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 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.     

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: 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.