The CpxA/R two-component signal transduction system of Escherichia coli can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random lacZ operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus, cpxP, whose transcription is stimulated by activation of the Cpx pathway. cpxP specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that cpxP transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that cpxP and cpx mutant strains display hypersensitivity to growth in alkaline conditions.
RpoS, an alternative primary sigma factor, has been shown to be regulated at multiple levels, including transcription, translation and protein stability. Here, we present evidence that suggests that RpoS is regulated at yet another level by the product of the crl gene. The crl gene was first thought to encode the major curlin subunit of curli (curli are surface structures that are induced by growth into stationary phase under conditions of low osmolarity and low temperature). Later, it was determined that crl actually contributes in a positive fashion to stimulate transcription of csgBA, the true locus encoding for the major subunit of curli. RpoS is also required for normal stationary-phase induction of csgBA. We found that lesions in crl, like lesions in rpoS, cause increased transcription of ompF during stationary phase. Taken together, these observations prompted us to analyse the effects of crl on an additional RpoS-regulated phenomenon. We found that a crl null allele influences expression of RpoS-regulated genes in a fashion similar to an rpoS null allele. Genetic evidence suggests that crl and rpoS function in a single pathway and that Crl functions upstream, or in concert with, RpoS. Although the effects of Crl on RpoS-regulated genes is entirely dependent on the integrity of RpoS, the presence of a crl null allele does not decrease the level of RpoS protein. Thus, we propose that Crl stimulates the activity of the RpoS regulon by stimulating RpoS activity during stationary phase.
We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of Escherichia coli. Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase. As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane. These mutant porins are toxic when secreted to the cell envelope. Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J. H. Carlson and T. J. Silhavy, J. Bacteriol. 175:3327-3334, 1993). We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism. Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane. Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA. The suppression mediated by TetA' is independent of the CpxA/CpxR regulon and the sigma E regulon, both of which are involved in regulating protein trafficking functions in the cell envelope.
EnvZ, a membrane receptor kinase-phosphatase, modulates porin expression in Escherichia coli in response to medium osmolarity. It shares its basic scheme of signal transduction with many other sensor-kinases, passing information from the amino-terminal, periplasmic, sensory domain via the transmembrane helices to the carboxy-terminal, cytoplasmic, catalytic domain. The native receptor can exist in two active but opposed signaling states, the OmpR kinase-dominant state (K+ P-) and the OmpR-P phosphatase-dominant state (K- P+). The balance between the two states determines the level of intracellular OmpR-P, which in turn determines the level of porin gene transcription. To study the structural requirements for these two states of EnvZ, mutational analysis was performed. Mutations that preferentially affect either the kinase or phosphatase have been identified and characterized both in vivo and in vitro. Most of these mapped to previously identified structural motifs, suggesting an important function for each of these conserved regions. In addition, we identified a novel motif that is weakly conserved among two-component sensors. Mutations that alter this motif, which is termed the X region, alter the confirmation of EnvZ and significantly reduce the phosphatase activity.
EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity. Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase. Conserved histidine-243 is critical for both autokinase and OmpR kinase activities. To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro. In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ. However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine. In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine. These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required. Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation. Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.
In Escherichia coli, the heat shock-inducible sigma-factor sigma(E) and the Cpx two-component signal transduction system are both attuned to extracytoplasmic stimuli. For example, sigma(E) activity rises in response to the overproduction of various outer-membrane proteins. Similarly, the activity of the Cpx signal transduction pathway, which consists of an inner-membrane sensor (CpxA) and a cognate response regulator (CpxR), is stimulated by overproduction of the outer-membrane lipoprotein, NlpE. In response to these extracytoplasmic stimuli, sigma(E) and CpxA/CpxR stimulate the transcription of degP, which encodes a periplasmic protease. This suggests that CpxA/CpxR and sigma(E) both mediate protein turnover within the bacterial envelope. Here, we show that CpxA/CpxR and sigma(E) also control the synthesis of periplasmic enzymes that can facilitate protein-folding reactions. Specifically, sigma(E) controls transcription of fkpA, which specifies a periplasmic peptidyl-prolyl cis/trans isomerase. Similarly, the Cpx system controls transcription of the dsbA locus, which encodes a periplasmic enzyme required for efficient disulfide bond formation in several extracytoplasmic proteins. Taken together, these results indicate that sigma(E) and CpxA/CpxR are involved in regulating both protein-turnover and protein-folding activities within the bacterial envelope.
