Tsuneharu Kuno

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: relationship between lattice formation and uniform salt forms.

Various uniform salt forms of an R‐form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN‐111 (O3‐:K1‐) were prepared and their ultra‐structure was examined. The LPS, which was extracted by the phenol‐water method, freed from contamination with RNA by treatment with RNase, and precipitated by addition of two volumes of 10 mM MgCl2‐ethanol, was used as the original preparation for uniform salt forms. The original LPS preparation formed a hexagonal lattice structure with a lattice constant of 14.9±0.2 nm. The LPS after electrodialysis retained the ability to form a hexagonal lattice structure, although its lattice constant was large (18.7±0.5 nm) and the lattice structure of the electro‐dialyzed LPS was labile at pH 8.0 in contrast to that of the original LPS preparation. The magnesium salt form of the LPS formed essentially the same ordered hexagonal lattice structure (lattice constant of 15.0±0.2 nm) as that of the original LPS preparation. The calcium and ammonium salt forms formed a hexagonal lattice structure, but the lattice constants of the calcium and ammonium salt forms were larger (18.6±0.6nm and 19.3±0.4nm, respectively) than that of the magnesium salt form. The sodium and potassium salt forms consisted of freely branching ribbon‐like structures with an average width of 13 nm and an average thickness of 9 nm. The triethylamine salt form consisted principally of short rods (10 nm × 9–13 nm).

In Vitro Hexagonal Assembly of Lipopolysaccharide of Escherichia coli K-12

We examined Escherichia coli K‐12 lipopolysaccharide (LPS), which is known to be an R‐form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K‐12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mm MgCl2‐ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2±0.3 nm and that of the calcium salt form 18.5±0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Structure of Various Uniform Salt Forms

Various uniform salt forms of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from culture supernatant were prepared as follows. Basic materials present in KO3 LPS were rigorously removed by electrodialysis and the electrodialyzed KO3 LPS was neutralized with NaOH, KOH, NH4OH, Ca(OH)2, tris(hydroxymethyl)aminomethane, or triethylamine. The ultrastructure of the uniform salt forms of KO3 LPS was examined using preparations stained with uranyl acetate. The sodium, potassium, ammonium, and trisaminomethane salt forms were structurally very similar to the natural form of KO3 LPS which consisted of a mixture of flat ribbon‐like structures (average width of 16 nm and average thickness of 7 nm) and spheres with various diameters, both covered with fine hairy structures. When KO3 LPS was converted to the triethylamine salt form, the ribbon‐like structures were distrupted into very small granules (7–9 nm × 9–15 nm). The calcium salt form consisted of particles and rods of various sizes and ribbon‐like structures which were markedly extended (maximum width of 50 nm) and presented irregular shapes. When converted to the calcium salt form, the ribbon‐like structures were extended and eventually divided into particles and rods. For reasons still unknown, the sodium salt of KO3 LPS was mostly stained positively with uranyl acetate in contrast to the natural form and the other uniform salt forms which were always negatively stained. In the positively stained preparation of the sodium salt form, it was clearly shown that the ribbon‐like structures consisted of a bilayer.

Staining of the O-Specific Polysaccharide Chains of Lipopolysaccharides with Alkaline Bismuth

granules occurred on the LPS (3). We also showed that Klebsiella O3 LPS and Salmonella minnesota S-form LPS were positively stained with ruthenium red, which is known to stain polysaccharides in the slime layer or the capsule of bacterial cells (5), whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-), Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by the treatment (4). Alkaline bismuth has been used for electron microscopic observation of glycogen granules in the liver cells (6). As far as we are aware, there has been no report dealing with application of alkaline bismuth to staining of the polysaccharide moiety of LPS. In the present study, we tried to stain LPS with alkaline bismuth and found that the staining procedure gave sufficient results for distinguishing between Sand R-form LPS lacking the

Stability of the Hexagonal Lattice Structure Formed by an R-Form Lipopolysaccharide of Klebsiella: Decrease in the Stability by Electrodialysis and Recovery by Addition of the Magnesium

The R‐form lipopolysaccharide (LPS) from Klebsiella strain LEN‐111 (O3‐:K1‐) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non‐electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon‐like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non‐electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Comparison with Other Lipopolysaccharides

Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed‐type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon‐like structures branching freely (average width 16 nm and average thickness 7 nm) and to contain a small proportion of spheres (diameter 20–50 nm), both structures covered with fine hairy structures (average length approximately 10 nm). When the polysaccharide of KO3 LPS was stained by the periodic acid‐thiosemicarbazide‐silver proteinate procedure, silver granules were deposited on the ribbon‐like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon‐like structures along their lateral margin.

Formation of a Hexagonal Lattice Structure by an R-Form Lipopolysaccharide of Klebsiella

An R‐form lipopolysaccharide (LPS) extracted from Klebsiella strain LEN‐111 (O3‐:K1‐) by the phenol‐chloroform‐petroleum ether method was compared with that extracted by the phenol‐water method in the ability to form a hexagonal assembly. The LPS which was extracted by the phenol‐water method and dialyzed against tap water to remove phenol showed ribbon‐like structures, and it formed a hexagonal lattice structure with a lattice constant of 14.5 ± 0.3 nm when it was precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. The LPS which was extracted by the phenol‐chloroform‐petroleum ether method and lyophilized consisted of ribbon‐like structures and their fragments and it often formed small pieces of a hexagonal lattice, although the LPS before lyophilization did not form such a lattice. When the LPS extracted by the phenol‐chloroform‐petroleum ether method was precipitated by addition of two volumes of 10 mM MgCl2‐ethanol, it formed essentially the same hexagonal lattice structure as that formed by the LPS extracted by the phenol‐water method. From these results it is concluded that the ability of the LPS to form a hexagonal lattice structure does not depend upon the method of its extraction from bacterial cells.

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.

Stability of the Hexagonal Lattice Structure Formed by an R-Form Lipopolysaccharide of Klebsiella: Study of Long-Range Stability

The R‐form lipopolysaccharide (LPS) from Klebsiella strain LEN‐111 (O3‐:K1‐) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2‐ethanol. The stability of this hexagonal lattice structure in long‐term incubation at 4 C was investigated. The hexagonal lattice structure was stable for at least 220 days when the LPS was suspended in distilled water, but it had been disintegrated into a rough mesh‐like structure when the LPS was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.5, at 4 C for 60 days. Half of the Mg bound to the LPS was released when the LPS was suspended in Tris buffer for 60 days, whereas Mg was not released when it was suspended in distilled water even for 220 days. By contrast, it was stable for at least 220 days in Tris buffer containing 5 mM MgCl2. The LPS suspended in Tris buffer for 60 days, at which time the structure had been disintegrated, could be restored to the original hexagonal lattice structure within 24 hr by addition of 5 mM MgCl2. From these results it is concluded that the hexagonal lattice structure of the LPS retains long‐range stability if Mg bound to the LPS is not released from the LPS.