Outer Membrane Pore Protein Research Articles

Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE.

The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene : Identification of an element, upstream from the promoter, required for efficient expression of phoE protein

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphatecontrolled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the −35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions −106 and −121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular −35 and −10 regions. A proper spacing between this upstream region and the −35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.

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

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.

Effects of linker insertions on the biogenesis and functioning of the Escherichia coli outer membrane pore protein PhoE.

To study the effects of small insertions on the biogenesis and functioning of outer membrane pore protein PhoE of Escherichia coli K12, oligonucleotides were inserted at five different sites in the phoE gene. The proteins encoded by the mutant alleles all appeared to be incorporated into the outer membrane since cells producing these proteins bound either phages specific for the PhoE protein or monoclonal antibodies or both. However, one mutant protein was apparently not incorporated normally since expression of this protein was lethal and the protein did not function as a pore. Amino acid residues 75 and 280 of the PhoE protein appear to be exposed on the cell surface because insertions at these sites interfere with the binding of PhoE-specific phages or monoclonal antibodies to whole cells, respectively.

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.

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage λ1059, using the Escherichia coli ompCgene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.

Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies.

Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.

Cross-linking analysis of yeast mitochondrial outer membrane

By enrichment of contact sites between the two mitochondrial boundary membranes it has been shown that this fraction contained a high activity of glutathione transferase and hexokinase which was bound to the outer membrane pore protein (Ohlendieck, K. et al. (1986) Biochim. Biophys. Acta 860, 672–689). Therefore, an interaction between the three proteins in the contact sites has been suggested. Cross-linking experiments with isolated outer membrane of yeast mitochondria show that glutathione transferase and the pore protein are already associated in the free outer membrane. Porin appeared to adopt four different oligomeric complexes in the membrane, including interactions with a 14 kDa polypeptide, which has glutathione transferase activity. The latter polypeptide could be phosphorylated by intrinsic or extrinsic protein kinases, while the porin itself was not phosphorylated. Yeast hexokinase, when bound to the outer membrane, was able to cross-link to the pore protein.

O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae

The accessibility of outer membrane pore protein PhoE to antibody molecules at the cell surface of intact cells of various Enterobacteriaceae was investigated. Significant antibody binding was observed for only two of the nine strains tested. Analysis of the lipopolysaccharide by sodium dodecyl sulphate gel electrophoresis revealed a clear correlation between the presence of an O-antigenic side chain and the inability to bind PhoE protein-specific antibodies. As mutants that lack the O-antigen chain appeared to have acquired the ability to bind antibody, it must be concluded that the presence of O-antigenic chains of lipopolysaccharide prevents binding of antibodies to PhoE protein at the surface of intact cells. The relevance of this conclusion for the potential use of enterobacterial outer membrane pore proteins as vaccine components is 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.

Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane.

Six monoclonal antibodies directed against the trimeric form of outer-membrane pore protein PhoE of Escherichia coli K-12 were isolated and characterized. All six antibodies bind to PhoE protein in intact cells and as isolated trimers, but not to dodecyl-sulphate-denatured monomers. Cross-reaction with the related pore proteins OmpF protein and OmpC protein was not observed. A hybrid pore protein in which the amino-terminal 74 amino acids of PhoE protein have been replaced by the corresponding part of OmpF protein is able to bind the six monoclonal antibodies. Five of the antibodies bind to the PhoE proteins of thirteen different Enterobacteriaceae when expressed in E. coli K-12, whereas the other antibody recognizes PhoE protein from nine of these strains. Four monoclonal antibodies are able to block adsorption of the PhoE-protein-specific phage TC45 to its receptor on whole cells. None of the antibodies has any effect on the uptake rate of the antibiotic cefsulodin through PhoE protein pores. Five antibodies are able to direct the complement-mediated killing of PhoE-protein-carrying cells. It is concluded that the six monoclonal antibodies recognize at least three distinct cell-surface-exposed epitopes whose specificity is determined by the carboxy-terminal 256 amino acids of PhoE protein.

Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

In Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The corresponding gene of Enterobacter cloacae was transferred to E. coli K-12 by using RP4 :: mini Mu plasmid pULB 113 and selecting for R-prime plasmids that carry the genes proA and proB, which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by γδ insertions. The E. cloacae phoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coli phoE gene. Functionally, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.

Gene encoding a hybrid OmpF--PhoE pore protein in the outer membrane of Escherichia coli K12.

To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2-73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, that colicin N uses OmpF protein as (part of) its receptor, that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.

