Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp.

Membranes were isolated and purified from nutrient broth-yeast extract- and hexadecane-grown cells of Acinetobacter sp. strain HO1-N. Two membrane fractions were isolated from nutrient broth-yeast extract-grown cells, the cytoplasmic membrane and the outer membrane. In addition to these two membrane fractions, a unique membrane fraction was isolated from hexadecane-grown cells (band 1) and characterized as a lipid-rich, low-density membrane containing high concentrations of hexadecane. The outer membrane preparations of Acinetobacter, obtained from nutrient broth-yeast extract- and hexadecane-grown cells, exhibited a low ratio of lipid phosphorus to protein and contained phospholipase activity and 2-keto-3-deoxyoctulosonic acid. Phosphatidic acid cytidyltransferase, adenosine triphosphatase, and reduced nicotinamide adenine dinucleotide oxidase were recovered almost exclusively in the cytoplasmic membrane fractions. The cytoplasmic membrane fractions contained 20 to 25 polypeptide species on sodium dodecyl sulfate-polyacrylamide gels, and the outer membrane fractions contained 15 to 20 polypeptide species. A major polypeptide species with an apparent molecular weight of approximately 42,000 to 44,000 was found for all outer membrane fractions. The buoyant densities of the cytoplasmic membrane fractions and the outer membrane fractions were closely similar, necessitating their separation by differential centrifugation. Band 1 of hexadecane-grown cells had a ratio of lipid phosphorus to protein that was almost twice that of cytoplasmic membrane and a correspondingly low buoyant density (1.086 g/cm3). Enzyme activities associated with band 1 were identical to those associated with the cytoplasmic membrane. The electrophoretic banding pattern of band 1 was essentially identical to the banding pattern of the cytoplasmic membrane. The phospholipid and neutral lipid compositions of the isolated membrane fractions were determined as qualitatively similar, with significant quantitative differences. The ultrastructure characteristics of the respective membrane fractions were examined by the negative-stain technique.     

Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis

Abstract Highly purified preparations of inner, i.e. cytoplasmic and intracytoplasmic, membranes and outer membranes were isolated from Nitrobacter hamburgensis strain X14 by sucrose density-gradient centrifugation of cell-free extracts. The two membrane fractions differed markedly in morphology, density, and protein composition as determined by polyacrylamide gel electrophoresis. The inner membrane fraction was enriched in NADH oxidase and nitrite oxidase activity. It contained four major protein bands of apparent M _rs of 28 000, 32 000, 70 000, and 116000. The outer membrane fraction was characterized by the presence of 2-keto-3-deoxyoctonate and contained two major proteins of apparent M _rs of 13 000 and 50 000. There was no evidence for differences between cytoplasmic and intracytoplasmic membranes.     

Heterogeneity in Lipid Composition of the Outer Membrane and Cytoplasmic Membrane of Pseudomonas BAL-31

The outer membranes and cytoplasmic membranes of the marine bacterium Pseudomonas BAL-31 were separated by washing the cells three times in 0.5 M NaCl and twice in 0.5 M sucrose. Electron microscopy during the removal of membranes revealed that the outer membranes fragmented in a regular manner to give rise to fairly uniform vesicles measuring approximately 140 nm in diameter. Isolated outer membranes had a buoyant density in sucrose of 1.230 g per cm(3), whereas the cytoplasmic membranes had a density of 1.194 g per cm(3). The removal of the outer membrane during the application of this procedure was monitored by measuring the release of 2-keto-3-deoxyoctulosonic acid and phospholipid. The cells lost 85.5% of their 2-keto-3-deoxyoctulosonic acid and 47.3% of their phospholipid during this treatment. Complete recovery of outer membrane material could be achieved. The removal of 25.5% of the 2-keto-3-deoxyoctulosonic acid and 0.9% of the phospholipid rendered the cells sensitive to lysis with Triton X-100. The phospholipid composition of the outer membrane was calculated to be 78.9% phosphatidylethanolamine and 16.1% phosphatidylglycerol. The phospholipid composition of the cytoplasmic membrane proved to be 71.5% phosphatidylethanolamine and 23.5% phosphatidylglycerol. The fatty acid composition was also found to be quantitatively heterogeneous between the two membranes.     

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.     

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.     

Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis

Summary A crude cell envelope suspension has been prepared from Proteus mirabilis after osmotic shock of penicillin-induced spheroplasts. Employing discontinuous sucrose gradients this cell envelope suspension can be fractionated into four fractions. Besides a pellet of remaining spheroplasts and an intermediate fraction with mixed composition a highly purified cytoplasmic membrane fraction and an outer membrane fraction have been obtained. The cytoplasmic membrane fraction is not contaminated with mucopeptide or outer membrane material. It has a buoyant density of 1.13 g/ml and a protein content of 38%. The specific activities of formate dehydrogenase and nitrate reductase and the content of cytochrome b₁ have increased sixfold in comparison with the crude cell envelope suspension. The outer membrane fraction contains only few contaminations with cytoplasmic membrane components and with mucopeptide.The gradient fractions have been characterized by electron microscopy and by polyacrylamide gel electrophoresis.     

Chitin synthetase activity is bound to the plasma membrane and to a cytoplasmic particulate fraction in Candida albicans germ tube cells

Abstract Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans . Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [³H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Electrochemical sterilization of bacteria adsorbed on granular activated carbon

Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans. Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [3H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Association of a photoreceptor-specific tetraspanin protein,ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.     

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.     

Collagen synthesis by human glomerular cells in culture

The intracellular localization of enterotoxin in Escherichia coli AP1, a strain of porcine origin which produces high levels of heat-labile, but no heat-stable enterotoxin, has been examined. The cytoplasmic and outer membranes of this strain both contained enterotoxin activity, while the membranes isolated from a serologically related non-enterotoxigenic strain (E. coli AP2) also of porcine origin, did not show enterotoxin activity. The periplasmic fraction isolated from the enterotoxigenic strain contained considerable enterotoxin activity, but this activity was associated with outer membrane fragments present in the periplasmic fraction. Thus, of the total cellular enterotoxin activity, about 55%, 15% and 30% were present in the outer membrane, cytoplasmic membrane and the cell cytoplasm, respectively. The specific activity of enterotoxin was 20 units per mg protein in the cytoplasm and 90 and 150 units per mg protein in the cytoplasmic and outer membranes, respectively.     

Membrane localization of motility,signaling, and polyketide synthetase proteins in Myxococcus xanthus

Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm(-3)). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm(-3)) and low buoyant density (1.169 to 1.171 g cm(-3)). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm(-3)) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.