer from RcsC (D1) to the conserved aspartate of RcsB (D2). Phosphorylated RcsB then serves as a transcriptional activator either alone or in concert with RcsA. The involvement of RcsA depends on the gene target in question because only a small subset of Rcs regulon members are regulated by the RcsAB complex.
In an effort to better understand the role of RcsA in RcsB-mediated regulation, researchers set out to characterize the RcsA binding site. Using sequence from Erwinia amylovora recognized by the RcsAB heterodimer, researchers identified a putative RcsA binding site in the promoters of rcsA and cps (57, 105). The following year, Wehland and Bernhard (195) found putative RcsAB binding sites in the main promoters of the exopolysaccharide operons in E. coli, serovar Typhi, and Klebsiella pneumoniae. The authors determined the core sequence required for RcsAB binding and called it the RcsAB box (195). An RcsB box was identified in the promoters of Rcs regulon members activated independently of RcsA (23, 43). Footprinting experiments suggest that RcsB binds alone as a dimer (178). Pristovsek et al. (151) presented evidence suggesting that an RcsAB heterodimer forms a more stable interaction with the RcsAB recognition sequence than RcsB alone. They argued that RcsA is not involved in promoter recognition, but stabilization of the complex (151). It has been suggested that RcsB ensures basal levels of gene expression, and that the introduction of an auxiliary factor facilitates high-level expression of the regulon members that are needed under specific environmental conditions (151).
Salmonella possesses RcsA, RcsB, and RcsC proteins that are homologous to those identified in E. coli (189). Interestingly, RcsA is not involved in the synthesis of its native CPS, the Vi antigen (189). Instead, an alternative auxiliary protein, TviA, interacts with RcsB at the promoter for genes involved in Vi antigen biosynthesis (189). The use of multiple auxiliary factors may be a conserved mechanism in Rcs-mediated capsular regulation as RcsB regulates the K2 capsule of K. pneumoniae in conjunction with another LuxR-like protein, RmpA (190). These findings suggest an interesting additional level of regulation for this system.
The mutations, proteins, and environmental conditions shown to induce the Rcs signaling cascade suggest that the major function of this pathway is to facilitate survival under conditions that disrupt the outer membrane. It seems sensible for a cell to initiate capsule formation in an environment that delivers significant damage to its outer surface. Mutations to rfa, mdoH, tolQRABpal, pmr, and surA all lead to upregulation of the Rcs phosphorelay system (27, 32, 58, 135, 145). These genes contribute to the synthesis of the LPS core (rfa) and membrane-derived oligosaccharides (mdo), are involved in maintaining envelope integrity (tol, surA), and facilitate resistance to membrane-damaging compounds (pmr) (27, 32, 58, 135, 145). In addition to the aforementioned mutations, overexpression of the envelope proteins DjlA, OmpG, and LolA also cause induction of the Rcs response (30, 104). At present it is not clear in what capacity they affect envelope integrity (30). The environmental stimuli shown to induce the Rcs phosphorelay include osmotic shock, low temperature, desiccation, high-level zinc exposure, and growth on solid surfaces (35, 64, 77, 173).
A recent report from the Ades laboratory suggests that the Rcs pathway also responds to peptidoglycan stress (113). This stems from microarray experiments in which cells were exposed to β-lactam antibiotics known to target penicillin-binding proteins (PBPs) found in the peptidoglycan layer. Using three scenarios, each of which target the peptidoglycan differently, they attempted to determine whether E. coli has a means to respond to peptidoglycan stress. Activation of the Rcs pathway was found to increase cell survival under all the conditions tested. RcsA and the capsule were not involved in this phenotype, suggesting that genes regulated by the RcsB homodimer facilitate survival when peptidoglycan synthesis is inhibited. The authors propose that Rcs senses peptidoglycan stress rather than the antibiotics themselves (113). This is supported by the fact that the Rcs response is activated by a subset of β-lactams that inhibit PBP transpeptidase activity and not those that simply interfere with protein synthesis and DNA replication (167). Additionally, mislocalization of the putative peptidoglycan hydrolases AmiA and AmiC lead to upregulation of cps expression (92). This is convincing evidence that the Rcs phosphorelay not only responds to general perturbations in the outer membrane, but also represents the first known stress response pathway involved in sensing changes to the peptidoglycan layer (113).
