Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli

The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompF and ompC genes, which respectively code for major outer membrane proteins OmpF and OmpC of Escherichia coli. The OmpR protein has been purified to homogeneity from an overproducing strain harboring an ompR gene-carrying plasmid. Throughout the purification the OmpR protein behaved as a single entity. The molecular weight determined on sodium dodecyl sulfate-polyacrylamide gel, the total amino acid composition, and the NH2-terminal amino acid sequence of the purified protein were essentially the same as those deduced from the nucleotide sequence of the ompR gene. Molecular weight determination and cross-linking study on the native protein revealed that the purified protein exists as a monomer. The purified OmpR protein was specifically bound to the promoter regions of the ompC and ompF genes. Experiments with a series of upstream deletions of the ompC and ompF promoters revealed that the region upstream from the -35 region was indispensable for OmpR binding to both the ompC and the ompF promoters. Although it has been proposed that depending on the medium osmolarity the OmpR protein may exist in two alternative structures, which respectively regulate functioning of the ompC and the ompF promoters, the purified OmpR protein appeared to be homogeneous and interacted with both promoters to the same extent.     

Interaction of OmpR, a positive regulator, with the osmoregulated ompC and ompF genes of Escherichia coli. Studies with wild-type and mutant OmpR proteins

The OmpR protein is a positive regulator involved in osmoregulatory expression of the ompC and ompF genes that specify the major outer membrane proteins OmpC and OmpF, respectively. We purified the OmpR protein not only from wild-type cells but also from two ompR mutants (ompR2 and ompR3) exhibiting quite different phenotypes as to osmoregulation of the ompC and ompF genes. The OmpR2 protein has an amino acid conversion in the C-terminal portion of the OmpR polypeptide, whereas the OmpR3 protein has one in the N-terminal portion. Comparative studies on these purified OmpR proteins were carried out in terms of their interaction with the ompC and ompF promoters. The nucleotide sequences involved in OmpR-binding were determined in individual promoter regions by deoxyribonuclease I footprinting. The OmpR3 protein as well as the wild-type OmpR protein appeared to bind, to similar extents, to both the ompC and ompF promoters. In contrast, the OmpR2 protein bound preferentially to the ompF promoter and failed to protect the ompC promoter against DNAse I digestion. These results support the view that the C-terminal portion of the OmpR protein is responsible for the binding of the OmpR protein to the ompC and ompF promoter DNAs. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.     

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.     

Evidence for multiple OmpR-binding sites in the upstream activation sequence of the ompC promoter in Escherichia coli: a single OmpR-binding site is capable of activating the promoter.

We present experimental evidence for the existence of multiple activator-binding sites in the upstream sequence of the ompC promoter, the expression of which is activated by the positive regulator OmpR in response to the osmolarity of the medium. We also found that a single OmpR-binding site can activate the ompC promoter, providing that the binding site is close and placed stereospecifically with respect to the canonical-35 and -10 regions.     

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.