MzrA-EnvZ interactions in the periplasm influence the EnvZ/OmpR two-component regulon

MzrA was identified as a modulator of the EnvZ/OmpR two-component signal transduction system. Previous evidence indicated that MzrA interacts with EnvZ and modulates its enzymatic activities to influence OmpR phosphate (OmpR～P) levels. Moreover, MzrA was shown to connect the bacterial envelope stress response systems CpxA/CpxR and σ(E) to EnvZ/OmpR to widen the defensive response regulatory network. In this study, experiments were carried out to establish whether the membrane or periplasmic domain of MzrA is critical for MzrA-EnvZ interactions and to reveal MzrA residues that play an important role in these interactions. Data obtained from chimeric constructs, in which the transmembrane domain of MzrA was replaced with the unrelated transmembrane domain of NarX or signal sequence of PhoA, showed that the transmembrane domain residues of MzrA do not play a critical role in MzrA-EnvZ interactions. The importance of the periplasmic domain of MzrA in MzrA-EnvZ interactions was revealed by characterizing bifunctional, fully soluble, and periplasmically localized MalE::MzrA chimeras. This was further corroborated through the isolation of loss-of-function, single-amino-acid substitutions in the conserved periplasmic domain of MzrA that interfered with MzrA-EnvZ binding in a bacterial two-hybrid system. Together, the data suggest that the binding of MzrA to EnvZ influences the ability of EnvZ to receive and/or respond to environmental signals in the periplasm and modulate its biochemical output to OmpR.     

EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12.

The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.     

Formation of the stoichiometric complex of EnvZ,a histidine kinase,with its response regulator,OmpR

EnvZ, a histidine kinase, and its cognate response regulator OmpR of Escherichia coli are responsible for adaptation to external osmotic changes by regulating the levels of the outer membrane porin proteins, OmpF and OmpC. The osmosensor, EnvZ, has dual enzymatic functions with OmpR kinase and OmpR-P phosphatase. Here, we demonstrate that the cytoplasmic kinase domain of EnvZ (EnvZc) and OmpR are able to form a 1:1 complex detected by native PAGE. This indicates that two OmpR molecules can bind to one EnvZc dimer. As this 1:1 EnvZc/OmpR complex is formed even in the presence of a large excess of EnvZc, OmpR binding to EnvZc is co-operative. The complex formation is also observed between EnvZc and phosphorylated OmpR for the phosphatase reaction. OmpR-P bound to EnvZc was readily released upon the addition of OmpR, indicating that OmpR and OmpR-P can compete for the binding to EnvZ. On the basis of these results, a model is discussed to explain how cellular OmpR-P concentrations are regulated in response to medium osmolarity.     

Interaction of EnvZ,a sensory histidine kinase,with phosphorylated OmpR,the cognate response regulator

EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein-labelled OmpR (Fl-OmpR) with EnvZc was determined to be 1.96 +/- 0.28 micro M. We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl-OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 +/- 0.25 micro M and 0.89 +/- 0.14 micro M, respectively, under the conditions used. The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl-OmpR or Fl-OmpR-P as well as with OmpR or OmpR-P as a substrate. We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.     

Phosphorylation alters the interaction of the response regulator OmpR with its sensor kinase EnvZ.

OmpR and EnvZ comprise a two-component system that regulates the porin genes ompF and ompC in response to changes in osmolarity. EnvZ is autophosphorylated by intracellular ATP on a histidine residue, and it transfers the phosphoryl group to an aspartic acid residue of OmpR. EnvZ can also dephosphorylate phospho-OmpR (OmpR-P) to control the cellular level of OmpR-P. At low osmolarity, OmpR-P levels are low because of either low EnvZ kinase or high EnvZ phosphatase activities. At high osmolarity, OmpR-P is elevated. It has been proposed that EnvZ phosphatase is the activity that is regulated by osmolarity. OmpR is a two-domain response regulator; phosphorylation of OmpR increases its affinity for DNA, and DNA binding stimulates phosphorylation. The step that is affected by DNA depends upon the phosphodonor employed. In the present work, we have used fluorescence anisotropy and phosphotransfer assays to examine OmpR interactions with EnvZ. Our results indicate that phosphorylation greatly reduces the affinity of OmpR for the kinase, whereas DNA does not affect their interaction. The results presented cast serious doubts on the role of the EnvZ phosphatase in response to signaling in vivo.     

