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.     

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: functional importance of the transmembrane regions of membrane-located protein kinase, EnvZ.

EnvZ is a membrane-located protein kinase which modulates expression of the ompF and ompC genes through phosphotransfer signal transduction in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of EnvZ in isolated cytoplasmic membranes, and demonstrated that this particular form of EnvZ exhibits the ability not only of OmpR phosphorylation but also OmpR dephosphorylation. Taking advantage of this in vitro system, in this study, to assess the structural and functional importance of the membrane-spanning (transmembrane) regions of EnvZ, a set of mutant envZ genes, each of which specifies a mutant EnvZ protein with a single amino acid replacement within or very near the transmembrane regions, were isolated and characterized in terms of their in vivo osmoregulatory phenotypes and in vitro EnvZ-OmpR phosphotransfer activities. On the basis of the results, it was suggested that the transmembrane regions of EnvZ play roles in transmembrane signaling and consequent modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an osmotic stimulus.     

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.     

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.     

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.     

Co-regulation of polysaccharide production,motility, and expression of type III secretion genes by EnvZ/OmpR and GrrS/GrrA systems in Erwinia amylovora

The EnvZ/OmpR and GrrS/GrrA systems, two widely distributed two-component systems in gamma-Proteobacteria, negatively control amylovoran biosynthesis in Erwinia amylovora, and the two systems regulate motility in an opposing manner. In this study, we examined the interplay of EnvZ/OmpR and GrrS/GrrA systems in controlling various virulence traits in E. amylovora. Results showed that amylovoran production was significantly higher when both systems were inactivated, indicating that the two systems act as negative regulators and their combined effect on amylovoran production appears to be enhanced. In contrast, reduced motility was observed when both systems were deleted as compared to that of grrA/grrS mutants and WT strain, indicating that the two systems antagonistically regulate motility in E. amylovora. In addition, glycogen accumulation was much higher in envZ/ompR and two triple mutants than that of grrS/grrA mutants and WT strain, suggesting that EnvZ/OmpR plays a dominant role in regulating glycogen accumulation, whereas levan production was significantly lower in the grrS/grrA and two triple mutants as compared with that of WT and envZ/ompR mutants, indicating that GrrS/GrrA system dominantly controls levan production. Furthermore, both systems negatively regulated expression of three type III secretion (T3SS) genes and their combined negative effect on hrp-T3SS gene expression increased when both systems were deleted. These results demonstrated that EnvZ/OmpR and GrrS/GrrA systems co-regulate various virulence factors in E. amylovora by still unknown mechanisms or through different target genes, sRNAs, or proteins, indicating that a complex regulatory network may be involved, which needs to be further explored.     

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.     

Regulatory Protein OmpR Influences the Serum Resistance of Yersinia enterocolitica O:9 by Modifying the Structure of the Outer Membrane

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.     

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.     

MzrA: a novel modulator of the EnvZ/OmpR two-component regulon

Analysis of suppressors that alleviate the acute envelope stress phenotype of a Δ bamB Δ degP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR.     

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.     

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.     

Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus

In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR. Modulation of the levels of OmpR-phosphate controls the differential expression of ompF and ompC . While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood. We have used a comparative approach to study the sensing function of EnvZ. During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ . Nucleotide sequence analysis revealed that EnvZ of X. nematophilus (EnvZ^X.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZ^E.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ moleculsshared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZ^E.c. was absent in EnvZ^X.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZ^X.n. was able to complement a Δ envZ strain of E. coli . OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain. Further genetic analysis established that heterologous phosphorylation between EnvZ^X.n. and OmpR of E. coli (OmpR^E.c.) accounted for the complementation of the Δ envZ strain. In addition we show that the OmpR molecules of X. nematophilus and E. coli share 78% amino acid sequence identity. These results indicate that the EnvZ protein of X. nematophilus was able to sense changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E. coli .     

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.