Characterization of a structural series of lipid A obtained from the lipopolysaccharides of Neisseria gonorrhoeae. Combined laser desorption and fast atom bombardment mass spectral analysis of high performance liquid chromatography-purified dimethyl derivatives

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of serum-sensitive strains of Neisseria gonorrhoeae was fractionated on a silicic acid column to yield the hexaacyl and pentaacyl MLAs. The dimethyl derivative of the hexaacyl MLA was analyzed by proton nuclear magnetic resonance spectroscopy. The dimethyl esters of hexaacyl and pentaacyl MLAs were further purified by reverse-phase high performance liquid chromatography, and all of the peaks were analyzed by laser desorption mass spectrometry. Considerable structural information was obtained by laser desorption mass spectrometry due to three kinds of specific fragmentations of the sugar at the reducing end. Two major fractions were also analyzed by positive ion fast atom bombardment mass spectrometry. High performance liquid chromatography was able to separate the dimethyl MLA according to number, nature, and position of the fatty acyl groups. Since almost no structural information is available, the mass spectra of the samples were interpreted on the basis of the established structure of a model lipid A (hexaacyl MLA derived from Salmonella minnesota). Thirteen different structures of dimethyl MLA were identified. The four prominent dimethyl MLAs found in the fractionated samples were M1 (Mr = 1463), M2 (Mr = 1479), M3 (Mr = 1661), and M4 (Mr = 1677). These MLAs appear to have a 1'----6 linked glucosamine disaccharide backbone. The most prominent hexaacyl MLA was M3. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage and lauroxymyristate at the 2- and 2'-positions in amide linkage of the glucosamine disaccharide. The most abundant pentacyl MLA was M2. We propose that it contains hydroxylaurate at the 3- and 3'-positions in ester linkage, lauroxymyristate at the 2'-position in amide linkage, and hydroxymyristate at the 2-position in amide linkage of the disaccharide. The lipid A of N. gonorrhoeae appeared to differ from that of the Salmonella strains by the presence of shorter-chain fatty acids and by the normal fatty acid distribution in the reducing and distal subunits.     

Location of fatty acids in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was initially purified by silicic acid column chromatography to yield a single major pentaacyl MLA fraction. This fraction was methylated and further purified by reverse-phase high performance liquid chromatography to yield three prominent peak fractions. Laser desorption mass spectrometry of these three fractions allowed us to complete the important structural analysis of lipid A from this source. Three structurally distinct forms of dimethyl MLA were identified where Mr = 1447, 1449, and 1451 atomic mass units. These forms differed only by the presence or absence of unsaturation and keto group in the fatty acids. We established that the acyloxyacyl group (either delta 7-tetradecenoyloxytetradecanoate or tetradecanoyloxytetradecanoate) and the 3-ketotetradecanoate or hydroxytetradecanoate occupied the 2'- and 2-positions of the glucosamine disaccharide, respectively. Analysis of several minor fractions suggests that there is considerable structural heterogeneity in the MLA. With this new knowledge, the study of the structure-to-function relationship of the reported lack of toxicity of lipopolysaccharide from R. sphaeroides can be completed.     

Nature, type of linkage, and absolute configuration of (hydroxy) fatty acids in lipopolysaccharides from Xanthomonas sinensis and related strains.

The fatty acids present in lipopolysaccharides from Xanthomonas sinensis were identified as decanoic, 9-methyl-decanoic, 2-hydroxy-9-methyl-decanoic, 2-hydroxy-9-methyl-decanoic, D-3-hydroxy-decanoic, D-3-hydroxy-9-methyl-decanoic, D-3-hydroxy-dodecanoic, and D-3-hydroxy-11-methyl-dodecanoic acid. These fatty acids occur in the lipid A component where they are bound through ester and amide linkages to glucosamine residues. All types of fatty acids are ester bound; however, part of D-3-hydroxy-dodecanoic and D-3-hydroxy-11-methyl-dodecanoic acid is also involved in amide linkage. The hydroxyl groups of ester-linked 3-hydroxy fatty acids are not substituted. Similar fatty acid patterns were obtained from lipopolysaccharides of nine other Xanthomonas species.     

