Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products.

OmpF and OmpC are major outer membrane proteins. Although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination. In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes. Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel. On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified. Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame. Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component. The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity. Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed.     

Use of a series of ompF-ompC chimeric proteins for locating antigenic determinants recognized by monoclonal antibodies against the ompC and ompF proteins of the Escherichia coli outer membrane

A method is presented for the efficient location of antigenic determinants using a series of chimeric proteins. By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S. (1985) J. Bacteriol. 164, 797-801, and this work). The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein. Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immunoblot analysis. The antigenic determinants recognized by three anti-OmpC antibodies and one anti-OmpF antibody were thus located. Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies. The peptides reacted with the corresponding antibodies when the former were chemically coupled with bovine serum albumin. Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised. But they did not react with the trimer, the native form. These results are discussed in relation to the structures and functions of the OmpC and OmpF proteins. The use of a series of monoclonal antibodies for studying the mechanism of protein translocation across the cytoplasmic membrane is also discussed.     

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.     

Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium. This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene. Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins. Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene. In some combinations, negative complementation was observed. For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes. Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein. These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.     

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.     

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV‐gp41 epitope on the surface loops

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657–664. © 2016 Wiley Periodicals, Inc.     

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.     

Regulatory mutations conferring constitutive synthesis of major outer membrane proteins (OmpC and OmpF) in Escherichia coli.

An ompB strain of Escherichia coli K-12 lacking major outer membrane proteins OmpC and OmpF was used to isolate a pair of mutants that have restored the ability to synthesize either OmpC or OmpF protein. These mutants were found to produce the respective proteins constitutively under the several conditions where the synthesis in the wild-type strain was markedly repressed; namely, in the absence of the ompB gene function, under restrictive medium conditions, or upon lysogenization with phage PA-2. The mutations ompCp1 and ompFp9 responsible for such synthesis were shown to be located in the close vicinity of the corresponding structural genes, ompC and ompF. Moreover, the mutations affect the expression of these genes in a cis-dominant fashion. Taken together with other evidence, it was suggested that ompCp1 and ompFp9 represent regulatory site mutations occurring at the promoter regions of ompC and ompF respectively. Relevance of these findings to the genetic control of outer membrane protein synthesis is discussed.     

Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.     

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes

Summary To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.     

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein. The mutant was viable but possessed much-reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.     

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation and to compare it with those of the constitutive pore proteins OmpF and OmpC. Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all. By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of Pi through the outer membrane, such as a pore protein deficient strain behaves as a Km mutant. Comparison of the rates of permeation of Pi, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of Pi and phosphorus-containing compounds. The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate. Possible reasons for the preference for Pi and Pi-containing solutes are discussed.     

Host range mutants of bacteriophage Ox2 can use two different outer membrane proteins of Escherichia coli K-12 as receptors.

The Escherichia coli K-12 outer membrane protein OmpA functions as the receptor for bacteriophage Ox2. We isolated a host range mutant of this phage which was able to grow on an Ox2-resistant ompA mutant producing an altered OmpA protein. From this mutant, Ox2h5, a second-step host range mutant was recovered which formed turbid plaques on a strain completely lacking the OmpA protein. From one of these mutants, Ox2h10, a third-step host range mutant, Ox2h12, was isolated which formed clear plaques on a strain missing the OmpA protein. Ox2h10 and Ox2h12 apparently were able to use both outer membrane proteins OmpA and OmpC as receptors. Whereas there two proteins are very different with respect to primary structures and functions, the OmpC protein is very closely related to another outer membrane protein, OmpF, which was not recognized by Ox2h10 or Ox2h12. An examination of the OmpC amino acid sequence, in the regions where it differs from that of OmpF, revealed that one region shares considerable homology with a region of the OmpA protein which most likely is required for phage Ox2 receptor activity.     

A comparative analysis of the spatial structure of nonspecific porins from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis> using optical spectroscopy and molecular modeling

The spatial organization of outer-membrane porins is studied by optical spectroscopy and molecular modeling. It was found that the OmpF and OmpC porins from Yеrsiniа ruckeri are β-structured membrane proteins typical of the pore-forming proteins of other Gram-negative bacteria. The spatial structures of monomers and trimers of the OmpC and OmpF porins from Y. ruckeri are simulated using methods of structural bioinformatics. It was found that the structural stability of the more thermostable OmpF trimer is sustained by a greater number of hydrogen bonds and hydrophobic interactions. The main differences of the spatial structures of the test porins are observed in the structure of their outer loops. There are three tryptophan residues in the molecules of the OmpC and OmpF porins of Y. ruckeri. It is demonstrated by moleculardynamics methods that after thermal denaturation the solvent accessibility of the Trp212 residue in OmpF porin increased by two times, while the solvent accessibility of a Trp184 residue in OmpC porin was not increased. It is hypothesized that the red-shifted tryptophan fluorescence spectrum of OmpF porin during thermal denaturation is due to the behavior of the Trp212 residue.     

Outer Membrane Protein PhoE Takes the Place of OmpC/OmpF in Maintenance of the Surface Structure of Escherichia coli Cells

OmpC and OmpF, outer membrane porin proteins, are important in the maintenance of the cell surface structure of Escherichia coli cells [T. Nogami and S. Mizushima, J. Bacteriol., 156, 402 (1983)]. Mutants lacking both proteins are unstable and frequently revert or mutate to strains which either have regained one or both of the proteins or constitutively produce PhoE, another porin protein. In the present work, the structural importance of PhoE was studied in relation to OmpC. and OmpF. The strain devoid of both OmpC and OmpF was highly susceptible to Tris-HCl buffer at a concentration of 120 mm in terms of viability and cell structure. This strain was also susceptible to osmotic shock. In contrast, the strain possessing PhoE in place of OmpC/OmpF was as stable as the strain possessing OmpC/OmpF against these treatments. PhoE, like OmpC and OmpF, was assembled into a hexagonal lattice with lipopolysaccharide that covered the peptidoglycan sacculus. These results suggest that PhoE can take the place of OmpC/OmpF in the maintenance of the cell surface structure. The importance of porins in general in the maintenance of the cell structure is discussed.     

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.