Structure,composition, and biogenesis of prasinophyte cell coverings

Summary The cell body and flagellar surfaces of certain green flagellates are covered by non-mineralized scales. Scale structure has been widely used in the systematics of this group of algae commonly known as the Prasinophyceae. The special importance of the flagellar hairs as a taxonomic marker is discussed. We summarize current knowledge about the structure and chemical composition of these scales with emphasis on thecate flagellates. Scales consist mainly of acidic polysaccharides involving unusual 2-keto sugar acids. Glycoproteins as minor components are mainly involved in mediating scale subunit and scale-membrane interactions and species specific glycosylation patterns exist. In thecate prasinophytes the elaboration of 3-deoxy-manno-2-octulosonic acid and galacturonic acid side chains presumably favours a complex of thecal scales with calcium ions and thus extracellular coalescence of the scales to a rigid cell wall. Scales are formed within the Golgi apparatus (GA) and especially in thecate prasinophytes scale formation (i.e., during flagellar regeneration) represents an excellent model system for GA function. Movement of developing scales through the GA requires cisternal progression. Biogenesis of scales involves mainly polysaccharide synthesis, whereas about 50% of the scale-associated glycoproteins are added from a pre-existing pool. Possible functions of prasinophyte scales are briefly discussed.Abbreviations Dha 3-deoxy-lyxo-2-heptulosaric acid - DSA Datura stramonium agglutinin - ER endoplasmic reticulum - GA Golgi apparatus - GNA Galanthus nivalis agglutinin - Kdo 3-deoxy-manno-2-octulosonic acid - MeKdo 3-deoxy-5-O-methyl-manno-2-octulosonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Isolation,purification, and characterization of flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae)

Summary Flagellar scales from the green flagellateTetraselmis striata (Prasinophyceae) were isolated, purified by isopycnic cesium chloride-gradient and zonal sucrose gradient centrifugation and their structure and biochemical composition investigated. Three types of flagellar scales were purified to more than 90% purity, a fourth type up to 75% purity. In addition to the previously known types of flagellar scales (pentagonal scales, rod-shaped scales, hair-scales), a novel scale type (i.e., the knotted scales) was discovered. New information about the asymmetric structure of the rod-shaped scales is presented and consequently they are renamed man scales. Flagellar scales consist mainly of carbohydrate (50–70%), significant amounts of protein (11% of dry weight) were found only in pentagonal scales. The main sugars (90%) of the pentagonal and man scales are the unusual 2-keto-sugar acids 3-deoxy-5-O-methyl-2-octulosonic acid (5 OMeKDO), 3-deoxy-2-heptulosaric acid (DHA), and 3-deoxy-2-octulosonic acid (KDO), the knotted scales contain as major sugars galactose and arabinose in addition to KDO and 5 OMeKDO but lack DHA. 13 major polypeptides were identified in flagellar scales by one-dimensional SDS-PAGE, 11 of these are of high molecular mass (>116 kDa). While the majority of polypeptides was found associated with pentagonal scales, at least 4 polypeptides were tentatively assigned to the hair-scales and knotted scales.Abbreviations CSF crude scale fraction - PS pentagonal scales - MS man scales - HS hair-scales - KS knotted scales - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DHA 3-deoxylyxo-2-heptulosaric acid - 5 OMeKDO 3-deoxy-5-O-methyl-manno-2-octulosonic acid - KDO 3-deoxy-manno-2-octulosonic acid - GA Golgi apparatus     

Identification of 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methyl-manno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid in the cell wall (theca) of the green alga Tetraselmis striata Butcher (Prasinophyceae)

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3-deoxy-manno-2-octulosonic acid (42%). In addition two other 2-keto-sugar acids are present, namely, 3-deoxy-5-O-methyl-manno-2-octulosonic acid (7%), the first methylated derivative of 3-deoxy-manno-2-octulosonic acid found in nature, and 3-deoxy-lyxo-2-heptulosaric acid (11%). The characterization of the three 2-keto-sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC-MS and 500-MHz 1H-NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC-MS. Other monosaccharides occurring in the cell wall are D-galacturonic acid (14%), D-galactose (4%), D-gulose (2%), D-glucose (1%) and L-arabinose (1%).     