Isolation, purification and properties of an intermediate in 3-deoxy-D-manno-octulosonic acid--lipid A biosynthesis.

Incomplete lipid A has been purified from a mutant of Salmonella typhimurium which is temperature-sensitive both in synthesis of 3-deoxy-D-manno-octulosonic acid 8-phosphate (dOclA-8-P) and in growth. Pulse-chase experiments have shown that the incomplete lipid A molecule is the intermediate in the biosynthesis of the dOclA-lipid A portion of lipopolysaccharides. The purification procedure included DEAE-cellulose chromatography and electrodialysis. A highly water-soluble precursor material was obtained, consisting of glucosamine, phosphate and 3-hydroxymyristic acid in a molar ratio of 1:1.2:2.1. Labeling experiments as well as chemical degradation procedures revealed the precursor molecule to be composed of a diphosphorylated glucosamine-disaccharide carrying two amide-linked and two ester-linked 3-hydroxymyristic acids. In contrast to the complete dOclA-lipid A part, the intermediate lacks 3-deoxy-D-manno-octulosonic acid as well as nonhydroxylated fatty acids. On the basis of these findings a pathway for the final steps in dOclA-lipid A biosynthesis is proposed.     

Analytical studies of lipopolysaccharide and its derivatives from Salmonella minnesota R595. III. Reappraisal of established methods

Established methods for analysis of components of lipopolysaccharides were assessed. Optimal release of glucosamine from lipopolysaccharide occurs after hydrolysis in 6 M hydrochloric acid at 100°C for 4 h and fatty acids are best released by treatment with boron trifluoride/methanol at 100°C for 6 h. The semicarbazide assay for 3-deoxy-d-manno-octulosonic acid was modified to give results comparable to those from the periodate/thiobarbituric acid method. It was concluded that each molecule of lipopolysaccharide from Salmonella minnesota R595 contains two octulosonic acid residues and only four fatty acids, on average. There are two amide-linked hydroxyacids, together with, on average, 0.5 residues of ester hydroxyacid and a total of 1.5 residues of ester-linked normal fatty acids. This conclusion differs from the accepted view of Salmonella lipid A, but is supported by NMR results.     

Lipid A mutants of Salmonella typhimurium. Purification and characterization of a lipid A precursor produced by a mutant in 3-deoxy-D-mannooctulosonate-8-phosphate synthetase.

We describe here the isolation, purification, and structural characterization of a lipid A precursor synthesized under nonpermissive conditions by a mutant of Salmonella typhimurium conditionally defective in the synthesis of the 3-deoxy-D-mannoctulosonate (2-keto-3-deoxyoctonate, KDO) region of the lipopolysaccharide. The precursor was isolated free from lipopolysaccharide, murein, and phospholipids by extraction of delipidated cells with 90% phenol/CHCL3/petroleum ether. The molecule was recovered from the phenol phase after precipitation of lipopolysaccharide with H2O and subsequently purified by DEAE-cellulose chromatography. Structural analyses showed that the lipid A precursor is a phosphorylated glucosamine disaccharide containing one ester and two amide-linked residues of beta-hydroxymyristate. In contrast to lipid A, the precursor disaccharide lacks ester-linked 12:0 and 14:0 fatty acids as well as KDO. The molecule contains 2 phosphate residues both of which were identified as phosphomonoesters by 31P NMR spectroscopy. One of the phosphomonoesters is located in position 1 of the reducing terminal glucosamine residue; the location of the other phosphomonoester was not determined. The structure of the precursor provides strong support for the conclusion that KDO incorporation occurs at an early stage in lipid A biosynthesis prior to the incorporation of ester-linked saturated fatty acids.     

