From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with diferent densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.Sphingomonas paucimobilis is an aerobic gram-negative rod formerly belonging to the genus Pseudomonas. The new genus Sphingomonas was recently proposed by Yabuuchi et al. (21) because many taxonomical data, including the base sequence of the 16S rRNA, revealed that S. paucimobilis is apart from the fluorescent group pseudomonads. The most remarkable difference of S. paucimobilis from other gram-negative bacteria is the presence of glycosphingolipid, which is usually a membrane component of eukaryotic cells (4), as the component of cellular lipids and the absence of lipopolysaccharide. Lipopolysaccharide is an amphiphilic molecule and a major component of the outer membrane of gram-negative bacteria, localizing at the outer leaflet of the membrane (17, 18). For this reason lipopolysaccharide was believed to be an essential component for all gram-negative bacteria. However, we reported in our previous paper (9) that S. paucimobilis harbored monosaccharide-type and tetrasaccharide-type glycosphingolipids and contained no lipopolysaccharide-like molecule. Those unexpected findings on the cellular lipids of S. paucimobilis prompted us to investigate the cell surface structure of this gram-negative bacterium and the physiological roles of the glycosphingolipids. In the present paper the separation of an outer and a cytoplasmic membrane and the biochemical and electron microscopic investigations for the localization of the glycosphingolipids are described. MATERUILS AND METHODSBacterial strains and culture conditions. S. paucimobilis 1AM 12576 (originally NCTC 11030, type strain) was used in this study. Escherichia coli C600 (1) was used for the control experiment. Cells were grown in a medium and under conditions described previously (10). 284Isolation and separation of cell membranes. Membrane fractions were prepared by the method of Yamada et a...

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A procedure was developed to purify dermonecrotic toxin (DNT) from a sonic extract of a serotype D strain of Pasteurella multocida. Sonic extract containing DNT was applied to a DEAE-Sephacel column and eluted by a linear gradient of NaCI. Upon rechromatographing, fractions with dermonecrotic activity for guinea pigs were applied on a second Sephacel column, and a pooled fraction with the toxic activity was filtered through a Sephadex G-200 column. Pooled fractions with the toxic activity were subjected to polyacrylamide disc gel electrophoresis (PAGE), and the toxic substance was eluted from each sliced gel. Eluted fractions with the toxic activity were rechromatographed on a second Sephadex G-200 column, and a pooled fraction with high dermonecrotic activity was referred to as a purified DNT. The activity of purified DNT was increased by 1,000 times, and the average yield was about 1.8%. The purified DNT was homogeneous as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and thin-layer isoelectric focusing in polyacrylamide gels and gave a single band on PAGE and sodium dodecyl sulfate-PAGE. The molecular weight of the toxin was ca. 160,000 as determined by sodium dodecyl sulfate-PAGE. The isoelectric point of the toxin was ca. 4.7 to 4.8. Amino acid analysis of the purified DNT revealed that the toxin was composed of characteristically high proportions of glutamic acid, aspartic acid, glycine, proline, alanine, and leucine. The minimal necrotizing dose of the toxin was about 1 ng of protein, and the 50% lethal dose per mouse was 0.2 jug. The purified DNT was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde.

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.