Our current knowledge of the Rcs regulon is limited by the fact that many of the members identified to date are relatively undescribed (osmC, bdm, katE, spy, etc.) (77). As with the Rcs-activating signals, the characterized regulon members suggest that the pathway functions to maintain outer membrane and peptidoglycan integrity. This is supported by microarray data from E. coli revealing that over half the genes influenced by Rcs activation are predicted to encode functions related to the bacterial surface (64). As discussed above, the Rcs phosphorelay responds to envelope stress in part by upregulating synthesis of the CPS in E. coli and the Vi antigen in Salmonella. In E. coli, this is accomplished through RcsAB regulation of 17 genes involved in capsular polysaccharide biosynthesis (77). In the case of Salmonella, RcsB-TviA positively regulates viaA (189) and RcsAB upregulates ugd, which is involved in LPS modification and polymyxin B resistance (134, 135). The Rcs pathway has also been shown to positively regulate ftsZ (23, 69). The FtsZ ring, or Z ring, of E. coli is required for localized peptidoglycan synthesis (78). Given the recent evidence that the Rcs pathway senses peptidoglycan stress, it may upregulate FtsZ in an effort to reinforce the structural integrity of the peptidoglycan layer.
In addition to combating envelope stress the Rcs pathway has also been implicated in the process of biofilm development. Not only is the pathway induced upon cell attachment to solid surfaces (64), it also upregulates colanic acid production, a requirement for proper biofilm architecture (38, 149, 150). In addition, the Rcs pathway has been shown to downregulate expression of several surface appendages in both E. coli and Salmonella (64, 188, 192). It has been suggested that, by inhibiting the expression of flagellar and curli genes, the Rcs pathway facilitates the transition from a motile state to a sessile existence (68, 192). Premature activation of the Rcs system would inhibit the initial stages of biofilm formation by blocking cell attachment. Therefore, the Rcs system plays an important role in the timing of biofilm development.
As with the Cpx envelope stress response, the exact role of the Rcs pathway in pathogenesis is unclear. While the Rcs pathway is required for a systemic infection by Salmonella, maximal activation leads to attenuation (133). This is supported by the fact that rcsCDB mutants are out-competed by wild-type Salmonella in a mouse model of infection (63). The authors found that RcsB regulates ydeI, a periplasmic gene believed to confer increased resistance to antimicrobials (63). This gene is coregulated by the stationary phase sigma factor RpoS, which is also subject to indirect regulation by the Rcs pathway (63, 146). In addition, Wang et al. (192) demonstrated that Rcs normally functions to positively regulate the SP1-2 pathogenicity island of serovar Typhimurium. SP1-2 encodes genes needed for intracellular survival and proliferation. Interestingly, SP1-2, SP1-1, and several other virulence genes are repressed when the Rcs pathway is maximally activated (192). It is possible that the Rcs pathway has evolved a similar role in pathogenesis as it performs in biofilm development, with Rcs pathway activity influencing the timing of virulence gene expression. Therefore, hyperactive mutants may be attenuated simply because they downregulate the expression of motility and virulence genes at the incorrect point during an infection. Serovar Typhimurium actually encodes an essential protein, intracellular growth attenuator A, or IgaA, that represses Rcs to enable virulence gene expression (21, 51). It has been suggested that IgaA ensures that the Rcs phosphorelay system is deactivated at the appropriate time in the host (192).
The most recent cellular process to be classified as an envelope stress response is that of vesiculation (122) (Chapter Outer Membrane Vesicles). These closed outer membrane blebs contain both periplasmic and outer membrane components and are generated by actively growing cells of gram-negative bacteria (111). The release of outer membrane vesicles has been correlated with protein accumulation in the envelope (122). Mutations causing increased vesiculation enable E. coli cells to better withstand envelope stress (122). There is evidence to suggest that outer membrane vesicles preferentially package unwanted envelope material, targeting it for expulsion from the cell (122). Because the outer membrane vesicle response is not regulated by the σE response, the Cpx and Bae two-component systems, or the Psp response it has been classified as an independent envelope stress response in both E. coli and Salmonella (121) (Chapter Outer Membrane Vesicles). Like the other envelope stress responses, the outer membrane vesicle response has also been implicated in pathogenesis (111). Some pathogens deliver their virulence determinants by means of outer membrane vesicles (111). For a complete review of this exciting new envelope stress response please refer to references 111 and 121.
Just as in the cytoplasm, numerous stress response pathways are in place to monitor the envelope and mediate adaptation to ever changing environmental conditions. The evolution of six separate envelope stress responses that respond to both overlapping and unique extracytoplasmic signals emphasizes the importance of envelope maintenance to cell survival. As we come to better understand the scope of influence these pathways have over various cellular functions, we will undoubtedly improve our understanding of bacterial physiology, evolution, and pathogenesis.
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Module5.4.7
