ompR Gene Research Articles

Characterization by deletion and localized mutagenesis in vitro of the promoter region of the Escherichia coli ompC gene and importance of the upstream DNA domain in positive regulation by the OmpR protein.

The ompC gene codes for a major outer membrane protein whose expression is regulated by the ompR and envZ genes. Two sets of promoter deletion mutants, with upstream and downstream deletions, were constructed on a plasmid in vitro, and their promoter activity was studied by connecting them with the lacZ gene. The DNA sequence for the ompC promoter, including the -35 and -10 regions and the mRNA start site, was defined at the region about 100 base pairs upstream from the ATG initiation codon for the pro-OmpC protein. An additional 61-base-pair sequence extending upstream from the -35 region was required for the ompC promoter to function fully. After targeting the upstream region of the ompC promoter fused to the lacZ gene on a plasmid, in vitro-localized mutagenesis was performed to isolate cis-dominant mutations that affect ompC transcription. Four mutant groups, each of which had common phenotypes for expression and regulation of the gene, were identified. The individual groups also had common base substitutions. In two of the groups, the common base substitutions were localized in the upstream region of the ompC promoter, whereas in the other two they were localized in the -35 region. From these results, the upstream region of the ompC promoter was considered to be the domain responsible for activation by the ompR gene product.

Growth phase and ompR regulation of transcription of microcin B17 genes.

The synthesis of the peptide antibiotic microcin B17 was shown to occur as the cells entered the stationary phase of growth. This type of growth phase regulation is commonly observed in the production of a number of different bacterial products such as toxins and antibiotics. Microcin B17 synthesis is also dependent on the product of the ompR gene. To determine the role of transcription in this double regulation of microcin B17 production, operon fusions with Mu d1 (Ap lac) were constructed. Insertions were obtained in all four plasmid genes involved in production of microcin B17 (mcbA-D) and in the immunity region. Three classes of fusions were obtained. Fusions into mcbA, mcbB, and mcbC (first class) exhibited an increase in their transcription as the cells approached the stationary phase. These increases as well as basal levels of transcription were dependent on OmpR. Expression of fusions in mcbD and in the immunity region (second class) was also dependent on OmpR, but their expression remained constant throughout growth. One fusion in mcbC (third class) was obtained which was transcribed in the opposite direction than the others. It showed no growth phase regulation and no OmpR dependence. The implications of these results in terms of the transcriptional organization of the mbc genes are discussed.

Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters.

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12.

The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant backgrounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC- phenotype in an ompR+ background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules and thereby support the idea that OmpR can exist as a multimeric protein.

Identification, Mapping, Cloning and Characterization of a Gene (sbmA) Required for Microcin B17 Action on Escherichia coli K12

We have identified mutations in three different chromosomal genes of Escherichia coli K12 which reduce sensitivity to microcin B17. Mutations in ompF and ompR genes affected production of an outer membrane porin protein, OmpF, and resulted in reduced sensitivity to a number of other agents (colicins, bacteriophages) besides microcin B17. The third class of mutants were specifically and highly resistant to microcin B17. The mutations in these strains were mapped to a gene (sbmA), located at 8.7 min on the E. coli K12 chromosome, which is closely linked to phoA. The wild-type sbmA allele was cloned into multiple copy number plasmids, and its location within the cloned DNA fragment was further defined by mutagenesis with MiniMudII1681. These insertion mutations resulted in in-frame fusions between the sbmA and lacZ genes, thereby allowing us to determine the direction of sbmA gene transcription. Plasmids carrying these gene fusions produced low levels of beta-galactosidase, indicating that the sbmA gene is poorly expressed. We have been unable to identify the sbmA gene product, but indirect evidence indicates that it might be an envelope protein involved in microcin uptake.

Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes coding for major outer membrane proteins is osmoregulated by solutes, such as sucrose and NaCl, in the growth medium. The OmpR protein, a positive regulator of these genes, is involved in the osmoregulation (Dairi et al. 1985; Nara et al. 1984). In the present work, five mutant ompR genes exhibiting different phenotypes of osmoregulation were cloned and sequenced. Three of them, ompR1, ompR2 and ompR20, were previously isolated mutants. The others, ompR3 and ompR4, were isolated in the present work. The ompR1 mutation resulted in the deletion of 19 amino acids near the C-terminus of the OmpR protein. The ompR3 and ompR4 mutations resulted in Arg15 to Cys and Arg71 to Thr conversions, respectively, at the N-terminal portion, whereas the ompR20 and ompR2 mutations resulted in Arg150 to Cys and Val207 to Met conversions, respectively, at the C-terminal portion. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.

Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system.