THE CELL WALL (THECA) OF TETRASELMIS STRIATA (CHLOROPHYTA): MACROMOLECULAR COMPOSITION AND STRUCTURAL ELEMENTS OF THE COMPLEX POLYSACCHARIDES

Isolated cell walls (thecae) from the scaly flagellate green alga Tetraselmis striata Butcher contain the unusual 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (Kdo), 3-deoxy-5-O-methyl-manno-2-octulosonic acid (5OMeKdo), and 3-deoxy-lyxo-2-heptulosaric acid (Dha). In addition, galacturonic acid, galactose, gulose, and arabinose are present. EDTA-extraction yielded an insoluble fraction that retains the shape of the cell walls and contains no 2-keto-sugar acids. Methylation analysis demonstrated the presence of terminal hexose, GalA, Dha, and Kdo as well as 2-substituted hexose, 4-or 8-substituted Kdo, and 4,8-disubstituted Kdo. However, most of the carbohydrate material (about 60%) was not methylated. Periodate oxidation of the cell wall preparation showed the presence of 2-substituted Gul, 4- or/and 5-substituted and 7- or/and 8-substituted Kdo, which is in agreement with the methylation analysis. Again, a significant amount of carbohydrate material was not degraded, indicating complex substitution patterns. Oligosaccharides were generated by partial hydrolysis and fractionated using gel permeation chromatography and high-pH anion-exchange chromatography. Oligosaccharides contained either GalA and Kdo, or Gal, Kdo, Dha, and Gul, respectively. The structure of a GalA and Kdo containing disaccharide was established using ¹ H NMR spectroscopy.     

Lipopolysaccharide of Providencia rettgeri

The chemical constitutional analysis of the lipopolysaccharide (LPS) isolated from Providencia rettgeri was carried out. Polyacrylamide gel electrophoresis using sodium dodecylsulfate or sodium deoxycholate showed that the lipopolysaccharide mostly consisted of short sugar chains. The lipid A was precipitated out after mild acid hydrolysis of LPS. From the supernatant degraded polysaccharide and unsubstituted core fractions were isolated. Compositional analysis of the core material revealed the presence of galacturonic acid, galactose, glucose, glucosamine, l-glycero-d-manno-heptose, 3-deoxy-d-manno-octulosonic acid, alanine and phosphorus. Methylation analysis of the core material indicated the presence of terminal units of glucose, galacturonic acid and glucosamine. The chemical structure of the lipid A was elucidated. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester and glycosidically-bond phosphate residues. The ester-bound phosphate was found to be substituted by a 4-amino-4-deoxy-l-arabinosyl residue. The amino groups of the backbone disaccharide are N-acylated by 3-O-(14:0)14:0 and 3-O-14:0.Two hydroxyl groups of the disaccharide are esterified by 3-O-(14:0)14:0 and 3-O-14:0. The taxonomical importance of these structural details will be discussed.Abbreviations LPS lipopolysaccharide - l-d-heptose l-glycero-d-manno-heptose - dOclA 3-deoxy-d-manno-octulosonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - PS degraded polysaccharide - glc-ms combined gas liquid chromatography-mass spectrometry     

Localization of the enzyme 3-deoxy-d-manno-2-octulosonic acid aldolase

Summary Several strains of Gram-negative microorganisms were screened for maximum 3-deoxy-d-manno-2-octulosonic acid (KDO) aldolase (EC 4.1.2.23) activity. Although this enzyme has been noted to be inducible on special medium, no induction was found. By centrifugation studies the KDO aldolase was found to be localized in the cell wall or membrane fraction. The enzyme activity was very susceptible to small amounts of detergent in solution. Offprint requests to: M.-R. Kula     

Scale-associated glycoproteins of Scherffelia dubia (Chlorophyta) form high-molecular-weight complexes between the scale layers and the flagellar membrane

Flagellar scales were isolated from the flagellate green alga Scherffelia dubia. The flagellar scales consist mainly of acidic polysaccharides (70%) and glycoproteins (10%), and monosaccharide analyses show that the scales contain high amounts of unusual 2-keto-sugar acids. Approximately, 72 mol% of total carbohydrate is 3-deoxy-manno-2-octulosonic acid, 3-deoxy-5-O-methylmanno-2-octulosonic acid and 3-deoxy-lyxo-2-heptulosaric acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of at least 18 different scale-associated proteins (SAPs), ranging in apparent molecular mass from 77 kDa to over 300 kDa. Lectin blot analyses performed in combination with glycosidase treatment, showed that SAPs contained N-glycans of the highmannose type and the hybrid type, as well as a complex type that was not immunologically related to higher-plant complex glycans. Most of the SAPs were present in two or possibly three high-molecular-weight complexes. In these complexes, individual polypeptides are cross-linked by disulfide bridges. A polyclonal antibody was raised against a SAP of 126 kDa (SAP 126), a glycoprotein present in a high-molecular-weight complex. The SAP126 antibody was used to localize the protein between scale layer and flagellar membrane. We suggest that these high-molecular-weight complexes link scales to the flagellar membrane.Abbreviations AAA Aleuria aurantia agglutinin - DSA Datura stramonium agglutinin - DTT dithiothreitol - GNA Galanthus nivalis agglutinin - RCA Ricinus communis agglutinin - SAP Scale-associated protein - TBS Tris-buffered saline Dedicated to Professor Eberhard Schnepf on the occasion of his 65th birthday.This work was supported by the Deutsche Forschungsgemeinschaft and an Alexander von Humboldt Foundation research award to L. Perasso. We thank G. Noat for providing the anti--glucosidase and anti-pectin methyl esterase antibodies.     

