Composition of the fractions separated by polyacrylamide gel electrophoresis of the lipopolysaccharide of a marine bacterium.

The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.     

Porins from marine bacteria of the genus Pseudoalteromonas (Gammaproteobacteria: Pseudoalteromonadaceae)

Outer membrane (OM) fractions were isolated from marine bacteria of the genus Pseudoalteromonas (P. haloplanktis, P. tetraodonis, and Pseudoalteromonas sp. KMM 223). The purity of OM fractions was confirmed by ultracentrifugation in a sucrose gradient. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the bilayer lipid membrane (BLM) technique, heat-modifiable porin-like proteins were identified among the OM proteins of marine pseudomonads. The pore-forming P-1 and P-2 proteins with molecular masses of 43 and 39 kDa, respectively, were obtained from the marine bacterium P. haloplanktis. The nature of current fluctuations in the BLM and the conductivity of pores formed by these proteins suggest that these isolated porins are not identical in their functional properties. A nonlinear dependence of channel conductivity on salt concentration in the aqueous phase was found for the P-2 protein, which is typical of marine bacterial porins.     

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.     

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Structure and biological relationships of Coxiella burnetii lipopolysaccharides

Lipopolysaccharides (LPSs) extracted from nine strains of Coxiella burnetii were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and lethal toxicities in galactosamine-sensitized mice. The structure of a unique disaccharide of hydrolyzed phase I LPS was determined to be galactosaminuronyl-alpha (1-6)-glucosamine (GalNU-alpha (1-6)-GlcN, C12H22N2O10) with an Mr of 354. The Mr of LPSs of C. burnetii intra- and interspecific strains and the content of GalNU-alpha (1-6)-GlcN and two sugars, virenose and dihydrohydroxystreptose, were used as biochemical markers of truncated LPSs. Smooth-phase I LPS contained all three compounds, semi-rough-phase I LPS did not contain virenose, and rough-phase II LPS contained none of the three compounds. These analyses indicate that the intermediate to larger Mr LPSs require the addition of GalNU-alpha (1-6)-GlcN and dihydrohydroxystreptose to obtain the major (10.5 kDa), the intermediate (between 10.5 and 27 kDa), and the minor (23 kDa) LPS bands. The addition of virenose to the major and the minor bands produced the large Mr phase I LPSs. Extreme microheterogeneity in the banding profile ranging in Mr from the 2.5 to 10.5 kDa may be due to unidentified components, while the microheterogeneity in Mr of the 10.5-kDa and larger LPS bands is related to variations in the compounds described here. All of the LPSs were toxic in galactosamine-sensitized mice, albeit they were 100-1000-fold less toxic than Escherichia coli and Salmonella typhimurium endotoxin.     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

Electrophoretic resolution of microheterogeneity inVibrio cholerae lipopolysaccharide

Lipopolysaccharide (LPS) fromVibrio cholerae has been analysed by sodium dodecyl sulfate-potyacrylamide gel electrophoresis. Under normal conditions of electrophoresis which resolveEscherichia coli LPS, V.cholerae LPS shows two diffuse and unresolved bands. However, on long gels at low concentration it can be resolved into two major band types. There are at least 10 slow moving, discrete bands of regular periodicity and three fast moving bands. Comparison with LPS fromE. coli indicates that the heterogeneity occurs over a much smaller range of molecular weight in V.cholerae LPS, with the entire spectrum of discrete bands being contained within the space of fourE. coli repeating units.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Isoenzymes of soluble starch synthetase from Oryza sativa grains

Two isoenzymic fractions of soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase were obtained from developing (non-waxy) rice grains by gradient elution through DEAE-cellulose. After Sephadex G-200 chromatography, fractions I and II were electrophoretically homogeneous and have MW values of 110000 and 69000, respectively. Sodium dodecyl sulfate gel electrophoresis of fraction I produced five bands with MW of 12000, 26000, 50000, 70000, and 105000 while fraction II gave two bands with MW of 12000 and 22000. Fraction II, which contains 1·7% carbohydrate, was active in the absence of added primer while fraction I, which does not contain carbohydrate, required primer.     

Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

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.     

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.     

Des activites photosynthetiques sans les complexes pigmentaires CP1 et CP2

Photosynthetic activity in the absence of the CP1 and CP2 pigmentary complexesVarious photochemical activities were tested on chloroplasts of Zea mays that received 4 s of light every 4 h during the culture period. Photosystem I and Photosystem II were functioning, as well as the photosynthetic electron transport. These chloroplasts exhibited upon sodium dodecyl sulphate gel electrophoresis neither Complex 1 (M_r 70 000) generally associated with Photosystem I nor Complex 2 M_r 25 000) generally associated with Photosystem II. Chlorophyll is indeed attached to polypeptides of molecular weight 21 000 and 29 000.These results lead us to question the functional role of chloroplast protein-pigment complexes observed by sodium dodecyl sulphate gel electrophoresis.     

Mitochondrial malate dehydrogenase from corn : purification of multiple forms

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.     

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.     

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell^{1, 2}. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli^3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence^6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods⁹. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann¹⁰, is ideal for LPS extraction.     

Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures

Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 ± 5000, 105,000 ± 5000, and 94,000 ± 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M_r, as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K_m values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and α-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.     

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.