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.     

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 .     

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.     

Expression of outer membrane proteins in Escherichia coli growing at acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

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.     

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.     

cis-acting sites required for osmoregulation of ompF expression in Escherichia coli K-12.

OmpF and OmpC are major outer membrane proteins which form passive diffusion pores in Escherichia coli K-12. The expression of the structural genes for these proteins, ompF and ompC, is influenced by medium osmotic strength and requires the products of two regulatory genes, ompR and envZ. We have constructed a series of ompF-lacZ fusions containing different regions of ompF to determine sites involved with osmoregulation. These fusions were crossed onto a specialized transducing phage and integrated into the bacterial chromosome in unit copy. By measuring the fluctuations of beta-galactosidase activity in lysogens grown in high versus low osmolarity, we have identified three regions which are necessary. Furthermore, we have determined that, although the OmpR activation site is not sufficient, OmpR is probably essential for ompF osmoregulation.     

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.     

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.