Fatty acid composition of Mesorhizobium huakuii lipopolysaccharides. Identification of 27-oxooctacosanoic acid

Lipopolysaccharides of three Mesorhizobium huakuii strains carried a number of amide-linked 3-hydroxylated fatty acids including: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0, 3-OH-i-21:0, 3-OH-22:0, 3-OH-23:0 and unsaturated 3-OH-22:1. The first three of the above mentioned acids are the main amide-linked fatty acids in the LPS preparations. The main ester-bound fatty acids comprise 16:0, i-17:0, 18:0, 20:0 and 27-OH-28:0. Among minor constituents of lipid A 25-OH-26:0 and 29-OH-30:0 together with some non-polar fatty acids were found. Additionally, the presence of 4-oxo-20:0, 4-oxo-i-21:0 and 4-oxo-22:0 amide-bound fatty acids as well as the 27-oxo-28:0 ester-linked fatty acid were proved. To our knowledge oxo fatty acids are rare constituents of lipopolysaccharides and 27-oxo-28:0 was found for the first time in the LPS preparations from members of Rhizobiaceae.     

Studies of lipid A fractions from the lipopolysaccharides of Pseudomonas aeruginosa and Pseudomonas alcaligenes

Lipid A fractions from Pseudomonas aeruginosa and Pseudomonas alcaligenes have similar compositions and structural features. By means of hydrazinolysis of the parent lipopolysaccharides and partial hydrolysis of the deacylation products, it was established that both lipids are derived from the β-(1→6)-linked disaccharide of glucosamine. Phosphorylated derivatives of the disaccharide from Ps. aeruginosa were also characterized. The lipids differ mainly in the absence of hexadecanoic acid and 2-hydroxydodecanoic acid from the lipid from Ps. alcaligenes. Evidence that in Ps. aeruginosa these acids are ester-linked to residues of 3-hydroxyalkanoic acids (including 3-hydroxydecanoic acid) was obtained. Heterogeneity of lipid A fractions was indicated by t.l.c., and by gel filtration of de-O-acylation products from mild alkaline methanolysis of the lipids.     

Biochemical studies on the cell wall lipopolysaccharides (O-antigens) of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba).

Lipopolysaccharides were isolated from the cell walls of Vibrio cholerae 569 B (Inaba) and El-tor (Inaba). Chemical analysis revealed the presence of glucose, fructose, mannose, heptose, rhamnose, ethanolamine, fatty acids and glucosamine. The lipopolysaccharides do not contain 2-keto-3-deoxyoctonate, the typical linking sugar of polysaccharide and lipid moieties of enterobacterial lipopolysaccharides. Galactose, a typical core polysaccharide component of many gram-negative bacteria was also absent from lipopolysaccharides of these organisms. By hydrolysis in 1% acetic acid, the lipopolysaccharides have been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Components of degraded polysaccharide and lipid A moiety were identified and determined. The lipid A fractions contained fatty acids, phosphorus and glucosamine. All the neutral sugars detected in lipopolysaccharides were shown to be the constituents of its polysaccharide moiety. The fatty acid analysis of lipopolysaccharide and lipid A showed the presence of both hydroxy and non hydroxy acids. They were different from those of lipids extracted from cell walls before the extraction of lipopolysaccharides. 3-Hydroxylauric and 3-hydroxymyristic acids predominated in lipopolysaccharide and lipid A of Vibrio cholerae and El-tor (Inaba).     