Previous evidence has indicated that a gene, proU, is involved in the response of bacterial cells to growth at high osmolarity. Using Mu-mediated lacZ operon fusions we found that transcription of the proU gene of Salmonella typhimurium is stimulated over 100-fold in response to increases in external osmolarity. Our evidence suggests that changes in turgor pressure are responsible for these alterations in gene expression. Expression of proU is independent of the ompR gene, known to be involved in osmoregulation of porin expression. Thus, there must be at least two distinct mechanisms by which external osmolarity can influence gene expression. We show that there are relatively few genes in the cell which are under such osmotic control. The proU gene is shown to encode a high-affinity transport system (Km = 1.3 microM) for the osmoprotectant betaine, which is accumulated to high concentrations in response to osmotic stress. Even when fully induced, this transport system is only able to function in medium of high osmolarity. Thus, betaine transport is regulated by osmotic pressure at two levels: the induction of expression and by modulation of activity of the transport proteins. We have previously shown that the proP gene encodes a lower-affinity betaine transport system (J. Cairney, I. R. Booth, and C. F. Higgins, J. Bacteriol., 164:1218-1223, 1985). In proP proU strains, no saturable betaine uptake could be detected although there was a low-level nonsaturable component at high substrate concentrations. Thus, S. typhimurium has two genetically distinct pathways for betaine uptake, a constitutive low-affinity system (proP) and an osmotically induced high-affinity system (proU).

Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins.

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.

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.

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.

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.

Selection of lac gene fusions in vivo: ompR-lacZ fusions that define a functional domain of the ompR gene product.

We describe a simple method for selecting Escherichia coli mutants that carry gene fusions between a cloned gene and lacZ. We test this technique with the ompR gene, which codes for a positive regulatory factor in porin synthesis. A number of OmpR-LacZ hybrid proteins are examined, and several unusual phenotypes associated with these protein fusions are described. Evidence is presented to support the two-domain model for ompR proposed previously (Hall and Silhavy, J. Mol. Biol. 151:1-15). In addition, one of the ompR-lacZ fusions exhibits a dominant OmpR- phenotype. The utility of isolating a series of lacZ gene fusions to any target gene is discussed.

Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.

Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2. II. Identification of the envZ gene product, a protein involved in the expression of the porin proteins.

We have isolated the envZ gene from a recombinant plasmid carrying the complete ompB region of Salmonella typhimurium (Liljeström et al. 1982). The gene was found to be situated very near the ompR gene in the ompB locus. Using minicells, the envZ gene product was identified as a 46 kd protein. Strains harboring either an ompR+ plasmid or an envZ+ ompR+ plasmid were found to differ in their response to environmental changes with respect to porin protein expression. The results obtained suggest that the EnvZ protein, together with ompR, ultimately determines the level of expression of the porin proteins.

Osmoregulation of gene expression. I. DNA sequence of the ompR gene of the ompB operon of Escherichia coli and characterization of its gene product.

The ompB region on the Escherichia coli chromosome codes for two genes, ompR and envZ, which are required for the osmolarity sensitive biosynthetic regulation of the outer membrane matrix proteins (porins), OmpF and ompC. A part of the ompB region containing the ompR gene has been cloned (Wurtzel, E. T., Movva, N. R., Ross, F. L., and Inouye, M. (1981) J. Mol. Appl. Genet. 1, 61-69). We have determined the DNA sequence, including the promoter and structural regions encompassed in a 1.3-kilobase pair Ava I-Eco RI subfragment. This fragment codes for the entire ompR gene as well as the 5' end of the envZ gene. The ompR gene codes for a protein of 32,489 daltons, consisting of 284 amino acid residues. This was confirmed by identifying the gene product by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determining a partial amino acid sequence of the NH2-terminal region of the gene product. A sequence of 57 amino acid residues located in the COOH-terminal region of the protein is extremely basic. It contains 10 arginine plus lysine residues in contrast to 1 glutamic acid residue in this region. In vitro transcription of the DNA from this region indicates that ampR and envZ are co-transcribed as a polycistronic mRNA from a promoter located 5' to the ompR gene. Translation of the am pR gene terminates at two tandem TAS codons and translation of the envZ gene initiates 29 nucleotides downstream. Cloning of the promoter region of ompB at a site 5' to the structural portion of the beta-galactosidase gene indicates that transcription of ompB is under positive control by cAMP.

Cloning of the regulatory genes (ompR and envZ) for the matrix proteins of the Escherichia coli outer membrane.

We have cloned the regulatory gene cluster of Escherichia coli which is composed of at least two distinct genes, ompR and envZ. These genes are known to regulate the production of the outer membrane matrix proteins. The newly formed plasmids were found to complement not only ompR mutations but also envZ mutations. The ompR gene product was identified as a protein of an apparent molecular weight of 28,500.

Genetic analysis of the ompB locus in Escherichia coli K-12

Fine-structure mapping and complementation analysis of the ompB locus indicate that this locus is comprised of two genes, envZ and ompR. These genes are ordered on the chromosome as follows: aroB-envZ-ompR-asd. A model is proposed for the ompB-mediated regulation of the major outer membrane porin genes, ompF and ompC. According to this model, the ompR gene product is a bifunctional regulatory protein. The envZ gene product is an envelope protein that senses the cell's external environment.

Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12

We report here on the cloning of a gene located within the ompB locus of E. coli K-12. This gene, designated ompR, resides on a 10.9-kilobase EcoRI fragment that we cloned, using a lambda vector and in vitro packaging techniques. By subcloning portions of this fragment into the high-copy-number plasmid pBR322, we have isolated the ompR gene on a 1.4-kilobase EcoRI-AvaI fragment. This fragment has been characterized physically and will facilitate a more detailed study of the role and mechanism of porin regulation by the ompB locus.