Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.     

Optical properties of an outer membrane lipoprotein from Escherichia coli

The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had and α-helical conformation but no sign for the existence of β-structures. From circular dichroism spectra of the lipoprotein, the α-helical content of the protein was found to be as high as 88% in 0.01–0.03% sodium dodecyl sulfate in the presence of 10^?5 M Mg²⁺ at pH 7.1 and 23° C. When sodium dodecyl sulfate concentration increased higher than 0.1%, the α-helical content of the lipoprotein decreased to about 57%. Divalent cations, such as Mg²⁺ and Mn²⁺, were found to increase the helical content of the lipoprotein. The high α-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55° C). The significance of the high α-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precursor pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [³⁵S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s.The [³⁵S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30–120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation.Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Preferential release of new outer membrane fragments by exponentially growing Escherichia coli

We have examined whether the outer membrane fragments released by normally growing Escherichia coli contain relatively old or new outer membrane.Double-label experiments show that after incorporation of radioactive leucine into E. coli protein, there is a preferential release of outer membrane material which contains a high percentage of newly labeled protein. This implies that outer membrane fragments are preferentially released from those regions where newly synthesized proteins are inserted into the outer membrane. We estimate that these insertion regions cover no more than 13% of the total outer membrane, and that newly inserted proteins diffuse in the plane of the outer membrane with a diffusion constant ? 5 · 10^?13 cm²/s.     

Phosphate transport in membrane vesicles from Escherichia coli

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate-phenazine methosulphate as electron donor. To a lesser extent also the oxidation of d-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K⁺ and Mg²⁺, the highest activities being observed with both ions present.Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or d-lactate.     

Organization of proteins in the native and reformed outer membrane of Escherichia coli

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1. The proteins of the outer membrane of Escherichia coli were characterized by polyacrylamide gel electrophoresis and their molecular weights determined.     

Isolation and identification of 5-methylthioribose from Escherichia coli B

5-Methylthioribose was isolated after incubation of Escherichia coli B in a glucose-salts medium. At least 60% of the radioactivity in absolute ethanol extracts of the residue from lyophilized medium supplemented with ³⁵SO₄²⁻ was located in two chromatographic areas that were identified as 5-methylthioribose and its sulfoxide. The sulfoxide was formed by oxidation of 5-methylthioribose during necessary processing of cultures and fractions. These compounds were characterized by functional group analysis and chromatographic comparison with authentic material. 5-Methylthioribose sulfoxide was isolated from 12 l of incubation medium of E. coli. After purification in three paper and one thin-layer chromatographic systems, 50 μg was obtained. The trimethylsilyl derivative of this compound was compared with that of authentic 5-methylthioribose sulfoxide. Gas chromatography and mass spectrometry confirmed the identity. This is the first report of 5-methylthioribose from a bacterium.     

Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110

Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs.     

Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12

The thiol-oxidizing agent “diamide” (CH₃)₂NCON=NCON(CH₃)₂ was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic processes. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in γ-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide range of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.     

Isolation of detergent-resistant membranes (DRMs) from Escherichia coli

Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm^?1 and an amide II band at 1548 cm^?1. The outer membrane had a broad amide I band at 1631–1657 cm^?1 and an amid II band at 1548 cm^?1 with a shoulder at 1520–1530 cm^?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm^?1, whereas the band at 1631 cm^?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.     

Architecture of the outer membrane of Escherichia coli K12 IV. Relationship between outer membrane particles and aqueous pores

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage λ receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and aqueous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.     

Protease inhibitors inhibit production of protein I of the outer membrane in Escherichia coli

Effects of protease inhibitors on composition of newly synthesized protein were studied by pulse-labeling E. coli cells with [³H]leucine and analyzing the labeled proteins by sodium dodecylsulfate gel electrophoresis. In addition to tosyl-lysine chloromethylketone that had been studied previously, antipain, leupeptin and diisopropyl fluorophosphate all inhibited production of a major outer membrane protein, protein I. Synthesis of protein I was specifically inhibited by antipain or leupeptin in strain K12, whereas several other proteins were also affected in strain B. Protein synthesis in strain B was generally more sensitive to inhibition by antipain than that in strain K12.     

Assembly and overexpression of membrane proteins in Escherichia coli

The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class. Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins. In addition, we will briefly review and discuss how E. coli has been used as a vehicle for the overexpression of membrane proteins.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.