Identification of ester-linked fatty acids of bacterial endotoxins by negative ion fast atom bombardment mass spectrometry

Negative ion fast atom bombardment mass spectrometry (NI-FAB/MS) was employed to characterize the fatty acids esterified to the lipid A backbone of lipopolysaccharides (LPS) of gram-negative bacteria. LPS and their chemically derived lipid A produced readily detectable fragment ions characteristic of fatty acids. The NI-FAB/MS method is specific, yielding ions indicative of ester- but not of amide-bound fatty acids. The mass spectra of Enterobacteriaceae LPS revealed the presence of lauric (m/z 199), myristic (m/z 227), palmitic (m/z 255), and 3-hydroxymyristic (m/z 243) acids. Pseudomonas aeruginosa LPS gave distinctive fragment ions indicative of 3-hydroxydecanoic (m/z 187), lauric, and 2-hydroxylauric (m/z 215) acids. The Neisseria gonorrhoeae LPS could be distinguished from the others due to the presence of ester-linked 3-hydroxylauric acid. All of the LPS gave abundant ions of m/z 177 and 159, which were derived from diphosphoryl substituents. The use of NI-FAB/MS thus allowed rapid identification of lipid A esterified fatty acids without chemical derivatization or gas chromatographic analysis.     

Nature, type of linkage, quantity, and absolute configuration of (3-hydroxy) fatty acids in lipopolysaccharides from Bacteroides fragilis NCTC 9343 and related strains.

The main fatty acids present in lipopolysaccharides from Bacteroides fragilis NCTC 9343 were identified as 13-methyl-tetradecanoic, D-3-hydroxypentadecanoic, D-3-hydroxyhexadecanoic, D-3-hydroxy-15-methyl-hexadecanoic, and D-3-hydroxyheptadecanoic acids. Of these, 13-methyl-tetradecanoic acid is exclusively ester bound, and 3-hydroxy-15-methyl-hexadecanoic acid is exclusively involved in amide linkage. The other 3-hydroxy fatty acids are both ester and amide bound. All 3-hydroxy fatty acids possess the D configuration, and the 3-hydroxyl group of ester-linked 3-hydroxy fatty acids is not substituted. Lipopolysaccharides of related Bacteroides species (B. thetaiotaomicron, B. ovatus, B. distasonis, and B. vulgatus) showed a fatty acid spectrum with both similar and distinct features compared to that of B. fragilis lipopolysaccharides.     

Determination of monosaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortia by capillary GC-MS of their dimethyl disulphide adducts

Monounsaturated fatty acid double-bond position and geometry have been determined for microbial monocultures and complex microbial consortia by capillary GC-MS of their dimethyl disulphide (DMDS) adducts. The technique has permitted (i) chromatographic separation and positive identification of adducts derived fromcis/trans isomers, (ii) characterization of long-chain monounsaturated components (up to 26:1), and (iii) the identification of a wide range of monounsaturated components derived from methanotrophic soil material. The methanotrophic soil sample contained a high relative proportion of the novel phospholipid ester-linked fatty acid 18:1Δ 10c. The DMDS procedure offers a simple and rapid approach that can be routinely applied to microbial fatty acids derived from environmental samples and monocultures.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

Interaction of spinach leaf adenosine diphosphate glucose alpha-1,4-glucan alpha-4-glucosyl transferase and alpha-1,4-glucan, alpha-1,4-glucan-6-glycosyl transferase in synthesis of branched alpha-glucan

Salmonella newington lipopolysaccharide extracted from a cell paste grown up from a single smooth clone was fractionated by chromatography on DEAE-cellulose in the presence of 1% Triton X-100 into seven lipopolysaccharide fractions which differed in their degrees of polymerization of the repeating unit of the O-antigen side chain and in their substitution with ester phosphate. Several of the lipopolysaccharide fractions were hydrolyzed in 1% acetic acid at 100 °C to cleave the linkage between the polysaccharide and lipid A parts of the structure. The polysaccharide fractions from each of the purified lipopolysaccharides could be further fractionated on DEAE-cellulose columns to yield a number of peaks of polysaccharide having monosaccharide ratios quite distinct from those of the parent lipopolysaccharide. The results show a high degree of structural heterogeneity in the original lipopolysaccharide.     

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.     

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.     

