Electron microscopic study of the I, II phases and R-forms of Sh. sonnei]

Electron microscopic study of the microbial cells of the I, II phases and the R-form was carried out. Intact cells were examined by negative contrasting, and morphological differences of various bacterial phases were shown: cells of the I phase had a relatively smooth surface, bacteria of the II phase had a smooth surface, but many cell wall fragments were split from them; the surface of the R-form cells was coarse, folded, and cell wall fragments were split from the majority of bacteria. Antigenic determinants responsible for phasic specificity in bacteria of the I and II phases were located at some distance from the external membrane of the cell wall; as to the R-form cells--they were localized on the wall.     

Disintegration of Mg2+ -induced hexagonal assembly of an R-form lipopolysaccharide from Klebsiella pneumoniae by treatment with CaCl2

R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-: K1-), which was precipitated by the addition of 2 volumes of ethanol containing 10 mM MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R-form LPS was suspended in 50 mM tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mM or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R-form LPS was disintegrated and changed to an irregular rough, mesh-like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg-bound R-form LPS result in a tighter binding of Ca2+ to the R-form LPS and the release of Mg2+ from the R-form LPS, and as a consequence, destroys the Mg2+ -induced ordered hexagonal lattice structure of the R-form LPS.     

Differential characteristics of the I and II phases and R-form of Sh. sonnei]

Characteristics of various phases of Sh. sonnei were determined on the standard strains of Sh. sonnei of phases I and II and R-form used for the industrial production of agglutinating monoreceptor sera. Bacteria of phase I displayed a distinct morphological, cultural and serological differences. For the differentiation of Sh. sonnei of the II phase and R-form, representing the greatest difficulty in this respect, it is recommended to use phages RFfm and 6-SR, and also indirect immunofluorescence method with the application of agglutinating monophasic serum against the II phase of Sh. sonnei. In addition, a study was made of over 20 various Sh. sonnei strains at different dissociative process phases. Verification data completely confirmed the specificity of phages RFfm and 6-SR for Sh. sonnei in the II phase. The efficacy of the immunofluorescence method was confirmed on 6 strains.     

Relationship between Mg2(+)-induced hexagonal assembly of R-form lipopolysaccharides and chemical structure of their R-cores

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.     

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.     

Comparison of R- and S-form lipopolysaccharides fractionated from Escherichia coli UKT-B lipopolysaccharide in pyrogen and Limulus tests

Different LPS was shown to have a relatively different proportion of O-specific chain-less (R-form) LPS by polyacrylamide gel electrophoresis (PAGE) with sodium deoxycholate (DOC). By using DOC-PAGE, S-form LPS having O-chain with approximately 11 repeating units on average (S-Fr) and O-chain-less LPS (R-Fr) were separated from Escherichia coli UKT-B S-form LPS. Significantly stronger pyrogenicity was observed in R-Fr than in S-Fr when measured on the weight basis. Similar result was observed in Limulus test. Comparing biological activities of different S-form LPS, attention should be given to the amounts of co-existing R-form LPS.     

Hexagonal assembly of the magnesium salt of an R-form lipopolysaccharide from Klebsiella pneumoniae: its lowered stability compared with original non-electrodialyzed preparation

The magnesium salt of R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm as the original non-electrodialyzed preparation of the R-form LPS. When the magnesium salt was suspended in 50 mM glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4 C for 24 hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh-like structure at pH 3.0 or higher. In the original non-electrodialyzed preparation of the R-form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4 C for 24 hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg2+ and in stabilizing the ordered hexagonal assembly of the R-form LPS.     

Antigenic composition of different forms of Sh. sonnei]

Data are presented on the study, with the aid of immunophoresis, of the antigenic composition of the I, II phases and R-forms of Sh. sonnei. From 5 to 7 antigens differing by thermoresistance, electrophoretic and diffuse mobility were found in the composition of microbial cells of various forms Sh. sonnei. Differences between the I phase on the one hand and cells of the II phase and R-form, on the other hand, consisted both in the quantitative composition of components of their thermostable O-antigens and in the structure of specific lipopolysaccharides of the forms.     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella sp.

We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Klebsiella sp. strain LEN-111 (O3-:KI-) and followed the changes in ultrastructure of the LPS during the extraction procedure. When the LPS was obtained from the water phase of an extract by addition of 2 volumes of 10 mM MgCI2-ethanol, it consisted of membrane pieces with a hexagonal lattice structure with a lattice constant of 14 to 15 nm. The lattice structure of the LPS was disrupted into short rods with sodium dodecyl sulfate, but the same hexagonal lattice structure was again formed by precipitation with 2 volumes of 10 mM MgCI2-ethanol. The LPS preparation after two cycles of treatment by the phenol-water method, which contained no detectable amounts of proteins, kept an unaltered ability to form the hexagonal lattice structure. Extensive treatment with pronase and extraction with chloroform did not impair the ability of the LPS preparation to form the lattice structure. When the other salts, NaCI, CaCI2 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgCI2, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCI-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgCI2-ethanol. From these results, it was concluded that the R-form LPS has the ability of in vitro self-assembly into a hexagonal lattice structure in the presence of Mg2+ without the help of other components such as proteins and free lipids from outer membrane.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Nature and linkage type of fatty acids present in lipopolysaccharides of phase I and II Coxiella burnetii