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC–electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3–18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.     

Der Rhizoplast vonTetraselmis cordiformis sichtbar auch im Lichtmikroskop

Summary The massive rhizoplast of the prasinophycean algaTetraselmis cordiformis, well known from the electron microscopy investigations, is shown on living cells by light microscopy using the phase-contrast. It appears as a dark girdle attached to the surface of the nuclear envelope.

     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

Extraction and Characterization of the Smooth-Type Lipopolysaccharide from Fusobacterium nucleatum JCM 8532 and Its Biological Activities

The lipopolysaccharide (LPS) of Fusobacterium nucleatum JCM 8532 was isolated by hot-phenol water extraction. Most of the LPS was extracted in the phenolic phase and shown to be the smooth-type, whereas the aqueous phase contained mainly rough-type LPS. The chemical composition of the LPS was similar to that reported in other studies, but D -quinovosamine, which may be a major component of O-antigenic polysaccharide, and 3-deoxy-D -manno-2-octulosonic acid (Kdo) were detected for the first time by gas chromatography-mass spectrometry. The biological activities of smooth-type LPS, including limulus activity, lethal toxicity, pyrogenicity, and B lymphocyte mitogenicity, were comparable to those of enterobacterial LPS. Smooth-type LPS inhibited the cell growth and DNA synthesis of adult and fetal human gingival fibroblasts in a dose-dependent manner, suggesting that LPS may play a role in the occurrence of human gingivitis.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

Microalgal growth enhancement by levoglucosan isolated from the green seaweed <Emphasis Type="Italic">Monostroma nitidum</Emphasis>

Microalgal growth was enhanced by the addition of levoglucosan to the culture medium. The growth-enhancing compound levoglucosan was isolated from the green seaweed Monostroma nitidum using water extraction, molecular fractionation, DEAE-cellulose column chromatography, and high-performance liquid chromatography. Yield of the compound from seaweed powder was 5 × 10⁻³% (w/w). At 10 mM concentration, levoglucosan enhanced cell growth and the specific growth rate of all feed microalgal species tested (Chaetoceros gracilis, Chlorella ellipsoidea, Dunaliella salina, Isochrysis galbana, Nannochloris oculata, Navicula incerta, Pavlova lutheri, Tetraselmis suecica) in most culture media by approximately 150%. Cellular fatty acid profiles and cell size differed marginally between cultures with and without levoglucosan.     

Microalgae for use in tropical aquaculture I: Gross chemical and fatty acid composition of twelve species of microalgae from the Northern Territory,Australia

Twelve species of microalgae, isolated from north Australian marine, freshwater and hypersaline environments, were grown under controlled conditions of temperature, pH, photon flux density and salinity, and analysed for ash, total protein, water soluble carbohydrates, chlorophylla, total lipids, total fatty acids and fatty acid composition. Highest levels of the polyunsaturated fatty acid eicosapentaenoic acid [20:5(n-3)] were found in the marine diatoms.Nitzschia (frustulum) andN. closterium (23.1% and 15.2% of total fatty acids, respectively). None of the species studied had levels of docosahexaenoic acid [22:6(n-3)] greater than 1.1 % of total fatty acids. None of the chlorophyte species contained significant levels of either 20:5(n-3) or 22:6(n-3). The highest total fatty acid concentration of all species in the study was found in the freshwater chlorophyte speciesScenedesmus dimorphus (105 mg g^–1 dry wt). The hypersaline speciesDunaliella salina had the highest total lipid content (28.1% dry wt), followed byN. closterium, N. (frustulum) andNavicula sp. (24.2–27.8% dry wt).Chlamydomonas sp. had the highest protein content (66.9% dry wt).N. (frustulum) was highlighted as a possible useful source of lipids and polyunsaturated fatty acids in mixed microalgal diets for mariculture organisms used in tropical aquaculture.Author for correspondence     

Interaction of a living system with an electromagnetic field

Irradiation of the inoculum with 50 Hz frequency electromagnetic field for 3 h practically did not influence the growth of suspension culture of one-celled green algae Dunaliella tertiolecta and Tetraselmis viridis. The irradiation for 4 h led to approximately 30% reduction of growth. After 6-hour irradiation the growth of Dunaliella tertiolecta was reduced by 50% and the growth of Tetraselmis viridis was reduced by 70%. The irradiation of hydras for 3 h showed no visible effect. The irradiation for 4 h stimulated a little the growth and regeneration of injured hydras. The effect of 24 h irradiation resulted in the shortening of lifetime of uninjured and injured hydras. The effect of regeneration was not observed.     

Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a -1, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3 and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3 differentiates E. carotovora lipid A from that of other gram-negative bacteria.Abbreviations LPS lipopolysaccharide - GlcN glucosamine - KDO 3-deoxy-d-manno-octulosonic acid - FAB-MS fast atom bombardment mass spectrometry - u atomic mass unit     

The genus-specific lipopolysaccharide epitope of Chlamydia is assembled in C. psittaci and C. trachomatis by glycosyltransferases of low homology

Chlamydiae possess a genus-specific epitope that is located on the lipopolysaccharide (LPS) and is composed of a 3-deoxy-d -manno-octulosonic acid (Kdo) trisaccharide of the sequence αKdo-(2→8)–αKdo–(2→4)-αKdo. In Chlamydia trachomatis, this trisaccharide is biosynthetically generated through the action of a multi-functional Kdo-transferase encoded by the gene gseA. gseA of Chlamydia psittaci 6BC was cloned and expressed in a rough mutant (Re chemotype) of Escherichia coli (strain F515) that contains an LPS with only two α2→4-linked Kdo residues. Recombinant strains were able to add the immunodominant Kdo residue in a α2→8-linkage to the parental LPS, as determined by SDS–PAGE and Western blot analysis using a monoclonal antibody against the genus-specific epitope. The DNA sequence of gseA was determined and aligned to that published recently for C. trachomatis serovar L2. Most surprisingly, the two deduced amino acid sequences shared only an overall homology of 67%. Thus, gseA exhibits species specificity at the DNA level, whereas its gene product results in the synthesis of a carbohydrate antigen with genus specificity.     

Chemical analysis of Azospirillum lipopolysaccharides

Lipopolysaccharides (LPS) were extracted by hot phenol-water from five strains each of Azospirillum lipoferum and Azospirillum brasilense. Rhamnose, glucose, glucosamine and 3-deoxy-d-mannooctulosonic acid were comon sugar constituents of all LPS preparations. 2-O-Mefucose, 3-O-Me-fucose, 3-O-Me-rhamnose and 2-O-Megalactose were found in LPSs of some A. brasilense strains. Fatty acid spectra from all LPSs studied were almost identical with predominance of 3-hydroxymyristic and 3-hydroxypalmitic acids. 3-Hydroxypalmitic acid was the only amide-linked fatty acid. Lipopolysaccharides isolated from A. brasilense showed higher heterogeneity in sugar composition than those from A. lipoferum.Abbreviations glc gas liquid chromatography - ms mass spectrometry - LPS lipopolysaccharide - dOclA 3-deoxy-d-mannooctulosonic acid - 3-OH-16:0 3-hydroxypalmitic acid - nir^- nitrite reductase negative - nir⁺ nitrite reductase positive     

IMMUNOLOCALIZATION OF CENTRIN IN OXYRRHIS MARINA (DINOPHYCEAE)1

A new polyclonal antibody was raised against centrin isolated from the flagellate green alga Spermatozopsis similis (Chlorophyta; anti-SSC). It stains by immunofluorescence and immunoelectron microscopy well-known reference systems for centrin like the nucleus–basal body connectors in Chlamydomonas reinhardtii (Chlorophyta) and the system II fibers (rhizoplasts) of Scherffelia dubia (Chlorophyta). In addition, it recognizes in immunoblots a single 20-kDa protein in isolated cytoskeletons of Spermatozopsis similis and Tetraselmis striata (Chlorophyta) as well as purified centrin isolated from Tetraselmis striata. Using this antibody, centrin was localized in whole cells and isolated cytoskeletons of Oxyrrhis marina Dujardin (Dinophyceae) by immunofluorescence and immunogold electron microscopy. In the flagellar apparatus of O. marina, five different structures were antigenic. Four short fibers (connectives 1–4) link the basal bodies to the four major fibrous flagellar roots, which do not cross-react with anti-centrin. The most prominent of the labeled structures (connective 5), a crescent-shaped fiber, extends from the flagellar canal of the transverse flagellum along the base of the tentacle to the flagellar canal of the longitudinal flagellum, interconnecting the distal parts of the microtubular roots/bands in the basal apparatus. For most of its length, it underlies and is connected to a transversely oriented subamphiesmal microtubular band. In immunoblot analyses, anti-SSC recognizes only a single 20-kDa protein in cytoskeletons of O. marina. Functional and phylogenetic aspects of centrin-containing structures in dinoflagellates are discussed.     

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.