Occurrence of an Unusual Hopanoid-containing Lipid A Among Lipopolysaccharides from Bradyrhizobium Species

The chemical structures of the unusual hopanoid-containing lipid A samples of the lipopolysaccharides (LPS) from three strains of Bradyrhizobium (slow-growing rhizobia) have been established. They differed considerably from other Gram-negative bacteria in regards to the backbone structure, the number of ester-linked long chain hydroxylated fatty acids, as well as the presence of a tertiary residue that consisted of at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative. The structural details of this type of lipid A were established using one- and two-dimensional NMR spectroscopy, chemical composition analyses, and mass spectrometry techniques (electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry and MALDI-TOF-MS). In these lipid A samples the glucosamine disaccharide characteristic for enterobacterial lipid A was replaced by a 2,3-diamino-2,3-dideoxy-d-glucopyranosyl-(GlcpN3N) disaccharide, deprived of phosphate residues, and substituted by an α-d-Manp-(1→6)-α-d-Manp disaccharide substituting C-4′ of the non-reducing (distal) GlcpN3N, and one residue of galacturonic acid (d-GalpA) α-(1→1)-linked to the reducing (proximal) amino sugar residue. Amide-linked 12:0(3-OH) and 14:0(3-OH) were identified. Some hydroxy groups of these fatty acids were further esterified by long (ω-1)-hydroxylated fatty acids comprising 26–34 carbon atoms. As confirmed by mass spectrometry techniques, these long chain fatty acids could form two or three acyloxyacyl residues. The triterpenoid derivatives were identified as 34-carboxyl-bacteriohopane-32,33-diol and 34-carboxyl-2β-methyl-bacteriohopane-32,33-diol and were covalently linked to the (ω-1)-hydroxy group of very long chain fatty acid in bradyrhizobial lipid A. Bradyrhizobium japonicum possessed lipid A species with two hopanoid residues.     

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species.

Two strains of Bacteroides asaccharolyticus and two strains of Bacteroides fragilis were analyzed for total fatty acid, total lipid fatty acid, and total bound fatty acid profiles. Extracted lipids and defatted cell residues were subjected to sequential alkaline and acid methanolyses to distinguish ester- and amide-linked fatty acids in each fraction. In the lipid fractions, all the ester-linked fatty acids were nonhydroxylated, whereas all of the amide-linked fatty acids were hydroxylated. In the nonextractable fractions, both hydroxy and nonhydroxy fatty acids were found in both ester and amide linkage, although hydroxy acids predominated. The fatty acid profiles of the bound fractions differed widely from those of the lipid fractions. Bound fatty acid represented approximately 10% of the total cellular fatty acids.     

Studies on the lipopolysaccharide of a virulent and an avirulent strain of Vibrio vulnificus.

Vibrio vulnificus is a marine bacterium associated with both primary septicemias and wound infections in humans. The lipopolysaccharides of a virulent and an avirulent strain of Vibrio vulnificus were compared with respect to their chemical constituents and electrophoretic characteristics. 2-Keto-3-deoxyoctonic acid, a normal constituent of the lipopolysaccharide of typical Enterobacteriaceae, was not found in the lipopolysaccharide of either strain. Hexadecenoate (C16:1) was the predominant fatty acid of the lipid A moiety of the lipopolysaccharides and of the membrane phospholipids of both strains. Hydroxy fatty acids composed 44% of the total fatty acids of the lipid A of the avirulent and 40% of those in the virulent strain. In addition, odd-numbered fatty acids were detected in both lipopolysaccharides. The electrophoretic profile was similar for both strains, but demonstrated no "ladder-like" pattern characteristic of "smooth" lipopolysaccharides. The result of this study showed no significant differences between the lipopolysaccharides of the virulent and avirulent strains of Vibrio vulnificus. The possible role for lipopolysaccharide in pathogenesis of Vibrio vulnificus infections is discussed.