Outer Membrane Proteins of Escherichia coli III. Evidence that the Major Protein of Escherichia coli O111 Outer Membrane Consists of Four Distinct Polypeptide Species

Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.     

A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.     

Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.     

衣原体主要外膜蛋白的研究进展

衣原体感染与多种慢性疾病密切相关,其主要外膜蛋白(MOMP)是一种多功能蛋白,分别与外膜结构的稳定性、生长代谢调节、抗原性和毒力密切相关。随着沙眼衣原体和肺炎衣原体基因组测序的完成,人们得以揭示其重要的生物合成、代谢途径,确定调控机制及其与致病的相关性。利用分子生物学技术在分子水平分析衣原体主要外膜蛋白的结构、抗原表位,对于免疫防御、免疫病理和免疫诊断均有重要意义。本文综述了衣原体主要外膜蛋白的分子结构、基因特性、抗原表位与应用前景。     

Membrane Topology of Outer Membrane Protein AlgE,Which Is Required for Alginate Production in Pseudomonas aeruginosa

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.Pseudomonas aeruginosa is an ubiquitous opportunistic human pathogen responsible for chronic infections of the lungs of patients with cystic fibrosis (CF), in whom it is the leading cause of mortality and morbidity (9). The establishment of a chronic infection in the lungs of patients with CF coincides with the switch of P. aeruginosa to a stable mucoid variant, producing copious amounts of the exopolysaccharide alginate; this is typically a poor prognostic indicator for these patients (24, 31). Alginate is a linear unbranched exopolysaccharide consisting of 1,4-linked monomers of β-d-mannuronic acid and its C-5 epimer, α-l-guluronic acid, which is known to be produced by only two bacterial genera, Pseudomonas and Azotobacter (34). The switch to a mucoid phenotype coincides with the appearance of a 54-kDa protein in the outer membrane; this protein has been identified and has been designated AlgE (13, 31).The genes encoding the alginate biosynthesis machinery are located within a 12-gene operon (algD-alg8-alg44-algK-algE-algG-algX-algL-algI-algJ-algF-algA). AlgA and AlgD, along with AlgC (not encoded in the operon), are involved in precursor synthesis (34). Alg8 is the catalytic subunit of the alginate polymerase located at the inner membrane (35). AlgG is a C-5 mannuronan epimerase (19). AlgK contains four putative Sel1-like repeats, similar to the tetratricopeptide repeat motif often found in adaptor proteins involved in the assembly of multiprotein complexes (3, 10). AlgX shows little homology to any known protein, and its role is unclear (14). Knockout mutants of AlgK, AlgG, and AlgX have nonmucoid phenotypes, although they produce short alginate fragments, due to the activity of the alginate lyase (AlgL), which degrades the nascent alginate (1, 14, 19-21, 36). AlgF, AlgI, and AlgJ are involved in acetylation of alginate, but they are not ultimately required for its production (12). The membrane-anchored protein, Alg44, is required for polymerization and has a PilZ domain for the binding of c-di-GMP, a secondary messenger essential for alginate production (16, 25, 33). The periplasmic C terminus of Alg44 shares homology with the membrane fusion proteins involved in the bridging of the periplasm in multidrug efflux pumps (11, 43). The periplasmic alginate lyase, AlgL, appears to be required for the translocation of intact alginate across the periplasm (1, 26). AlgE is an outer membrane, anion-selective channel protein through which alginate is presumably secreted (30). A protein complex or scaffold through which the alginate chain can pass and be modified and which spans the periplasm bridging the polymerase located (Alg8) at the outer membrane pore (AlgE) has been proposed (21). Indeed, it has been demonstrated that both the inner and the outer membranes are required for the in vitro polymerization of alginate (35).The requirement of AlgE for the biosynthesis of alginate in P. aeruginosa was first observed by complementation of an alginate-negative mutant derived by chemical mutagenesis with a DNA fragment containing algE (8) Secondary structure predictions suggested that AlgE forms an 18-stranded β barrel with extended extracellular loops. Several of these loops show high densities of charged amino acids, suggesting a functional role in the translocation of the anionic alginate polymer (29, 30). Preliminary analysis of AlgE crystals has been reported (48).In this study, the role of AlgE in alginate biosynthesis was investigated and the membrane topology of AlgE was assessed by site-directed insertion mutagenesis.     

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB¹⁵³-YMT and LamB¹⁵³-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd²⁺ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.     

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N‐terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW‐GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW‐GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW‐GFP into OMVs through the increased OmpW‐GFP expression on the ΔnlpI cells. Finally, the amount of OmpW‐GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW‐GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51–57, 2018     

Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles

Mutations in the tol-pal genes induce pleiotropic effects such as release of periplasmic proteins into the extracellular medium and hypersensitivity to drugs and detergents. Other outer membrane defective strains such as tolC, lpp, and rfa mutations are also altered in their outer membrane permeability. In this study, electron microscopy and Western blot analyses were used to show that strains with mutations in each of the tol-pal genes formed outer membrane vesicles after growth in standard liquid or solid media. This phenotype was not observed in tolC and rfaD cells in the same conditions. A tolA deletion in three different Escherichia coli strains was shown to lead to elevated amounts of vesicles. These results, together with plasmid complementation experiments, indicated that the formation of vesicles resulted from the defect of any of the Tol-Pal proteins. The vesicles contained outer membrane trimeric porins correctly exposed at the cell surface. Pal outer membrane lipoprotein was also immunodetected in the vesicle fraction of tol strains. The results are discussed in view of the role of the Tol-Pal transenvelope proteins in maintaining outer membrane integrity by contributing to target or integrate newly synthesized components of this structure.     

Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Microbial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation of Escherichia coli cells by overexpression of the bcsB gene was investigated. The flocculation induced by overexpression of bcsB was consistent among the various E. coli strains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeled E. coli cells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) in E. coli flocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore, bcsB-induced E. coli flocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbA and ΔdsbB strains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production of E. coli cells.     

Lambda Receptor in the Outer Membrane of Escherichia coli as a Binding Protein for Maltodextrins and Starch Polysaccharides

The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane lambda receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coli in a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the lambda receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as lambda receptor is present. (iv) Using the inhibition of lambda receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the lambda receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (K(d) for maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM.     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.     

Outer Membrane Proteins of Escherichia coli VII. Evidence That Bacteriophage-Directed Protein 2 Functions as a Pore

Protein 1, a major protein of the outer membrane of Escherichia coli, has been shown to be the pore allowing the passage of small hydrophilic solutes across the outer membrane. In E. coli K-12 protein 1 consists of two subspecies, 1a and 1b, whereas in E. coli B it consists of a single species which has an electrophoretic mobility similar to that of 1a. K-12 strains mutant at the ompB locus lack both proteins 1a and 1b and exhibit multiple transport defects, resistance to toxic metal ions, and tolerance to a number of colicins. Mutation at the tolF locus results in the loss of 1a, in less severe transport defects, and more limited colicin tolerance. Mutation at the par locus causes the loss of protein 1b, but no transport defects or colicin tolerance. Lysogeny of E. coli by phage PA-2 results in the production of a new major protein, protein 2. Lysogeny of K-12 ompB mutants resulted in dramatic reversal of the transport defects and restoration of the sensitivity to colicins E2 and E3 but not to other colicins. This was shown to be due to the production of protein 2, since lysogeny by phage mutants lacking the ability to elicit protein 2 production did not show this effect. Thus, protein 2 can function as an effective pore. ompB mutations in E. coli B also resulted in loss of protein 1 and similar multiple transport defects, but these were only partially reversed by phage lysogeny and the resulting production of protein 2. When the ompB region from E. coli B was moved by transduction into an E. coli K-12 background, only small amounts of proteins 1a and 1b were found in the outer membrane. These results indicate that genes governing the synthesis of outer membrane proteins may not function interchangeably between K-12 and B strains, indicating differences in regulation or biosynthesis of these proteins between these strains.     

Electron Microscopy of the Major Outer Membrane Protein of Campylobacter jejuni

The surfaces of the disrupted-cell surfaces of the Campylobacter jejuni strains FUM158432 and M1 were examined using the negative-staining technique and electron microscopy. The surfaces of the whole cells and the outer membranes were covered with small dark dots which, in some areas, were arranged in hexagonal patterns. The hexagonal arrangement was more clearly seen in extracted outer membrane. The size of each structure was measured based on a center-to-center distance with the adjacent structure, and was determined to be 9.9±0.9 nm. A profile of the proteins in the outer membrane by SDS-PAGE, performed in 0.1% SDS and at 100 C, showed 42 kDa proteins to comprise the major outer membrane protein of this bacterium. Digestion of the outer membrane materials with proteinase reduced this protein band in the SDS-PAGE, and the amount of dark dots on the electron micrograph indicated the structure to be the major outer membrane protein (porin) of this bacterium. The power spectrogram of a computer-assisted Fourier transformation of the hexagonally arranged porin proteins suggests that the porin has a trimeric structure rather than a monomeric one.     

Impairment of Temperate Bacteriophage Adsorption by Brief Treatment of Escherichia coli with Dilute Solutions of Ethylenediaminetetraacetate

Protass, Jay J. (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.), and David Korn. Impairment of temperate bacteriophage adsorption by brief treatment of Escherichia coli with dilute solutions of ethylenediaminetetraacetate. J. Bacteriol. 91:143-147. 1966.-Cells of Escherichia coli K-12 treated for 2 min with 2 x 10(-4)m ethylenediaminetetraacetate (EDTA) are unable to adsorb the temperate bacteriophages lambdavir and 434 but show no impairment of their ability to adsorb T-even phages or T5. This finding is consistent with the hypothesis that there are basic structural differences between the cell-wall receptors involved in the adsorption of the temperate and T classes of coliphages.     

Production of a Monoclonal Antibody Specific for the Major Outer Membrane Protein of Campylobacter jejuni and Characterization of the Epitope

Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 × 10³ bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence ²¹⁶GGQFNP²²¹, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.     

Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.     

Display of Polyhistidine Peptides on the Escherichia coli Cell Surface by Using Outer Membrane Protein C as an Anchoring Motif

A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 μmol of Cd²⁺ per g (dry weight) of cells.