EnvZ controls the concentration of phosphorylated OmpR to mediate osmoregulation of the porin genes.

Osmoregulation of the bacterial porin genes ompF and ompC is controlled by a two-component regulatory system. EnvZ, the sensor component of this system, is capable both of phosphorylating and dephosphorylating OmpR, the effector component. Mutations were isolated in envZ that abolish the expression of both porin genes. These mutants appear to have lost the kinase activity of EnvZ while retaining their phosphatase activity, so that in their presence OmpR is completely unphosphorylated. The behavior of these mutants in haploid, and in diploid with other envZ alleles, is consistent with a model in which EnvZ mediates osmoregulation by controlling the concentration of a single species. OmpR-P.     

Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [³H]-NaBH₄ indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.     

Transmembrane signal transduction and osmoregulation in Escherichia coli. Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ

The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation. In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions. It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane. Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity. It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected. These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus.     

A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation

In bacteria and lower eukaryotes, adaptation to changes in the environment is often mediated by two-component regulatory systems. Such systems provide the basis for chemotaxis, nitrogen and phosphate regulation and adaptation to osmotic stress, for example. In Escherichia coli, the sensor kinase EnvZ detects a change in the osmotic environment and phosphorylates the response regulator OmpR. Phospho-OmpR binds to the regulatory regions of the porin genes ompF and ompC, and alters their expression. Recent evidence suggests that OmpR functions as a global regulator, regulating additional genes besides the porin genes. In this study, we have characterized a previously isolated OmpR2 mutant (V203M) that constitutively activates ompF and fails to express ompC. Because the substitution was located in the C-terminal DNA-binding domain, it had been assumed that the substitution would not affect phosphorylation of the N-terminal domain of OmpR. Our results indicate that this substitution completely eliminates phosphorylation by a small phosphate donor, acetyl phosphate, but not phosphorylation by the kinase EnvZ. The mutant OmpR has altered dephosphorylation kinetics and altered binding affinities to both ompF and ompC sites compared to the wild-type. Thus, a single amino acid substitution in the C-terminal DNA-binding domain has dramatic effects on the N-terminal phosphorylation domain. Most strikingly, we have identified a single base change in the OmpR binding site of ompC that restores high-affinity binding activity by the mutant. We interpret our results in the context of a model for porin gene expression.     

Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli.

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.     

Re-examination of the role of the periplasmic domain of EnvZ in sensing of osmolarity signals in Escherichia coli

In Escherichia coli , EnvZ senses changes in the osmotic conditions of the growth environment and controls the phosphorylated state of the regulatory protein, OmpR. OmpR-phosphate regulates the expression of the porin genes, ompF and ompC . To investigate the role of the periplasmic domain of EnvZ in sensing of osmolarity signals, portions of this domain were deleted. Cells containing the EnvZ mutant proteins were able to regulate normally the production of OmpF and OmpC in response to changes in osmolarity. The periplasmic domain of EnvZ was also replaced with the non-homologous periplasmic domain of the histidine kinase PhoR of Bacillus subtilis . Osmoregulation of OmpF and OmpC production in cells containing the PhoR–EnvZ hybrid protein was indistinguishable from that in cells containing wild-type EnvZ. Identical results were obtained with an envZ – pta/ack strain, which could not synthesize acetyl phosphate. Thus, acetyl phosphate was not involved in the regulation of ompF and ompC observed in this study. These results indicate that the periplasmic domain of EnvZ is not essential for sensing of osmolarity signals.     

Dynamic Interaction between the CpxA Sensor Kinase and the Periplasmic Accessory Protein CpxP Mediates Signal Recognition in E. coli

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

Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins.

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.