Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media

Fusions to the beta-lactamase (bla) gene were employed to analyze the presence of localization information in the mature part of OmpC, a major pore-forming outer membrane protein in Escherichia coli K-12. Six translational ompC-bla gene fusions were constructed, the shortest of them containing only part of the ompC signal sequence and the largest approximately 90% of the sequence encoding mature OmpC protein. Export of the hybrid proteins to a non-cytoplasmic location was a prerequisite for ampicillin resistance. Localization of the hybrid proteins by cell fractionation and solid phase iodination of whole cells suggested that the exported hybrid proteins possibly interacted with the outer membrane in vivo. No specific sequence of the mature OmpC protein, however, was found to promote this interaction.     

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.     

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.     

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.     

Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector.

Summary The isolation and properties of a hybrid plasmid carrying the Y gene of the lac operon of Escherichia coli are described. The lactose carrier protein, coded for by the Y gene, is readily identified upon lac operon induction in strains carrying the plasmid. The protein comprises about 15% of the cytoplasmic membrane protein synthesized in the first generation after induction, compared with a wild type strain induced under the same conditions where lactose carrier protein comprises 1.4% of the cytoplasmic membrane protein.     

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.     

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.     

New pore protein produced in cells lysogenic for Escherichia coli phage HK253hrk

Outer membrane pore protein OmpC was identified as the receptor for the temperate Escherichia coli phage HK253hrk. The part of OmpC protein recognized by the phage was identified by using hybrid proteins in which parts of OmpC protein are replaced by the corresponding parts of the related PhoE protein. In contrast to other OmpC-specific phages, HK253hrk recognizes a part of OmpC within the C-terminal 50 amino acids of the protein. E. coli strains lysogenic for HK253hrk produce reduced amounts of OmpC protein, and produce a new pore protein instead. Expression of this new protein was temperature-dependent, i.e. low at 30 degrees C. The functioning of this new pore protein was characterized both in vivo by studying the uptake of beta-lactam antibodies and in vitro after reconstitution of the protein in black lipid films. Its effective pore size was larger than that of the OmpF pores of E. coli B. The new porin appears to be cation-selective. A comparison with the selectivity of the known OmpC and OmpF pores of E. coli showed that the new pore has a higher selectivity than OmpF but is less selective than OmpC. The new pore protein appears to function in E. coli K12 lysogens as the receptor for the phages HK187, HK189 and HK332.     

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.     

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.     

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

Summary OmpC and OmpF are major outer membrane proteins and although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine their differences, we have constructed a series of ompC-ompF chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The recombination sites in the chimeric genes were localized by means of restriction endonuclease analysis and nucleotide sequence determination. Most of the chimeric gene products were accumulated in the outer membrane. One of the chimeric gene products, with a fusion site in a central region between the OmpC and OmpF proteins, was normally expressed but not accumulated in the outer membrane. The trimeric structures of some of the chimeric gene products appeared to be extremely unstable in a SDS solution. From these results, domains contributing to the formation of specific structures in which the OmpC and OmpF proteins differ were identified. Bacterial cells possessing the chimeric gene products were also investigated as to their sensitivity to phages that require either OmpC or OmpF as a receptor component. With the aid of the chimeric gene products, the immunogenic determinants for three anti-OmpC monoclonal antibodies were found to be localized at different portions of the OmpC polypeptide: the N-terminal, central and C-terminal portions, respectively.     

Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?

An E. coli strain carrying a fusion of the malE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of β-galactosidase, by addition of maltose to cells. The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the β-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane. Thus the protein becomes stuck to the cytoplasmic membrane. Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences. This effect is observed within 15 min after high levels of induction are achieved. The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins. These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme. If this interpretation is correct, we can estimate that an E. coli cell has roughly 2 × 10⁴ such sites in the cytoplasmic membrane. A system is described for detecting the precursor of any exported protein.     

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 P_i through the outer membrane, such that a pore protein deficient strain behaves as a K_m mutant. Comparison of the rates of permeation of P_i, 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 P_i 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 P_i and P_i-containing solutes are discussed.     

Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.     

Transmembrane signal transduction and osmoregulation in Escherichia coli. Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ

The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation. In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions. It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane. Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity. It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected. These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus.     

Translational control of exported proteins that results from OmpC porin overexpression.

The regulation of synthesis and export of outer membrane proteins of Escherichia coli was examined by overexpressing ompC in multicopy either from its own promoter or from an inducible promoter in an expression vector. Overexpression of OmpC protein resulted in a nearly complete inhibition of synthesis of the OmpA and LamB outer membrane proteins but had no effect on synthesis of the periplasmic maltose-binding protein. Immunoprecipitation of labeled proteins showed no evidence of accumulation of uncleaved precursor forms of OmpA or maltose-binding protein following induction of OmpC overexpression. The inhibition of OmpA and LamB was tightly coupled to OmpC overexpression and occurred very rapidly, reaching a high level within 2 min after induction. OmpC overexpression did not cause a significant decrease in expression of a LamB-LacZ hybrid protein produced from a lamB-lacZ fusion in which the fusion joint was at the second amino acid of the LamB signal sequence. There was no significant decrease in rate of synthesis of ompA mRNA as measured by filter hybridization of pulse-labeled RNA. These results indicate that the inhibition is at the level of translation. We propose that cells are able to monitor expression of exported proteins by sensing occupancy of some limiting component in the export machinery and use this to regulate translation of these proteins.     

Membrane-associated assembly of a phage T4 DNA entrance vertex structure studied with expression vectors

The DNA entrance vertex of the phage head is critical for prohead assembly and DNA packaging. A single structural protein comprises this dodecameric ring substructure of the prohead. Assembly of the phage T4 prohead occurs on the cytoplasmic membrane through a specific attachment at or near the gp20 DNA entrance vertex. An auxiliary head assembly gene product, gp40, was hypothesized to be involved in assembling the gp20 substructure. T4 genes 20, 40 and 20 + 40 were cloned into expression vectors under lambda pL promoter control. The corresponding T4 gene products were synthesized in high yield and were active as judged by their ability to complement the corresponding infecting T4 mutants in vivo. The cloned T4 gene 20 and gene 40 products were inserted into the cytoplasmic membrane as integral membrane proteins; however, gp20 was inserted into the membrane only when gp40 was also synthesized, whereas gp40 was inserted in the presence or absence of gp20. The gp20 insertion required a membrane potential, was not dependent upon the Escherichia coli groE gene, and assumed a defined membrane-spanning conformation, as judged by specific protease fragments protected by the membrane. The inserted gp20 structure could be probed by antibody binding and protein A-gold immunoelectron microscopy. The data suggest that a specific gp20-gp40-membrane insertion structure constitutes the T4 prohead assembly initiation complex.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

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.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.