OmpR Protein Research Articles

Molecular analysis of OmpR binding sequences involved in the regulation of ompF in Escherichia coli.

OmpR, the transcriptional regulatory protein of ompF, had not been previously shown to specifically bind to the -70 to -60-bp region of ompF. We show that the -102 to -76-bp sequence of ompF has a high affinity binding site for OmpR and produced a single OmpR/ompF complex (complex b). Extension of this DNA fragment to include an inverted repeat sequence located between the -71 and -64-bp region resulted in the formation of a second, slower migrating complex (complex a). A -102 to -58-bp fragment containing a substitution of the -70 CG bp was able to form complex b, but not complex a. A mutant OmpR protein derived from a strain that can not repress ompF was unable to form complex a, while complex b was formed normally. Deletion of the -70 CG bp resulted in incomplete repression of OmpF. These results suggest that OmpR binds to the -71 to -64-bp region and that this sequence plays a role in the regulation of ompF in Escherichia coli.

Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR.

During the search for unknown factors involved in motility, we have found that expression of the flagellar master operon flhDC is affected by mutations of the pta and ackA genes, encoding phosphotransacetylase and acetate kinase, respectively (S. Shin, J. Sheen, and C. Park, Korean J. Microbiol. 31:504-511, 1993). Here we describe results showing that this effect is modulated by externally added acetate, except when both pta and ackA are mutated, suggesting the role of acetyl phosphate, an intermediate of acetate metabolism, as a regulatory effector. Furthermore, the following evidence indicates that the phosphorylation of OmpR, a trans factor for osmoregulation, regulates flagellar expression. First, in a strain lacking ompR, the expression of flhDC is no longer responsive to a change in the level of acetyl phosphate. Second, an increase in medium osmolarity does not decrease flhDC expression in an ompR mutant. It is known that such an increase normally enhances OmpR phosphorylation. Third, OmpR protein binds to the DNA fragment containing the flhDC promoter, and its affinity is increased with phosphorylation by acetyl phosphate. DNase I footprinting revealed the regions of the flhDC promoter protected by OmpR in the presence or absence of phosphorylation. Therefore, we propose that the phosphorylated OmpR, generated by either osmolarity change or the internal level of acetyl phosphate, negatively regulates the expression of flagella.

Mechanism of gene activation by the Escherichia coli positive regulator, OmpR: A mutant defective in transcriptional activation

The OmpR protein is an activator specific for the E. coli ompC and ompF genes. This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase. In this study we isolated OmpR mutants which were suggested to be defective for transcriptional activation, but not for DNA binding. Two such mutants, that we isolated, have a single amino acid alteration at positions 131 [P131S], and 179 [P179L], respectively, of OmpR, comprising 239 amino acids. These altered amino acids in OmpR may be implicated, directly or indirectly, in the presumed RNA polymerase/OmpR interaction that is important for transcriptional activation.

The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation.

In Escherichia coli the ompF gene encodes a major outer membrane porin protein that is differentially regulated by the OmpR protein. OmpR acts as a positive as well as a negative regulator of ompF expression by binding to DNA sequences in the ompF promoter region. The DNA binding activity of OmpR is itself regulated by phosphorylation through the kinase protein EnvZ. Phosphorylation is believed to change the function of OmpR from an activator to a repressor molecule. By using purified OmpR and various regions of the ompF promoter we show that phosphorylation causes binding of OmpR to a DNA region between the -40 to -100 region of the ompF promoter previously shown to be important for ompF expression. As the amount of OmpR-phosphate increases, a binding site located at a further upstream -360 to -380 region was occupied. This latter site has been reported to be important for ompF repression. Further experiments indicate that the -70 to -100 region is a high affinity site, while the -45 to -60 and -360 to -380 regions are low affinity sites. We also provide evidence that OmpR binding at the -360 to -380 region requires previous binding at downstream sequences, which is indicative of long range interactions between OmpR molecules. We interpret our results in terms of a model for ompF regulation involving hierarchical binding by phosphorylated OmpR and potential DNA looping.

The alpha subunit of RNA polymerase specifically inhibits expression of the porin genes ompF and ompC in vivo and in vitro in Escherichia coli.

Overproduction of the alpha subunit of RNA polymerase in Escherichia coli resulted in inhibition of transcription of two osmoregulated porin genes, ompF and ompC, but not of constitutively expressed housekeeping genes. Overproduction of the sigma subunit did not have any inhibitory effects. The specific inhibitory effect of the alpha subunit was also found to depend upon the OmpR protein, the transcriptional activator for ompF and ompC. These results are in general agreement with other biochemical and genetic evidence suggesting that the alpha subunit is the subunit of RNA polymerase that directly interacts with certain transcriptional activators to initiate transcription.

Crystallization and X-ray Studies of the DNA-binding Domain of OmpR Protein, a Positive Regulator Involved in Activation of Osmoregulatory Genes in Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively. The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method. X-ray studies show that the crystals belong to the trigonal space group P3 121 (or P3 221) with a = b = 60·4 Å, c = 58·8 Å and γ = 120°. The asymmetric unit contains one molecule. The crystals diffract to at least 3 Å resolution and are suitable for X-ray structure analysis.