Immunochemical analysis of the membrane proteins of rat liver and Zajdela hepatoma mitochondria

The contents of mitochondrial inner membrane protein complexes were compared in normal liver and in Zajdela hepatoma mitochondria by the immunotransfer technique. Antibodies against core proteins 1 and 2, cytochrome c 1, the iron-sulfur protein of Complex III, subunits I and II of cytochrome oxidase, and the α and β subunits of the F 1-ATPase were used. In addition, antibodies against a primary dehydrogenase, β-hydroxybutyrate dehydrogenase, as well as the outer membrane pore protein were used. The results indicate that the components of the cytochrome chain and porin are greatly enriched in hepatoma mitochondria compared to normal rat liver mitochondria. This enrichment was also reflected in the rates of respiration in tumor mitochondria using a variety of substrates. Enrichment of porin may partially account for increased hexokinase binding to tumor mitochondria. In contrast to the respiratory chain components, the F 1-ATPase and F 0 (measured by DCCD binding) were not increased in tumor mitochondria. Thus, Zajdela hepatoma mitochondria components are nonstoichiometric, being enriched in oxidative capacity but relatively deficient in ATP synthesizing capacity. Finally, β-hydroxybutyrate dehydrogenase, which is often decreased in hepatoma mitochondria, was shown here by immunological methods to be decreased by only 40%, whereas enzyme activity was less than 5% of that in normal rat liver.

Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene.

The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.

Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12

The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established. The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension. This peptide has the general characteristics of a signal sequence. The promoter region of phoE has no homlogy with the consensus sequence of E. coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed. The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782. A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed. Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212. When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found. Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.

Outer membrane permeation of beta-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae.

Mutant strains lacking outer membrane protein(s) were isolated from Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. The outer membrane protein(s) of P. mirabilis and E. cloacae corresponding to E. coli porin were identified on the basis of their function, namely, their ability to allow the permeation of glucose as demonstrated by [14C]glucose uptake by intact cells. P. mirabilis has only one outer membrane pore protein (molecular weight, 40,000), but E. cloacae has at least two such proteins (molecular weights, 37,000 and 39,000 to 40,000). When the bacteria lost these proteins or porin, the outer membrane permeation of cefazolin was found to be greatly reduced in these three species. Such a change in the outer membrane permeation closely correlated with a significant decrease in the bacterial susceptibility to cephalosporins, including cefoxitin. These results suggested that the main pathway for cephalosporin permeation is the pore made up of these proteins. The 39,000- to 40,000-molecular-weight protein in E. cloacae was also assumed to play an important role in the outer membrane permeation of tetracycline and chloramphenicol. On the other hand, the permeation route of penicillins was obscure. The susceptibility to penicillins, except in some cases, was little influenced by the absence of the proteins. Ampicillin was found to pass through the outer membrane via the same route as the cephalosporins, but the possibility that ampicillin and other penicillins possess another unknown route for outer membrane permeation could not be ruled out.

Cloning of ompF, the structural gene for an outer membrane pore protein of E. coli K12: Physical localization and homology with the phoE gene

The ompF gene of Escherichia coli K12, which is the structural gene for an outer membrane pore protein has been cloned into the multicopy plasmid vector pACYC184. Expression of the cloned ompF gene results in overproduction of the OmpF protein and in strongly decreased production of the other two pore proteins, OmpC protein and PhoE protein, whereas production of OmpA protein is much less affected. Expression of the cloned ompF gene is still dependent on the ompB+ gene product, but is hardly influenced by the osmolarity of the growth medium. The gene was localized on the hybrid plasmids by the analysis of in vitro constructed deletion plasmids and mutant plasmids generated by Tn5 insertions. Heteroduplex analysis revealed an extensive DNA homology of the ompF gene with the phoE gene. Since the direction of transcription of the phoE gene is known, the direction of transcription of the ompF gene could be deduced.

Cloning of phoE, the structural gene for the Escherichia coli phosphate limitation-inducible outer membrane pore protein.

The hybrid plasmid pLC44-11 from the Clarke and Carbon collection, which was known to carry the proA gene, was shown also to contain the phoE gene. In vitro recombination techniques were used to subclone a 4.9-kilobase-pair DNA fragment of pLC44-11 into the plasmid vectors pACYC184 and pBR322. Expression of this fragment in a minicell system showed that it codes for the PhoE protein and for polypeptides with apparent molecular weights of 47,000 and 17,000. These results supply definite proof for the earlier supposition that the phoE gene is the structural gene for the outer membrane PhoE protein. Overproduction of the PhoE protein in a phoS strain resulted in reduced amounts of OmpF and LamB proteins.

In vivo effects of local anesthetics on the production of major outer membrane proteins by Escherichia coli

Synthesis of a major outer membrane pore protein (the OmpF protein) by Escherichia coli K-12 was specifically and reversibly inhibited by low doses of procaine and other local anesthetics. The treated cells maintained the same total number of pores in their outer membrane by increased synthesis of the OmpC pore protein. Procaine also inhibited synthesis of the OmpF protein by Salmonella typhimurium and by E. coli B, although in the latter case, some OmpF protein was still detected in the outer membrane of treated cells. Experiments in which transcription was blocked by pretreatment with rifampicin indicated that procaine did not inhibit translation of the stable OmpF mRNA and that there was no pool of preformed OmpF mRNA in cells grown in the presence of procaine. Procaine did not affect biosynthesis of the lipopolysaccharide core and did not inhibit the association of OmpF protein with the peptidoglycan. These results are discussed in terms of the known effects of procaine on membrane molecular packaging.