Disruption of normal protein trafficking in the Escherichia coli cell envelope (inner membrane, periplasm, outer membrane) can activate two parallel, but distinct, signal transduction pathways. This activation stimulates the expression of a number of genes whose products function to fold or degrade the mislocalized proteins. One of these signal transduction pathways is a two-component regulatory system comprised of the histidine kinase CpxA and the response regulator, CpxR. In this study we characterized gain-of-function Cpx* mutants in order to learn more about Cpx signal transduction. Sequencing demonstrated that the cpx* mutations cluster in either the periplasmic, the transmembrane, or the H-box domain of CpxA. Intriguingly, most of the periplasmic cpx* gain-of-function mutations cluster in the central region of this domain, and one encodes a deletion of 32 amino acids. Strains harboring these mutations are rendered insensitive to a normally activating signal. In vivo and in vitro characterization of maltose-binding-protein fusions between the wild-type CpxA and a representative cpx* mutant, CpxA101, showed that the mutant CpxA is altered in phosphotransfer reactions with CpxR. Specifically, while both CpxA and CpxA101 function as autokinases and CpxR kinases, CpxA101 is devoid of a CpxR-P phosphatase activity normally present in the wild-type protein. Taken together, the data support a model for Cpx-mediated signal transduction in which the kinase/phosphatase ratio is elevated by stress. Further, the sequence and phenotypes of periplasmic cpx* mutations suggest that interactions with a periplasmic signaling molecule may normally dictate a decreased kinase/phosphatase ratio under nonstress conditions.
In Escherichia coli, levels of the two major outer membrane porin proteins, OmpF and OmpC, are regulated in response to a variety of environmental parameters, and numerous factors have been shown to influence porin synthesis. EnvZ and OmpR control porin-gene transcription in response to osmolarity, and the antisense RNA, MicF, influences ompF translation. In contrast to these characterized factors, some of the components reported to influence porin expression have only modest effects and/or act indirectly. For others, potential regulatory roles, although intriguing, remain elusive. Here we review many of the components that have been reported to influence porin expression, address the potential regulatory nature of these components, and discuss how they may contribute to a regulatory network controlling porin synthesis.
In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.
The secretion of proteins from the cytoplasm of Escherichia coli requires the interaction of two integral inner membrane components, SecY and SecE. We have devised a genetic approach to probe the molecular nature of the SecY-SecE interaction. Suppressor alleles of secY and secE, termed prlA and prlG, respectively, were analyzed in pair-wise combinations for synthetic phenotypes. From a total of 115 combinations, we found only seven pairs of alleles that exhibit a synthetic defect when present in combination with one another. The phenotypes observed are not the result of additive defects caused by the prl alleles, nor are they the consequence of multiple suppressors functioning within the same strain. In all cases, the synthetic defect is recessive to wild-type secY or secE provided in trans. The recessive nature argues for a defective interaction between the Prl suppressors. The extreme allele specificity and topological coincidence of the mutations represented by these seven pairs of alleles identify domains of interaction between SecY/PrlA and SecE/PrlG.
The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming. This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure. To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct. We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery. This suggests that jamming is caused by LacZ folding. We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel. Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm. Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.
DegP is a heat-shock inducible periplasmic protease in Escherichia coli. Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by sigma 32. Rather, the degP gene is transcriptionally regulated by an alternate heat shock sigma factor, sigma E. Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter. To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion. Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP. Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP. These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein. Cpx-mediated transcription of degP is partially dependent on the activity of E sigma E, suggesting that the Cpx pathway functions in concert with E sigma E and perhaps other RNA polymerases to drive transcription of degP.
The SecA protein of Escherichia coli is required for protein translocation from the cytoplasm. The complexity of SecA function is reflected by missense mutations in the secA gene that confer several different phenotypes: (i) conditional-lethal alleles cause a generalized block in protein secretion, resulting in the cytoplasmic accumulation of the precursor forms of secreted proteins; (ii) azi alleles confer resistance to azide at concentrations up to 4 mM; and (iii) prlD alleles suppress a number of signal sequence mutations in several different genes. To gain further insights into the role of SecA in protein secretion, we have isolated and characterized a large number of prlD mutations, reasoning that these mutations alter a normal function of wild-type SecA. Our results reveal a striking coincidence of signal sequence suppression and azide resistance: the majority of prlD alleles also confer azide resistance, and all azi alleles tested are suppressors. We suggest that this correlation reflects the mechanism(s) of signal sequence suppression. There are two particularly interesting subclasses of prlD and azi alleles. First, four of the prlD and azi alleles exhibit special properties: (i) as suppressors they are potent enough to allow PrlD (SecA) inactivation by a toxic LacZ fusion protein marked with a signal sequence mutation (suppressor-directed inactivation), (ii) they confer azide resistance, and (iii) they cause modest defects in the secretion of wild-type proteins. Sequence analysis reveals that all four of these alleles alter Tyr-134 in SecA, changing it to Ser, Cys, or Asn. The second subclass consists of seven prlD alleles that confer azide supersensitivity, and sequence analysis reveals that six of these alleles are changes of Ala-507 to Val. Both of the affected amino acids are located within different putative ATP-binding regions of SecA and thus may affect ATPase activities of SecA. We suggest that the four azide-resistant mutations slow an ATPase activity of SecA, thus allowing successful translocation of increased amounts of mutant precursor proteins.
