A synthetic compound (506), 0(1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at * Corresponding author. (08:K27, Re-mutant) in both effective dose and degree of stimulation. However, compound 406, like the biosynthetic lipid A precursor, was far less active in characteristic endotoxic properties such as pyrogenicity and the activity to prepare the local Shwartzman reaction, though the compound had as high a lethal toxicity as F515 lipid A for mice loaded with galactosamine (5). Galanos et al. (6) have reported essentially the same results in experiments with the same synthetic preparations. The marked differences noted between the synthetic compound, 406, and the natural product, F515 lipid A, regarding biological activities that more or less characterize endotoxic lipopolysaccharide (LPS) and its lipid A moiety have been assumed to be attributable to the following fact: all four acyl groups bound to the amino groups at C-2 and C-2' positions 225

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The endotoxin of a heptoseless mutant of Salmonella minnesota R595 was extracted with phenol-water. Most of this material was found distributed in the insoluble fraction of the extract. The results showed that the R595 endotoxin behaved as a lipid rather than as a lipopolysaccharide (LPS). The preparation, although it does not contain 0-specific polysaccharides, does contain 2-keto-3-deoxyoctonic acid (KDO), hexosamine, and several other unidentified compounds. Therefore, the term "glycolipid" is used in this paper instead of lipopolysaccharide. The crude glycolipid fraction, which was soluble in a mixture of chloroform-methanol (8:2), was purified by a procedure including fractionation with organic solvents and by different-column chromatographic methods. Although a chromatographic fraction of the glycolipid showed homogeneity in most systems investigated, the presence of contaminants could not be excluded. Chemical analysis of the glycolipids showed the absence of hexoses and heptoses. Constituents which were found were hexosamine, KDO, fatty acids, and phosphorus, which showed a relatively simple chemical composition. Partial acidic hydrolysis of the glycolipid yielded hexosamine-phosphates, as described in "Lipid A" fractions of smooth LPS preparations. Thin-layer chromatography of the partially hydrolyzed glycolipid showed a pattern similar to "Lipid A" fractions of other strains. The biological activity of the glycolipid was at the same level as that of other gram-negative endotoxins. Pyrogenicity, Shwartzman reactivity, and chick embryo LD5, values were as high or higher than those of purified Serratia marcescens endotoxin preparations, but mouse LD50 measurements gave significantly lower results. MATERIALS AND METHODS Bacterial strain. The heptoseless mutant strain, S. minnesota R595, used in this experiment was obtained through the courtesy of Otto Luderitz. Cultivation of bacteria. The bacterial cells of the R595 strain were cultivated in a broth culture medium consisting of 1.5% tryptone (Difco), 0.5% beef extract (Difco), 0.3% sodium chloride, 0.23% Na2HPO4, 0.5% yeast extract (Difco), and 0.3% glucose, which was described by Schlecht and Westphal (22). A 10-liter portion of the culture medium, adjusted to pH 7.4, was transferred to a 15-liter bottle; it was inoculated with 500 ml of 18-hr broth culture of the R595 strain, and then incubated at 37 C for 6.5 hr. The mixtures in the bottles were stirred at 250 rev/min, and the aeration rate was 4 liters/min. A 1-liter portion of 5% phenol was added to the culture fluid after it had been cooled with tap water and allowed to stand overnight. The cells, harvested by centrifugation, were washed with saline and kept in a freezer. The yield of the wet cells was about 115 g/10 liters.

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.Lipopolysaccharide (LPS) bearing some different chemical features from Salmonella LPS has been found in several species of oral gram-negative bacteria. In the earlier studies by Hofstad et al (5, 6) and Kasper et al (9,12), LPS that lacks heptose and 3-deoxy-D-manno-octulosonic acid (KDO) has been isolated from Bacteroides species. Recently, an unusual fatty acid composition including branched and odd-numbered long chain 3-hydroxy acids was identified in the LPS of B. fragilis and of related Bacteroides species by Wollenweber et al (23). A fatty acid spectrum similar to that of B. fragilis LPS was also shown in a purified LPS preparation of B. gingivalis by Nair et al (13). More recently, another unusual LPS preparation containing only nonhydroxy fatty acids was isolated from Eikenella corrodens by Progulske et al (15,16).

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