Genetic and physiological regulation of intrinsic proteins of the outer membrane of Salmonella typhimurium.

Four major outer membrane polypeptides, accounting for approximately 20% of the total protein of the outer membrane of Salmonella typhimurium, were induced by growth in minimal medium. The polypeptides were tightly bound membrane components. Physiological and genetic evidence indicates that the four polypeptides fall in two separate regulation groups. Synthesis of one of these groups was coordinately regulated by the concentration of iron in the medium, and a mutant strain has been identified in which there is constitutive synthesis of this group of major outer membrane proteins.     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.     

Outer membrane proteins of Escherichia coli. II. Heterogeneity of major outer membrane polypeptides

When E. coli outer membrane protein is dissolved in sodium dodecyl sulfate (SDS) solution and boiled briefly, a single major peak (peak B) with a molecular weight of 42,000 daltons is observed on SDS-containing polyacrylamide gels. If the protein is dissolved in SDS solution at 37 °C and applied to gels without further treatment, peak B disappears and two other major peaks appear: Peak A, which is composed of aggregates and migrates more slowly than peak B, and peak C which is composed of monomeric protein not fully reacted with SDS and which migrates faster than peak B. When cyanogen bromide peptides of protein from peak A and peak C were compared, it was evident that peak A and peak C contained entirely different polypeptides. This was further confirmed by differential labeling studies with methionine and leucine. The cyanogen bromide peptide profiles of protein from peak A suggested that this peak was composed of two polypeptides, and this was confirmed by electrophoresis in an alkaline gel system which resolves peak B into three subcomponents. Two of these were derived from peak A and the third was derived from peak C. These results indicate that the outer membrane of E. coli contains at least three nonidentical major polypeptides, each of which has a nearly identical molecular weight of about 42,000 daltons. These polypeptides are present in identical proportions in the soluble and insoluble fractions obtained when the outer membrane is treated with Triton X-100 plus EDTA.     

Characterization of an outer membrane mannanase from Bacteroides ovatus

Bacteroides ovatus utilizes guar gum, a high-molecular-weight branched galactomannanan, as a sole source of carbohydrate. No extracellular activity was detectable. Approximately 30% of the total cell-associated mannanase activity partitioned with cell membranes. When inner and outer membranes of B. ovatus were separated on sucrose gradients, the mannanase activity was associated mainly with fractions containing outer membranes. Enzyme activity was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or by Triton X-100 at a detergent-to-protein ratio of 1:1. The enzyme was stable for only 4 h at 37 degrees C and for 50 to 60 h at 4 degrees C. Analysis of the products of the CHAPS-solubilized mannanase on Bio-Gel A-5M and Bio-Gel P-10 gel filtration columns indicated that the enzyme breaks guar gum into high-molecular-weight fragments. The CHAPS-solubilized mannanase was partially purified by chromatography on a FPLC Mono Q column. The partially purified mannanase preparation contained three major polypeptides (Mr 94,500, 61,000, and 43,000) and several minor ones. High mannanase activity was seen only when B. ovatus was grown on guar gum. Cross-absorbed antiserum detected two other guar gum-associated outer membrane proteins: a CHAPS-extractable 49,000-dalton polypeptide and a 120,000-dalton polypeptide that was not solubilized by CHAPS. Neither of these polypeptides was detectable in the partially purified mannanase preparation. These results indicate that there are at least two guar gum-associated outer membrane polypeptides other than the mannanase.     

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.     

Insecticidal activity associated with the outer membrane vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.     

Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium.

Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.     

Demonstration of ferric L-parabactin-binding activity in the outer membrane of Paracoccus denitrificans.

