Crystallization of R-form lipopolysaccharides from Salmonella minnesota and Escherichia coli

Abstract

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...

Electron Microscopic Observation of Crystals of Escherichia coli K‐12 Lipopolysaccharide

“…2C). The spacing of the lattice fringes of the solid column crystals was very similar to that of crystals of S. minnesota Ra LPS (7). The spacing of the lattice fringes was found to correspond with the length of the c-axis value of LPS crystals (7).…”

Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

“…From the analyses of crystals by electron diffraction and X-ray diffraction it was suggested that the LPS crystals consist of hexagonal lattices with the lattice constant of 4.62 A for all these three kinds of LPSs and the long axis of 58.5, 84.7, and 87.5 A 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 the R core exhibit disordered structure or are in a random orientation (8). In contrast, S. minnesota S-form LPS possessing the O-antigen-specific polysaccharide and S. minnesota free lipid A obtained by acid hydrolysis of Re LPS did not crystallize under the same experimental conditions (8). Then E. coli K-12 LPS treated in the same way was found to form discoid crystals as well as hexagonal plate crystals and solid column crystals (9).…”

“…Previously we demonstrated that Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed threedimensional crystals, either hexagonal plates or solid columns, 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 4 C for 10 days (8). From the analyses of crystals by electron diffraction and X-ray diffraction it was suggested that the LPS crystals consist of hexagonal lattices with the lattice constant of 4.62 A for all these three kinds of LPSs and the long axis of 58.5, 84.7, and 87.5 A 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 the R core exhibit disordered structure or are in a random orientation (8).…”

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution

“…The LPS molecules appeared to have a tendency to associate with each other and form structures that looked ribbonlike when viewed face on and trilaminar when viewed edge on. More recently, Kato et al (19,20), who studied the packing order and conformational properties of LPS, showed that isolated R-form LPSs from Escherichia coli and a Klebsiella sp. formed regular arrays.…”