Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238.

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.

The characterization and ultrastructure of two new strains of Butyrivibrio.

Strains B-385-1 and 2-33 are numerically important components rumen bacterial populations , but they have remained (taxonomically) undefined. In spite of some resemblance to Selenomonas ruminantium in their cell size and in their formation of tufts of flagella, they more closely resemble Butyrivibrio fibrisolvens in the subpolar location of their flagella, in their guanine + cytosine content, and in most biochemical characteristics, including butyrate formation. Cells of these strains stain Gram negative, as do both Selenomonas and Butyrivibrio, but their cell walls closely resemble those of Butyrivibrio in their Gram-positive type of molecular architecture and in their cleavage pattern in freeze-etching. Cells of these strains and of B. fibrisolvens have a very thin (ca. 12 nm) peptidoglycan cell wall; thus, they fail to retain the crystal violet complex of the Gram stain and stain Gram negative. This important structural characteristic of their cell walls places strains B-385-1 and 2-33 within the genus Butyrivibrio and certain morphological and biochemical characteristics distinguish them from B. fibrisolvens.

4-O-(1-carboxyethyl)-D-galactose. A new acidic sugar from the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain 49.

The structure of a new acidic sugar from the extracellular polysaccharide of Butyrivibrio fibrisolvens strain 49 was determined as 4-O-(1-carboxyethyl)-D-galactose on the basis of 13C-n.m.r. and 1H-n.m.r. spectroscopy, m.s. and chemical degradation studies.

Lipopolysaccharide-stimulated PGE2 release from human monocytes. Comparison of lipopolysaccharides prepared from suspected periodontal pathogens.

Lipopolysaccharides (LPS) prepared from the suspected periodontal pathogens Actinobacillus actinomycetemcomitans (A. a.), Bacteroides gingivalis, B. intermedius and Wolinella recta were compared to Salmonella typhimurium LPS for their capacity to stimulate prostaglandin E2 (PGE2) release from human monocytes. Counterflow isolated monocytes were cultured with control medium or media containing 10 micrograms/ml LPS. Media were then exchanged every 24 hours for a total of 72 hours. Salmonella and Wolinella LPS preparations demonstrated seven-fold greater PGE2 release than B. gingivalis and two-fold greater than A. a. and B. intermedius. PGE2 release was found to decrease over time with all LPS preparations except Wolinella. The potency of the LPS preparations is tentatively ranked as follows: Wolinella greater than or equal to Salmonella greater than A. a. greater than B. intermedius greater than or equal to B. gingivalis. These findings demonstrate that LPS preparations from suspected periodontal pathogens are capable of stimulating PGE2 release from human monocytes. The high potency and prolonged stimulation of PGE2 release with Wolinella LPS suggests unusual toxic properties that may exert a greater influence in the pathogenesis of destructive periodontal diseases.

Characterization of Wolinella spp., Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens by polyacrylamide gel electrophoresis.

The small asaccharolytic, nonpigmenting gram-negative rods of the human oral cavity are difficult to differentiate from each other. Protein profiles of sonicated cells of Wolinella species, Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens were obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with a silver stain. The gels were scanned with a laser densitometer, and the similarity of strains was computed by determining correlation coefficients of normalized densities along the gels. The strains were grouped by cluster analysis of the correlation coefficients. All species were distinct from each other. Several groups were found within E. corrodens. A colored silver stain was found to highlight species differences and appears to be useful in the rapid identification of fresh isolates.

Fatty acid composition of oral isolates of Selenomonas.

Fatty acids of 16 strains of Selenomonas isolated from the human oral cavity were examined by gas-liquid chromatography. The strains showed similar patterns, characterized by the presence of straight-chain fatty acids in the range C11 to C18. Fatty acids of odd-numbered carbon atoms dominated and the major acids were n-pentadecanoate and 3-hydroxytridecanoate. The general fatty acid pattern of Selenomonas differed distinctly from those of other previously analysed anaerobic or microaerophilic Gram-negative bacilli.

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria.

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.

Purification and quantitative chemical analysis of cell wall peptidoglycans of Leptotrichia buccalis.