Under low-iron conditions, Paracoccus denitrificans excretes a catecholamine siderophore, L-parabactin, to sequester and utilize iron. In this report, we demonstrate the presence of stereospecific high-affinity ferric L-parabactin-binding activity associated with P. denitrificans membranes grown in low-iron medium. Isolated outer membrane components were shown to be three to four times higher in specific activity for ferric L-parabactin. The same amount of binding activity existed whether or not the radiolabel was present in the metal (55Fe) or the ligand (3H) portion of ferric parabactin chelate, suggesting that binding was to the intact complex. Ion-exchange chromatography of a Triton X-100-solubilized outer membrane mixture on DEAE-cellulose resulted in a 10-fold increase in binding activity relative to that present in whole membranes. Polypeptide profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of each stage of the purification showed that binding activity copurified with one or more of the low-iron-induced outer membrane proteins in the 80-kilodalton (kDa) region. Membrane proteins and [55Fe]ferric L-parabactin electrophoresed in nondenaturing gels demonstrated the presence of membrane component(s) which stereo-specifically bound ferric L-parabactin, thus providing independent confirmation of the binding assay results. Moreover, when the band labeled by [55Fe]ferric L-parabactin was excised and profiled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 80-kDa polypeptides were the major components present. These results demonstrate the presence of a high-affinity ferric L-parabactin receptor in P. denitrificans membranes and suggest that one or more of the 80-kDa low-iron-induced polypeptides are components of the ferric L-parabactin receptor.     

The Tom channel in the mitochondrial outer membrane: alive and kicking

The targeting of newly-made polypeptides to specific membranes, and the subsequent ability of a membrane to allow only certain polypeptides into its compartment, are essential to maintain the ultrastructure of Eukaryotic cells. Distinct oligomeric protein complexes in each cellular membrane catalyse these translocation processes. A recent report [Hill K et al. Nature 1998;395:516-521 (Ref. 1)] of the reconstitution of the translocation channel from the mitochondrial outer membrane, after producing the major structural component of the channel by recombinant means, promises a system to dissect in molecular detail the exact working of one of these protein translocation machines.     

Amidination of the outer and inner surfaces of the human erythrocyte membrane

We have synthesized a novel imidoester, isethionyl acetimidate, which is unable to penetrate the membrane of the human erythrocyte. It has the same specificity for amino groups as ethyl acetimidate, which penetrates the membrane. Either reagent can be labeled with ³H or ¹⁴C and, thus, be used to convert amines to radioactive amidines. An erythrocyte membrane saturated with either compound functions nearly normally. Therefore, the membrane can be double labeled if the amino groups on the outer surface of a cell are saturated with isethionyl acetimidate (e.g. labeled with ¹⁴C) and the remaining active sites are saturated with ethyl acetimidate (labeled with ³H). Alternatively, the membrane can be isolated after saturation with [¹⁴C]isethionyl acetimidate and treated with [³H]isethionyl acetimidate. From quantitative experiments of this kind we conclude that there are more than ten times as many reactive amino groups in protein on the inner surface than on the outer surface of the membrane. Nearly all of the reactive amino groups in lipid are on the inner surface. The localization of individual polypeptides confirms and extends assignments made previously by other techniques; as many as four major components may span the membrane. The proteins and lipids react to the same extent with ethyl acetimidate in the intact cell as they do in isolated membranes; this implies that the isolation does not load to major structural rearrangements.     

The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles

The yeast mitochondrial outer membrane was isolated and 10 of its major polypeptides were identified (mol. wts. 109, 70, 57, 45, 45, 42, 33, 29, 25 and 14 kd). The membrane has no major polypeptide in common with either mitochondrial inner membrane or rough microsomes. Protease treatment and immunochemical techniques showed that virtually all of the isolated outer membrane vesicles are sealed and display the same surface orientation as in the intact mitochondrion.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli.

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed alpha and beta (Mrs of 60,000 and 53,000, respectively). The beta polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70 degrees C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both alpha and beta polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual alpha and beta polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both alpha and beta polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.     

Identification of Bordetella pertussis virulence-associated outer membrane proteins

Bordetella pertussis virulence-associated 30-, 32-, 90- and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30- and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.