Mutations that Affect Separate Functions of OmpR the Phosphorylated Regulator of Porin Transcription in Escherichia coli

OmpR is a member of a family of bacterial transcriptional regulators whose activity is controlled by phosphorylation. It regulates the transcription of two genes, serving as an activator of ompC, and as both an activator and a repressor of ompF. A previously isolated collection of ompR mutations was analyzed for the effect of each on the expression of both genes simultaneously. The results of this analysis indicate that the activation, repression, and DNA binding functions of OmpR can be disrupted independently, and that mutations interfering with each of these functions cluster within the sequence of the OmpR protein. The nature of these mutations is discussed in terms of the mechanisms by which OmpR regulates transcription, and potentially similar mechanisms operating within closely related response regulators.

Activation of the osmoregulated ompF and ompC genes by the OmpR protein in Escherichia coli: a study involving chimeric promoters.

The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity. The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites. In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid. It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene. In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472). It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur. These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein.

Molecular analysis of the signaling pathway between EnvZ and OmpR in Escherichia coli.

OmpR is a DNA-binding protein that regulates transcription of ompF and ompC. The activity of OmpR is controlled by the inner membrane osmosensor, EnvZ. In order to study the signaling process between EnvZ and OmpR, we analyzed two different envZ strains: the envZ473 strain, in which OmpC is constitutively produced and OmpF is fully repressed, and the envZ3 strain, in which the production of OmpC is greatly reduced and OmpF is not fully repressed by high-osmolarity growth conditions. Using direct sequencing of DNA derived from the polymerase chain reaction amplification method, we identified the mutation in the envZ473 strain as a Val-241-to-Gly substitution and the mutation in the envZ3 as an Ala-219-to-Val substitution. The relative DNA-binding affinity of OmpR derived from the envZ473 strain was dramatically increased for the upstream sequence of both ompF and ompC. In contrast, OmpR derived from the envZ3 strain was not converted to the high-affinity form. The intracellular levels of OmpR-phosphate, as analyzed by the in vivo phosphorylation approach, significantly increased in the envZ473 strain, while in the envZ3 strain the levels were considerably reduced, relative to those found in the parent strain. The intracellular level of OmpR protein in the envZ473 strain was also found to be markedly elevated relative to that of the parent strain. These results are discussed in relation to the role of phosphorylation and relative DNA-binding affinity of OmpR in the expression of ompF and ompC.

Activation of the Osmoregulated ompC Gene by the OmpR Protein in Escherichia coli: A Study Involving Synthetic OmpR-Binding Sequences1

Expression of the Escherichia coli outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. The OmpR and EnvZ proteins are transcriptional factors involved in this osmotic regulation of the ompC and ompF genes. In particular, expression of the ompC gene is activated by the positive regulator, OmpR, in response to high osmolarity of the medium. In this study, we succeeded in defining a functional OmpR-binding sequence by analyzing a set of synthetic oligonucleotides, and propose a consensus motif for OmpR-binding. It was also demonstrated that the asymmetric OmpR-binding sequence, thus identified, can activate the canonical ompC promoter in an orientation independent-manner, providing that this sequence is placed closely and stereo-specifically with respect to the -35 region.

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.

Osmoregulatory expression of the porin genes inEscherichia 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 phosphotransger (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 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 OmpR1

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, OmpR1

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 porin genes inEscherichia coli: a comparative study on strains B and K-12

Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium. In contrast, E. coli B produces only the OmpF porin, regardless of the medium osmolarity. In this study, it was revealed that there is an extensive deletion within the ompC locus of the E. coli B chromosome. Cloning and nucleotide sequencing of the regulatory gene, ompR, of E. coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E. coli K-12. It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E. coli B.

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli

The OmpR of Escherichia coli is a positive regulator specific for the ompF and ompC genes, which encode outer membrane proteins OmpF and OmpC, respectively. The EnvZ protein is a protein kinase which specifically phosphorylates the OmpR protein. In this study, the results of in vitro transcription experiments revealed that the phosphorylation of the OmpR protein by the EnvZ protein stimulates the transcription of both the ompF and ompC genes.

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.

Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli

The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.

Identification of the DNA-binding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting

Expression of the ompF and ompC genes of Escherichia coli requires the OmpR protein for transcriptional activation. In vivo binding of the OmpR protein to the ompF and ompC promoter regions was observed using an in vivo dimethyl sulfate DNA footprinting technique. Two different sequence motifs were found to be protected by OmpR in both the ompF and ompC promoter regions. This technique was further used to localize the DNA-binding domain of OmpR to be within the C-terminal 117 amino acid residues. Binding of the C-terminal portion OmpR to the ompF and ompC promoter regions, however, did not result in activation of transcription. Our results, together with sequence homologies between OmpR and other regulatory proteins, suggests that OmpR has separable domain structures: the C-terminal portion for binding-specific DNA sequences and the N-terminal portion for interacting with RNA polymerase and/or other transcription factors.

Molecular Analysis by Deletion and Site-Directed Mutagenesis of the cis-Acting Upstream Sequence Involved in Activation of the ompF Promoter in Escherichia coli1

Expression of the ompF gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the ompF promoter. We previously demonstrated that this cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix. To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the ompF promoter were examined after they had been connected to the lacZ gene. The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the ompF promoter. It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the ompF expression. A local structural alteration in the ompF promoter was observed in some of the base-substitution mutants. Based on the results, the structure and function of the upstream sequence of the ompF promoter are discussed in relation to activation of the ompF promoter by the OmpR protein.