Peptidoglycans of Leptotrichia buccalis ATCC 14201 and ATCC 19616 were isolated by extraction with sodium dodecyl sulfate and subsequent digestion of the sodium dodecyl sulfate-insoluble residue with proteases and alpha-amylase. Cell wall fractions obtained by sodium dodecyl sulfate extraction and protease digestion were highly contaminated by a glucose polymer. The polyglucose was removed by alpha-amylase treatment, and the peptidoglycans were left behind. Analyses with amino acids and amino sugars of the cell wall fractions and peptidoglycan specimens revealed that D-glutamic acid, D-alanine, L-alanine, meso-2,6-diaminopimelic acid (A2pm), muramic acid, and glucosamine were the principal components. The dinitrophenylation method revealed that about half of the A2pm residue had a free amino group, and analysis by hydrazinolysis showed that a small part of alanine and A2pm was located at the C terminal. The above results indicate that one of the amino groups of the A2pm residue at one strand of the stem peptide subunit cross-linked to the carboxyl group of alanine of the neighboring strand. It was thus revealed that the peptidoglycans of L. buccalis belong to the A1 gamma type of the classification by Schleifer and Kandler (Bacteriol. Rev. 36:407-477).

Composition of peptidoglycans in Bacteroidaceae: determination and distribution of lanthionine.

Peptidoglycans of organisms belonging to the strictly anaerobic family Bacteroidaceae were investigated for the presence of lanthionine. Different procedures for the quantitation of lanthionine were compared. Performic acid and peroxide oxidation procedures on 35S-labeled peptidoglycan from Fusobacterium nucleatum Fev1 resulted in low yields of cysteic acid (42 and 60%, respectively) and many other additional unidentified oxidation products. Lanthionine was, however, recovered in high yield (89% or more) from acid hydrolysates of unoxidized peptidoglycans. Lanthionine was found exclusively in some species of Fusobacterium, in particular F. nucleatum, F. necrophorum, F. russi, and F. gonidiaformans, for which lanthionine may be ascribed a function as a taxonomic marker. Peptidoglycans of these bacteria are thus proposed to belong to a new chemotype, assigned A1 delta. One strain of Fusobacterium, F. mortiferum VPI 0473 contained both lanthionine and diaminopimelic acid in about equal proportions. Species of F.plauti had a composition atypic of gram-negative cells. Chemotypic differences were also indicated among the species of Bacteroides investigated. Thus, some species contained lysine and not diaminopimelic acid as the major dibasic amino acid (e.g., F. asaccharolyticus). It is concluded that peptidoglycans of gram-negative organisms constitute a somewhat more heterogeneous group than hitherto assumed.

Lysozyme digestion and chemical characterization of the peptidoglycan of Fusobacterium nucleatum Fev 1.

The amino acids in the peptidoglycan of Fusobacterium nucleatum Fev 1 are D-glutamic acid, meso-lanthionine, and D- (42%) and L-alanine (58%). About 70% of the lanthionine residues were not susceptible to dinitrophenylation, evidently because they are involved in cross-linkages. Consequently, lysozyme digestion of the peptidoglycan yielded 20 to 25% uncross-linked disaccharide tri- and tetrapeptides. A chemical analysis of isolated glycopeptides indicated that the structure of the building block of this peptidoglycan is N-acetylgucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid-meso-lanthionine(-D-alanine). I present evidence which supports the classification of the F. nucleatum Fev 1 peptidoglycan as a new A1 delta, directly cross-linked, meso-lanthionine-containing peptidoglycan.

Lipopolysacharides from anaerobic gram-negative rods.

The chemical composition and antigenic specificity of the cell wall lipopolysaccharides of the two main groups of Bacteroidaceae, Fusobacterium and Bacteroides species, are reviewed. Cell wall lipopolysaccharides from Fusobacterium species contain 2-keto-3-deoxy-octonate and heptose, but lipopolysaccharides isolated from Bacteroides species do not.

Identification and characterization of species of the family Bacteriodaceae by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was used to compare protein profiles of members of the family Bacteriodacceae. Cell-free extracts were prepared and the protein components separated by multistage polyacrylamide gel electrophoresis. The profiles were distinct and reproducible thus allowing identification of species, subspecies, and minor strain differences. Lyophilization of the cell-free extracts did not alter the major protein components. The results indicate that the techniques used may be useful in conjunction with conventional tests in identification at the species level.