OmpR, the transcriptional regulator of the ompF and ompC porin genes, is a member of a novel class of DNA-binding proteins. The mechanism(s) by which this class of proteins interacts with target DNA sites is not understood. To address this issue, we investigated the nature of the DNA sequences recognized by OmpR. A 36 bp DNA fragment was identified that is capable of supporting OmpR-DNA interaction in vivo. The base pairs within this region of DNA that are critical to this interaction were identified by isolating mutations within the fragment that hinder normal OmpR-DNA binding. The results obtained provide insights concerning the nature of the sequences recognized by OmpR and also support a model in which co-operative binding is involved in OmpR-DNA interaction.
Mutations in the secretory (sec) genes in Escherichia coli compromise protein translocation across the inner membrane and often confer conditional-lethal phenotypes. We have found that overproduction of the chaperonins GroES and GroEL from a multicopy plasmid suppresses a wide array of cold-sensitive sec mutations in E. coli. Suppression is accompanied by a stimulation of precursor protein translocation. This multicopy suppression does not bypass the Sec pathway because a deletion of secE is not suppressed under these conditions. Surprisingly, progressive deletion of the groE operon does not completely abolish the ability to suppress, indicating that the multicopy suppression of cold-sensitive sec mutations is not dependent on a functional groE operon. Indeed, overproduction of proteins unrelated to the process of protein export suppresses the secE501 cold-sensitive mutation, suggesting that protein overproduction, in and of itself, can confer mutations which compromise protein synthesis and the observation that low levels of protein synthesis inhibitors can suppress as well. In all cases, the mechanism of suppression is unrelated to the process of protein export. We suggest that the multicopy plasmids also suppress the sec mutations by compromising protein synthesis.
The processing-defective outer membrane porin protein LamBA23D (Carlson and Silhavy, 1993) and a tripartite fusion protein, LamB-LacZ-PhoA (Snyder and Silhavy, 1995), are both secreted across the cytoplasmic membrane of Escherichia coli, where they exert an extracytoplasmic toxicity. Suppressors of these toxicities map to a previously characterized gene, cpxA, that encodes the sensor kinase protein of a two-component regulatory system. These activated cpxA alleles, designated as cpxA*, stimulate transcription of the periplasmic protease DegP (Danese et al., 1995), which in turn catalyses degradation of the tripartite fusion protein. In contrast, degradation of precursor LamBA23D is not significantly stimulated in a cpxA* suppressor background. In fact, increased levels of DegP in a wild-type background stabilized this protein. While a functional degP gene is required for full cpxA*-mediated suppression of both toxic envelope proteins, residual suppression is seen in cpxA* degP::Tn10 double mutants. Furthermore, cpxA* mutations suppress the toxicity conferred by the LamB-LacZ hybrid protein, which exerts its effects in the cytoplasm, sequestered from DegP. Together, these observations suggest that the activated Cpx pathway regulates additional downstream targets that contribute to suppression. A subset of these targets may constitute a regulon involved in relieving extracytoplasmic and/or secretion-related stress.
The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [3H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.
Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
In Escherichia coli, OmpR and EnvZ comprise a two component regulatory system that controls the relative expression of the outer membrane porin proteins, OmpF and OmpC. In this system, OmpR functions as a transcriptional regulator, serving as an activator of ompC, and as both an activator and a repressor of ompF. Previous evidence suggests that OmpR-mediated transcriptional activation involves direct interaction between OmpR and the C-terminal domain of the alpha subunit of RNA polymerase. However, it has remained unclear what region(s) of OmpR is directly involved in this proposed interaction. Moreover, little else is known about how OmpR activates transcription. To identify residues important for transcriptional activation, we screened for mutations in ompR that render the protein specifically defective in its ability to activate transcription. The isolated ompR alleles were characterized through haploid and diploid analyses at both the ompF and ompC promoters, and through an in vivo DNA binding assay. Through this approach, we have identified five amino acid residues in OmpR that are specifically required for transcriptional activation; R42, P179, E193, A196 and E198. We propose that these mutations define a region(s) in OmpR that may contact the C-terminal domain of alpha to mediate transcriptional activation.