Isolation, properties, and reassembly of outer sheath carrying a polygonal array from an oral treponeme.

The outer sheath carrying a polygonal array was isolated from an oral treponeme, Treponema sp. strain E-21, by disruption of cells by means of repeated freeze-thawing and by removal of flagella under acidic conditions followed by linear sucrose density gradient centrifugation. Electron microscopy revealed that the outer sheath was isolated as a triple-layered vesicle having a polygonal array, free of flagella and wall membrane complex. Using optical diffraction, negatively stained preparations of the outer sheath fragments showed that the polygonal array appeared to be composed of a hexagonal pattern with a predominant spacing of about 16.3 nm. The isolated outer sheath contained 49.7% protein, 30.8% total lipid, and 11.0% carbohydrate. Phospholipid comprised about 95% of the total lipid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the outer sheath was composed primarily of one major protein with an apparent molecular weight of about 62,000. The material from the isolated outer sheath solubilized with 1% sodium deoxycholate was reassembled into vesicles having a roughly polygonal array upon removal of the detergent by dialysis against 10 mM Tris-hydrochloride buffer with or without Mg2+.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

MsbA is not required for phospholipid transport in Neisseria meningitidis

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.     

Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J.

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.     

Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath)

Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.8 g ml-1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23 - 1.24 g ml-1) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.     

Ultrastructure and chemical composition of the sheath of Leptothrix discophora SP-6.

Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diameter fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-negative cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a negative charge. Cell-free sheaths were isolated by treatment with a mixture of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl- and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approximately 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9% N, 6% H, and 2.1% S and approximately 34 to 35% carbohydrate (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorganic ash. Gas-liquid chromatography showed that the polysaccharide was an approximately 1:1 mixture of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected. Amino acid analysis showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath (D. Emerson and W.C. Ghiorse, J. Bacteriol. 175:7819-7827, 1993).     

Characterization of the periplasmic flagellum proteins of Leptospira interrogans.

The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.     

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%.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

Fine Structure of Spirochaeta stenostrepta, a Free-living, Anaerobic Spirochete

The fine structure of Spirochaeta stenostrepta strain Z1, a free-living anaerobic spirochete, was studied by electron microscopy. The organism possessed a coiled protoplasmic cylinder, an axial filament inserted subterminally, and a loosely fitting sheath which enclosed both the protoplasmic cylinder and the axial filament. The axial filament consisted of two fibrils partially overlapping in a 1-2-1 arrangement. The axial fibrils appeared to possess a sheath surrounding an inner core. Both inner core and sheath were apparently enclosed in a cross-striated tubular structure, which was itself surrounded by an outer sheath. The axial filament exhibited a basal hook. A disc- or mushroom-shaped structure, possibly consisting in part of cytoplasmic membrane, was observed at the insertion end of isolated filaments. The protoplasmic cylinder had a distinctive surface structure consisting of an array of tightly packed, longitudinally arranged helices measuring 2.0 to 2.5 nm in diameter. This layer of helices lay below the outer cell sheath and the axial filament. Ballistic disintegration loosened the helical array, causing individual helices or segments of helices to become separated from the cell. The function of this layer of helices is still obscure.     

Outer Membrane Changes in a Toluene-Sensitive Mutant of Toluene-Tolerant Pseudomonas putida IH-2000

We isolated a toluene-sensitive mutant, named mutant No. 32, which showed unchanged antibiotic resistance levels, from toluene-tolerant Pseudomonas putida IH-2000 by transposon mutagenesis with Tn5. The gene disrupted by insertion of Tn5 was identified as cyoC, which is one of the subunits of cytochrome o. The membrane protein, phospholipid, and lipopolysaccharide (LPS) of IH-2000 and that of mutant No. 32 were examined and compared. Some of the outer membrane proteins showed a decrease in mutant No. 32. The fatty acid components of LPS were found to be dodecanoic acid, 2-hydroxydodecanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxydecanoic acid in both IH-2000 and No. 32; however, the relative proportions of these components differed in the two strains. Furthermore, cell surface hydrophobicity was increased in No. 32. These data suggest that mutation of cyoC caused the decrease in outer membrane proteins and the changing fatty acid composition of LPS. These changes in the outer membrane would cause an increase in cell surface hydrophobicity, and mutant No. 32 is considered to be sensitive to toluene.     

Presence of exposed phospholipids in the outer membrane of Vibrio cholerae.

Vibrio cholerae 569B was found to be highly sensitive to a wide range of chemicals, particularly hydrophobic compounds and neutral and anionic detergents. The phospholipid profile of the outer membrane was similar to that reported for other Gram-negative bacteria. The lipopolysaccharide (LPS) contained O-antigenic sugars and exhibited heterogeneity. In addition, the LPS moiety was characterized by a relatively low negative charge. Analysis by topological probes revealed the presence of a significant amount of exposed phospholipids in the outer membrane. The reduced negative charge of LPS molecules and the exposed phospholipids present in the outer membrane could be important in the increased permeation of exogenous compounds in V. cholerae.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad.

The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.     

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.     

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.     

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.