SalmoneUa minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates (preferential growth along the a axis) or solid columns (preferential growth along the c axis), when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4TC for 10 days.Analyses of crystals suggested that they consist of hexagonal lattices with the a axis (a side of the lozenge as a unit cell on the basal plane) of 0.462 nm for all these three kinds of LPSs and the c axes (perpendicular to the basal plane) of 5. 85, 8.47, and 8.75 nm for S. minnesota Re and Ra LPSs and E. coli K-12 LPS, respectively, and that hydrocarbon chains of the lipid A portion play the leading part in crystallization, whereas the hydrophilic part of the lipid A (the disaccharide backbone) and R core exhibit a disordered structure or are in a random orientation. The phenomenon of doubling of the a axis to 0.924 nm was observed with crystals of S.minnesota Re LPS when they were incubated in 70% ethanol for an additional 180 days, but not with crystals of S. minnesota Ra LPS or E. coli K-12 LPS. S. minnesota S-form LPS possessing the 0-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions. Bacterial lipopolysaccharide (LPS) is the constituent of the outer membrane of gram-negative bacteria and consists of the polysaccharide (O antigen) which is linked to the R core consisting of oligosaccharide, which in turn is linked to the lipid portion termed lipid A (49). LPS strongly elicits a variety of host reactivities through interactions with humoral and cellular factors of the host. It has been widely accepted that the lipid A portion is mostly responsible for the biological activities of LPS (14,17,47), although the polysaccharide or the R core can modify the strength of action in some biological activities. Recently, studies on the relationship between chemical structure and biological activity of lipid A components have progressed greatly, since synthetic preparations of lipid A components and related compounds have been available (16,18,20,22,23,26,32,33,46). Although several studies have been done on the three-dimensional structure of LPS (4,10,11,34,35,39,40,56) and a schematic model of LPS has been proposed (34), the conformation of LPS has not been determined conclusively. We have found (29-31) that certain of the R-form LPSs form ordered two-dimensional hexagonal structures in the presence of MgCl2. During the experiments, we noticed that R-form LPSs from Salmonella minnesota and Escherichia coli K-12 form three-dimensional crystals when they are precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and suspended in 70% ethanol containing 250 mM MgCl2 and incubated at 4°C. We present here analyses of crystals of these R-form LPSs. This report is the first one that has de...

In culture fluid, Klebsiella pneumoniae type I Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type-specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type-specific capsular polysaccharide was isolated from ultraviolet-irradiated cells of K. pneumoniae type I Kasuya strain which had been labeled with leucine-requiring marker by selecting unagglutinable cells with the antiserum to the type-specific capsular polysaccharide. Serological tests showed that the type-specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the 0 antigen of K. pneumoniae was discussed.Previous studies from this laboratory have demonstrated that the polysaccharide prepared from culture supernatant fluid of Klebsiella pneumoniae type 1 Kasuya strain exhibits an extraordinarily strong adjuvant effect on antibody responses in mice to various antigens (11)(12)(13)(14)(15)(16)(17). The polysaccharide of K. pneumoniae prepared from its culture fluid is fractionated into the type-specific acidic polysaccharide fraction (major antigenic component) and the neutral polysaccharide fraction (minor antigenic component) by the addition of cetyl-pyridinium chloride (7). The active substance responsible for the strong adjuvant effect of the polysaccharide of K. pneumoniae type 1 Kasuya strain is its neutral polysaccharide frac-939

We extracted an R-form lipopolysaccharide (LPS) by the phenol-water method from Kkebsiella sp. strain LEN-111 (03-: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 MgCl2-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 MgCl2-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, NaCl, CaC12 or Zn(CH3COO)2, were used for precipitation of the LPS with ethanol in place of MgC92, the LPS did not form the hexagonal lattice structure. However, if the LPS precipitated with NaCl-ethanol was converted to the magnesium salt form after it was electrodialyzed, it formed the same hexagonal lattice structure as the LPS precipitated with MgC92-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. The cell wall lipopolysaccharides (LPS) of smooth gramnegative bacteria consist of the 0-specific polysaccharide, which is linked to the core polysaccharide, which in turn is linked to the lipid moiety termed lipid A. R mutants are blocked in the biosynthesis of the 0-specific polysaccharide in various steps. We previously studied the ultrastructure of Klebsiella 03 LPS isolated from Klebsiella sp. strain LEN-1 (03:Kl-) and showed that it consists principally of flat ribbonlike structures branching freely (average width, 16 nm; average thickness, 7 nm) and of a small proportion of spheres (diameter, 20 to 50 nm), both structures covered with fine hairy structures of an average length of approximately 10 nm (5). When the polysaccharide of Klebsiella 03 LPS was stained by periodic acid-thiosemicarbazide-silver proteinate (11), silver granules were deposited on the ribbonlike structures and around the spheres, suggesting that the 0-specific polysaccharide chain is located on their surface and that the fine hairy structures consist of the polysaccharide chain (5). Later we isolated a mutant strain LEN-111

Various uniform salt forms of an R-form lipopolysaccharide (LPS)

The molecular species composition of a-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of a-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatogramns were determined from the [M]+, [M-15]+, and [M-90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]', due to C2zC3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3-C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]'. The concentration of odd-and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different a units (C22:0, C24:0, and C26:0) were clearly determined by mass chromatography monitoring [M-15]+ ions and [B-29]+ ions, respectively. The molecular species composition of a-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of a-mycolic acid composition, and based on this the species were classified approximately into eight groups: (i) C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 a-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; (ii) C72 to C78 (av. cn. 75), dienoic with both C22 and C2A a units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); (iii) C72 to C80 (av. cn. 75 to 77), dienoic with C24 a-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); (iv) C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C2A a unit for M. agri and M. thermoresistibile (rapid growers); (v) C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C2M a unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and "M. novum") (slow growers); (vi) C76 to C84 (av. cn. 79 to 81), even-carbon-numbered dienoic with C24 a unit for MAIS tomplex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 a unit for M. szulgai, M. gordonae, and M. kansasii (chromnogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 a unit for M. bovis Ravenel and BCG and M. tuberculosis H37Rv. This study demonstrated that gas cht-omatography-mass spectrometric analysis of the molecular species composition of a-mycolic acid ca...