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.     

Evidence for the physiological importance of the phosphotransfer between the two regulatory components, EnvZ and OmpR, in osmoregulation in Escherichia coli

Previously, the transfer of the phosphoryl group between the EnvZ and OmpR proteins, which are involved in activation of the ompF and ompC genes in response to the medium osmolarity, has been demonstrated in vitro. In this study, we characterized mutant EnvZ and OmpR proteins in terms of their in vitro phosphorylation and dephosphorylation. The proteins isolated from the mutants, envZ11 and ompR3, were found to be defective in seemingly the same aspect, i.e. OmpR dephosphorylation. The protein isolated from the ompR77 mutant, which is a suppressor mutant specific for envZ11, was found to be defective in another aspect, i.e. OmpR phosphorylation. These results imply that the phosphotransfer reactions observed in vitro play roles in the mechanism underlying the osmoregulatory expression of the ompF and ompC genes in vivo. We provide evidence that the EnvZ protein is involved not only in OmpR phosphorylation but also in OmpR dephosphorylation.     

Signal transduction and osmoregulation in Escherichia coli. A single amino acid change in the protein kinase, EnvZ, results in loss of its phosphorylation and dephosphorylation abilities with respect to the activator protein, OmpR

The EnvZ protein is a bacterial protein kinase, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli. The phosphotransfer between the EnvZ and OmpR proteins was postulated to be involved in the signal transduction in response to an environmental osmotic stimulus. In this study, we isolated a novel type of envZ mutant, in which a base substitution resulted in a Tyr-to-Ser conversion at amino acid residue 351 of the EnvZ protein. This single amino acid conversion was found to dramatically affect the functions of the EnvZ protein. The mutant EnvZ protein was defective in its ability not only as to OmpR-phosphorylation but also as to OmpR-dephosphorylation. The envZ mutant, termed envZ30, was isolated as a pseudorevertant, which phenotypically suppresses an ompR3-type mutant exhibiting an OmpF- OmpC-constitutive phenotype. These results will be discussed in relation to the structure and function of the protein kinase, EnvZ.     

Transmembrane signal transduction and osmoregulation in Escherichia coli: I. Analysis by site-directed mutagenesis of the amino acid residues involved in phosphotransfer between the two regulatory components, EnvZ and OmpR

Previously, the transfer of a phosphoryl group between the EnvZ and OmpR proteins, which are involved in expression of the ompF and ompC genes in response to the medium osmolarity, was demonstrated in vitro. In this study, the histidine (His) residue at position 243 of the EnvZ protein, and the aspartate (Asp) residues at positions 12 and 55 of the OmpR protein were changed, respectively, by means of site-directed mutagenesis. We characterized the mutant proteins in terms of not only their in vitro phosphotransfer reactions but also their in vivo osmoregulatory phenotypes. The mutant EnvZ protein was defective in its in vitro ability not only as to EnvZ-autophosphorylation but also OmpR-phosphorylation and OmpR-dephosphorylation. This particular mutant EnvZ protein seemed to exhibit null functions as to the in vivo osmoregulatory phenotype. The mutant OmpR protein with the amino acid change at position 12 was clearly phosphorylated in vitro, but at a very low rate as compared with the wild-type OmpR protein. In vitro phosphorylation of the mutant OmpR protein with the amino acid change at position 55 was more severely affected. This mutant OmpR protein appeared to exhibit null functions as to the in vivo osmoregulatory phenotype. These results suggest that the histidine residue at position 243 of the EnvZ protein and the aspartate residues at positions 12 and 55 of the OmpR protein are deeply involved in the phosphotransfer between the EnvZ and OmpR proteins.     

Transmembrane signal transduction and osmoregulation in Escherichia coli: II. The osmotic sensor, EnvZ, located in the isolated cytoplasmic membrane displays its phosphorylation and dephosphorylation abilities as to the activator protein, OmpR

The EnvZ protein is presumably a membrane-located osmotic sensor, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli. In this study, we developed an in vitro system for analyzing the intact form of the EnvZ protein located in the isolated cytoplasmic membrane. This particular form of the EnvZ protein exhibited its in vitro ability not only as to OmpR-phosphorylation but also OmpR-dephosphorylation. It was found that when a high concentration of a mono-cation (K+, Na-, or Li+) was present during the in vitro reactions, OmpR-dephosphorylation was preferentially inhibited and consequently the phosphorylated from of the OmpR protein was accumulated under the in vitro conditions used, although the K+ ion appears to be essential for the OmpR-phosphorylation reaction. Procaine, a local anesthetic, is known to affect the osmotic regulation of the ompF and ompC genes in vivo. In this study, procaine was also found to preferentially inhibit OmpR-dephosphorylation mediated by the EnvZ protein in vitro.     

Osmoregulatory expression of the porin genes in Escherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer

The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.     

Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling

Summary
The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity. The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein. To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities. One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) In TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity. This EnvZ mutant conferred a phenotype of OmpF⁻/OmpC-constitutive. For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF⁺/OmpC⁺, was isolated. These suppresor mutants were revealed to have an additional amino acid change within either TM1 or TM2. Furthermore, they exhibited restored phosphatase activity (i.e., both kinase⁺ and phosphatase⁺ activities). It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation. These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane–spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus.     

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability

The Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs.     

Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli

EnvZ is a cytoplasmic membrane protein which is involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli possibly by sensing the environmental osmotic signal. A truncated form of the EnvZ protein (EnvZ*), comprising 82% of EnvZ starting from the C terminus, was purified to homogeneity. The purified EnvZ* was autophosphorylated with ATP. The phosphoryl group on EnvZ* could then be rapidly transferred to OmpR, which is a positive regulator of the ompF and ompC genes and which was proposed to interact with EnvZ in the process of osmoregulation. In the presence of ATP, the phosphorylated OmpR was rapidly dephosphorylated. These results suggest that the transfer of the phosphoryl group between EnvZ and OmpR plays an important role in the signaling pathway in osmoregulation.     

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.     

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.     

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.     

EnvZ-independent phosphotransfer signaling pathway of the OmpR-mediated osmoregulatory expression of OmpC and OmpF in Escherichia coli

The Escherichia coli EnvZ-OmpR regulatory system is a paradigm of intracellular signal transduction mediated by the well-documented phosphotransfer mechanism, by which the expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. Although it is clear that the EnvZ histidine(His)-kinase is the major player in the phosphorylation of OmpR, it has been assumed for some time that there may be an alternative phospho-donor(s) that can phosphorylate OmpR under certain in vitro and in vivo conditions. In this study, to address this long-standing issue, extensive genetic studies were done with certain mutant alleles, including delta envZ, delta(ackA-pta), and delta sixA, as well as delta ompR. Here, for the first time, genetic evidence is provided that, in addition to EnvZ, acetyl phosphate and an as yet unidentified sensor His-kinase can serve as alternative in vivo phospho-donors for OmpR, even in the envZ+ background. A model for the alternative phosphotransfer signaling pathway involved in the phosphorylation of OmpR is proposed.     

The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm

Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.     

Differential Expression of the OmpF and OmpC Porin Proteins in Escherichia coli K-12 Depends upon the Level of Active OmpR

We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.