ompF Promoter Research Articles

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.

Altered osmoregulation of ompF in integration host factor mutants of Escherichia coli.

Osmoregulation of the porin protein OmpF was strongly altered in integration host factor (IHF) mutants. These mutants produced approximately 15-fold more OmpF than did the parent strain when grown in media of intermediate osmolarity. At high osmolarity IHF mutants continued to produce considerable amounts of OmpF, although this protein was undetectable in the parent grown under these conditions. Experiments with an ompF-lacZ chromosomal fusion strain suggested that these changes in osmoregulation in large part involve alterations in transcriptional activity of the ompF promoter. These results add to the growing list of genes whose expression is modified in IHF mutants.

Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid.

We constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, beta-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.

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.

Static bend of DNA helix at the activator recognition site of the ompF promoter in Escherichia coli

Expression of the ompF gene coding for a major outer membrane protein of Escherichia coli is regulated by osmolarity of the medium. The ompF expression is controlled by a transcriptional activation mechanism which requires the ompR gene product acting on a region located upstream from the canonical −35 and −10 regions of the ompF gene. Evidence is presented that the upstream region of the ompF promoter contains a static bend of DNA having two sets of oligo (dA · dT) tracts with periodical spacing. It was demonstrated that the bending region overlaps with the recognition site for the activator protein, OmpR.

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.

Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli.

A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.

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.

Uncoupling of osmoregulation of the Escherichia coli K-12 ompF gene from ompB-dependent transcription.

The expression of the genes for the Escherichia coli K-12 outer membrane proteins, ompF and ompC, is subject to osmoregulation and responds to changes in the osmolarity of the growth medium. The transcription of these genes is dependent on the products of the regulatory locus ompB (comprising the genes ompR and envZ). The native promoter of ompF was replaced with an inducible lpp promoter to eliminate this transcriptional dependence of ompF expression on ompB. As a result, it was possible for the OmpF protein to be produced in an ompB mutant strain that does not normally express ompF. Surprisingly, the expression of ompF under the lpp promoter was still osmoregulated not only in the ompB+ strain but also in two ompB strains tested. These results indicate the involvement of a factor(s) besides the ompR and envZ gene products in the osmoregulation of OmpF production. This factor may interact with a sequence downstream of the ompF promoter. In addition, we show that the expression of ompF under the lpp promoter has no direct effect on ompC expression.

Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli

The ompF gene codes for a major outer membrane protein whose expression is positively regulated by the ompR and envZ genes. Two sets of promoter deletions, upstream deletions and downstream deletions, were generated in vitro, and the promoter function was studied by connecting them with the tet genes. One of the hybrid genes thus constructed had a functioning ompF-tet hybrid promoter. The 107 base-pair fragment was found to be functioning as the ompF promoter, 90 nucleotides upstream and 17 nucleotides downstream of the mRNA start site that was also determined in this study. The start site was preceded by a convenient Pribnow box. Although the sequence at the −35 region had a low degree of homology to the consensus sequence, analyses of the hybrid promoter suggested that this region is involved in the promoter function in relation to the Pribnow box. They also indicated that the domain responsible for regulation by the ompR gene is located within the −35 region and its upstream region.

Mechanism of localization of major outer membrane lipoprotein in Escherichia coli. Studies with the OmpF-lipoprotein hybrid protein.

A chimera gene consisting of the ompF promoter, the coding regions for the signal peptide and the NH2-terminal 11 amino acid residues of outer membrane OmpF protein, and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal 7 amino acid residues was constructed. Escherichia coli carrying the cloned chimera gene produced a hybrid protein with the predicted chemical structure. The protein was localized in the periplasmic space with an interaction with the peptidoglycan layer. These results indicate that the hybrid protein was expressed, secreted across the cytoplasmic membrane, and processed for the signal peptide normally. The hybrid protein, however, was not incorporated into the outer membrane, suggesting the importance of the lipid domain in the assembly of the lipoprotein into the outer membrane. Although a larger part of the protein was extractable with sodium dodecyl sulfate, a part of the hybrid protein was covalently bound to the peptidoglycan layer as the lipoprotein is. Upon treatment with lysozyme of the envelope the hybrid protein became water soluble. The solubilized protein most probably existed as a trimer. These results most likely suggest that the major lipoprotein exists as a trimer in the periplasmic space with interactions with the peptidoglycan layer through the protein domain on one side and with the outer membrane through the lipid domain on the other side.

Regulated OmpF porin synthesis in Escherichia coli K-12

The effects of growth conditions on the expression of the Escherichia coli K-12 major outer membrane porin gene ompF were determined by measuring the amount of OmpF protein incorporated into the outer membrane, β-galactosidase activity in cells in which lacZ was regulated by the ompF promoter, and by studying their effects in the presence of the general transcription inhibitor rifampicin. A variety of conditions reduced ompF transcription. Common features among the inhibitors of ompF expression could not be identified; another protease inhibitor and another uncoupler did not specifically affect ompF expression. It is therefore concluded that the inhibitors interact directly or indirectly with a sensory system (possibly EnvZ protein) which affects the porin gene regulon accordingly.

Regulated OmpF porin synthesis inEscherichia coliK-12

The effects of growth conditions on the expression of the Escherichia coli K-12 major outer membrane porin gene ompF were determined by measuring the amount of OmpF protein incorporated into the outer membrane, β-galactosidase activity in cells in which lacZ was regulated by the ompF promoter, and by studying their effects in the presence of the general transcription inhibitor rifampicin. A variety of conditions reduced ompF transcription. Common features among the inhibitors of ompF expression could not be identified; another protease inhibitor and another uncoupler did not specifically affect ompF expression. It is therefore concluded that the inhibitors interact directly or indirectly with a sensory system (possibly EnvZ protein) which affects the porin gene regulon accordingly.