Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12.

Expression of the ompF and ompC genes coding for major outer membrane proteins OmpF and OmpC is regulated in opposite directions by medium osmolarity. Chimera genes were constructed by a reciprocal exchange of the promoter-signal sequence region between the two genes. The chimera gene construction was designed so that the proteins synthesized by these genes were essentially the same as the OmpC and OmpF proteins. Studies with the chimera genes demonstrated that the osmoregulation of the OmpF-OmpC synthesis was promoter dependent. They also showed that cells grew normally even when the osmoregulation took place in opposite directions. The effects of the ompR2 and envZ mutations, which suppress ompC and ompF expression, respectively, also became reversed. The reduced expression was still subject to the promoter-controlled osmoregulation. Based on these observations, the mechanism of regulation of the ompF-ompC gene expression and its physiological importance are discussed.     

Positive control of transcription initiation in Escherichia coli. A base substitution at the Pribnow box renders ompF expression independent of a positive regulator

Expression of the ompF gene coding for a major outer membrane protein of Escherichia coli is positively regulated by the product of the ompR gene, OmpR. Using an ompF-tet chimera gene, ompF promoter mutants that render the ompF expression independent of the OmpR protein were isolated. In all of the four mutants that were isolated separately, the first base of the Pribnow box was changed from A to T. The mutant promoter did not require the upstream domain of the -35 region that is required for the OmpR-dependent functioning of the wild-type promoter. It is concluded that the domain upstream from the -35 region plays a role in the positive regulation by the OmpR protein. A statistical survey of the E. coli promoter sequence revealed that almost all of the genes that do not require an activator protein for their expression possess T at the first position of the Pribnow box, while the position is occupied by other bases in almost all of the positively regulated genes. Based on these facts, the mechanism of positive regulation of the gene expression by an activator protein is discussed.     

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.     

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.     

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.     

Expression of micF involved in porin synthesis in Escherichia coli: two distinct cis-acting elements respectively regulate micF expression positively and negatively

micF RNA, whose sequence is highly complementary to a 5'-portion of ompF mRNA, has been implicated in the osmoregulation and thermoregulation of the ompF porin gene in Escherichia coli. To define and characterize cis-acting regulatory regions upstream of the micF promoter, a series of deletions of the micF promoter fused to the lacZ gene were constructed. Two distinct regions, which function differently, were identified as cis-acting regulatory elements, namely, one responsible for OmpR-dependent activation and the other for OmpR-independent repression of micF expression. The former contains the OmpR-binding site, which simultaneously regulates both the genes, micF and ompC, in response to the medium osmolarity. The latter may be involved in an unknown regulatory process of micF expression.     

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.