The constituent fatty acids of lipopolysaccharides (LPS) of Coxiella burnetii (phase I and II) were qualitatively and quantitatively analysed by combined gas-liquid chromatography/mass spectrometry. The total fatty acid content (per mg LPS) was determined as 90.0 nmol (2.3 wt%) for LPS of phase I cells (LPS I) and 179.1 nmol (4.8 wt%) for LPS of phase II cells (LPS II). Of the 24 different acyl residues characterized (12 to 18 carbon atoms), nine were 3-hydroxy fatty acids (normal, iso- and anteiso-branched) which quantitatively predominated. All 3-hydroxylated fatty acids were found to possess the (R)-configuration, to be exclusively amide-linked and to be acylated at their 3-hydroxyl group. Ester-linked nonhydroxylated fatty acids (normal, iso- and anteiso-branched) were present but ester-bound 3-hydroxy- or 3-acyloxyacyl residues were lacking from C. burnetii LPS I and LPS II. As the major acyl group (R)-3-(12-methyl-tetradecanoyloxy)-12-methyl-tetradecanoic acid was identified. Our results show that the complex fatty acid spectrum of C. burnetii differs considerably from that of LPS of other Gram-negative bacteria. They further suggest an enormous heterogeneity of the lipid A component of C. burnetii LPS I and LPS II.     

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.     

Regulation of MHC expression in vivo. Bacterial lipopolysaccharide induces class I and II MHC products in mouse tissues by a T cell-independent, cyclosporine-sensitive mechanism

The effect of injections of bacterial LPS on the expression of class I and II products of the MHC in mouse tissues was investigated. MHC products were assessed in tissue homogenates by radiolabeled antibody binding and in tissue sections by indirect immunoperoxidase (IIP) staining. In mice given two i.p. injections of LPS from Escherichia coli or Salmonella minnesota, there were increases in class I and II MHC products in kidney, liver, heart, lung, and pancreas. Focusing on the changes in kidney, we demonstrated that the increase in MHC expression occurred in tubules and, in the case of class I, in glomeruli. LPS treatment also increased the deposition of Ig in glomeruli. Expressed on a standard curve, the total kidney class I and II expression was elevated approximately 10-fold. Time course studies indicated that increased class I expression could be induced by a single LPS injection, whereas class II induction required a second injection. The induction was influenced by the LPS sensitivity of the mice, being much greater in LPS-sensitive C3H/HeSn mice than in LPS-resistant C3H/HeJ mice. LPS induced class I and II Ag in nude mice and in mice with severe combined immunodeficiency, indicating that T cells were not required. Nevertheless, the effect of LPS was inhibitable by cyclosporine and by a mAb against IFN-gamma indicating that IFN-gamma was required for the MHC induction. We conclude that LPS induces an increase in expression and a redistribution of MHC products in kidney and in other tissues by a T cell-independent, cyclosporine-sensitive pathway. These findings are probably related to the known ability of LPS to mediate release of IFN-gamma and other cytokines.     

Outer membrane vesiculation of acinetobacter calcoaceticus

For intact cells of A. calcoaceticus 69V susceptibility to hydrophobic agents (antibiotics, dyes) was established. The composition of its outer membrane and comparison with that of a reference strain, A. calcoaceticus CCM 5593 with a blocked hydrophobic pathway, gave no indication of phospholipid bilayer domains as the structural basis of these permeability characteristics. The outer membrane composition together with the data of time-resolved fluorescence anisotropy measurements is indicative of a high state of order of the hydrocarbon region. A. calcoaceticus 69V releases lipopolysaccharide (LPS)-rich membrane vesicles into the growth medium when grown on a hydrophobic carbon source. While the cells contain both R-form and S-form LPS, the LPS released with the vesicles is exclusively of the R-type. The same selectivity with respect to LPS composition was observed when LPS was removed from intact cells by EDTA-NaCl treatment which leads to a break-down of the barrier to hydrophobic agents in A. calcoaceticus CCM 5593. We propose that due to its physical properties, R-form LPS forms tightly packed structures within the membrane which, under certain conditions, become destabilized and liberated into the surrounding medium. As a consequence, a disturbance of the highly ordered lateral molecular arrangement might lead to altered permeability properties of the outer membrane as suggested in one of the two alternative models existing to explain permeability changes observed in deep rough mutant strains of Enterobacteriaceae.     

Immunochemical Characterization of the Capsular Polysaccharide of Azospirillum irakense KBC1

The repeating unit structure of Azospirillum irakense KBC1 capsular polysaccharide (CPS) was established and was found to be identical to that of the O polysaccharide of A. irakense KBC1 lipopolysaccharide (LPS). The antigenic heterogeneity of the LPS and the CPS was shown to be related to differences in the macromolecular organization of these glycopolymers. After an immune response activation, R-form CPS molecules were found to be predominant.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

Heterogeneity and Distribution of Lipopolysaccharide in the Cell Wall of a Gram-Negative Marine Bacterium

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.     

Staining of O-specific polysaccharide chains of lipopolysaccharides with ruthenium red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.     

Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.