Minor Glycolipids Research Articles

Salinigranum marinum sp. nov. and Halohasta salina sp. nov., halophilic archaea isolated from sediment of a marine saltern and inland saline soil.

Two halophilic archaeal strains, ZS-10T and GSL13T, were isolated from the Zhoushan marine saltern in Zhejiang, and an inland saline soil from the Tarim Basin, Xinjiang, PR China, respectively. The cells of strain ZS-10T were pleomorphic while those of strain GSL13T were rod-shaped. Both of them stained Gram-negative and formed red-pigmented colonies on agar plates and their cells lysed in distilled water. The optimum growth of strain ZS-10T was observed at 40 °C, 3.4 M NaCl, 0.03 M MgCl2 and pH 7.5, while that of strain GSL13T was at 37 °C, 3.1 M NaCl, 0.5 M MgCl2 and pH 7.5. Phylogenetic and phylogenomic analyses indicated that these two strains were related to Salinigranum and Halohasta, respectively. Strains ZS-10T and GSL13T could be differentiated from the current members of Salinigranum and Halohasta based on the comparison of diverse phenotypic characteristics. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values among strain ZS-10T and current species of Salinigranum were 75.8-78.6 %, 80.6-81.9 % and 24.3-26.1 %, respectively. These values between strain GSL13T and current species of Halohasta were 78.4-80.8 %, 79.8-82.8% and 22.7-25.7 %, respectively, clearly below the threshold values for species demarcation. The polar lipids of strain ZS-10T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulphated mannosyl glucosyl diether (S-DGD-1), while those of strain GSL13T were phosphatidic acid, PG, PGP-Me, phosphatidylglycerol sulphate and S-DGD-1. The polar lipid profile of strain GSL13T was identical to those of Halohasta, whereas strain ZS-10T did not contain the minor glycolipids detected in the current Salinigranum species. The phenotypic, phylogenetic and genome-based results suggested that strains ZS-10T (=CGMCC 1.12868T=JCM 30241T) and GSL13T (=CGMCC 1.15214T=JCM 30841T) represent two novel species, for which the names Salinigranum marinum sp. nov. and Halohasta salina sp. nov. are proposed.

Natronomonas halophila sp. nov. and Natronomonas salina sp. nov., two novel halophilic archaea.

Two halophilic archaeal strains, C90T and YPL13T, were isolated from a salt lake and a salt mine in PR China. The two strains were found to form two clusters (97.5 and 89.5 % similarity between them, respectively) separating them from the three current members of the genus Natronomonas (95.4-97.0 % and 86.6-89.3 % similarity, respectively) on the basis of the 16S rRNA and rpoB' gene sequence similarities and phylogenetic analysis. Diverse phenotypic characteristics differentiate strains C90T and YPL13T from current Natronomonas members. The polar lipids of strain C90T were phosphatidic acid, phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulphate, two unidentified glycolipids, a major glycolipid and a minor glycolipid, while those of strain YPL13T were PG, PGP-Me, two unidentified phospholipids and a glycolipid. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between the two strains were 79.8 and 27.1 %, respectively, which were much lower than the threshold values proposed as a species boundaries (ANI 95-96 % and isDDH 70 %), which revealed that the two strains represent two novel species; these values (ANI 76.6-80.0 % and isDDH 21.6-27.0 %) of the strains examined in this study and the current members of Natronomonas are much lower than the recommended threshold values, suggesting that strains C90T and YPL13T represent two genomically different species of Natronomonas. These results showed that strains C90T (=CGMCC 1.13738T=JCM 32961T) and YPL13T (=CGMCC 1.13884T=JCM 31111T) represent two novel species of Natronomonas, for which the names Natronomonas halophila sp. nov. and Natronomonas salina sp. nov. are proposed.

Salinibaculum litoreum gen. nov., sp. nov., isolated from salted brown alga Laminaria.

A novel Gram-stain-negative, aerobic and rod-shaped halophilic archaeon, designated HD8-45T, was isolated from the red brine of salted brown alga Laminaria produced at Dalian, PR China. According to the results of 16S rRNA gene and rpoB' gene sequence comparisons, strain HD8-45T showed the highest sequence similarity to the corresponding genes of Salinirussus salinus YGH44T (95.1 and 85.2 % similarities, respectively), Halovenus aranensis EB27T (91.2 and 86.0 % similarities, respectively). The low sequence similarity and the phylogeny implied the novel generic status of strain HD8-45T. Genomic relatedness analyses showed that strain HD8-45T were clearly distinguished from other species in the order Halobacteriales, with average nucleotide identity, amino acid identity and in silico DNA-DNA hybridization values not more than 75.1, 65.6 and 21.5 %. The polar lipid pattern contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two major glycolipids and two minor glycolipids. The two major glycolipids and a minor glycolipid were chromatographically identical to disulfated mannosyl glucosyl diether, sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. The major respiratory quinones were menaquinone MK-8 and MK-8(H2). The DNA G+C content was 62.0 mol% (Tm) and 61.9 mol% (genome). All these results showed that strain HD8-45T represents a novel species of a new genus in the order Halobacteriales, for which the name Salinibaculum litoreum gen. nov., sp. nov. is proposed. The type strain of Salinibaculum litoreum is HD8-45T (=CGMCC 1.15328T=JCM 31107T).

Proposal of Pedobacter nototheniae sp. nov., isolated from the spleen of a black rock cod (Notothenia coriiceps, Richardson 1844) from the Chilean Antarctica

A pink pigmented, Gram-negative, rod-shaped, non-spore-forming bacterium (strain 36B243T), was isolated from the spleen of a black rock cod (Notothenia coriiceps, Richardson 1844) in the Chilean Antarctica. Strain 36B243T has a 5.26Mb chromosome with a DNA G + C content of 35.4mol%. The draft genome includes the prediction and annotation of 4585 coding genes, and 46 tRNA, 1 tmRNA, and 2735 hypothetical proteins. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain 36B243T into the genus Pedobacter with high sequence similarity to the type strains of Pedobacter sandarakinus (97.5%) and Pedobacter petrophilus (97.1%). Sequence similarities to type strains of all other current Pedobacter species were below 97.1%. Predominant fatty acids are summed feature 3 (C16:1ω7c and/or C16:1ω6c) and iso-C15:0 followed by iso-C17:0 3-OH and C16:0. The major respiratory quinone was menaquinone MK-7. The polar lipid profile contained the major lipids phosphatidylethanolamine, five unidentified aminolipids, two lipids lacking a functional group and two minor glycolipids and one lipid lacking a functional group. An alkali-stable lipid was present. The polyamine pattern contained the predominant compound sym-homospermidine. Characterization by 16S rRNA gene sequence analysis, physiological parameters, pigment analysis, ubiquinone, polar lipid, and fatty acid composition revealed that strain 36B243T represents a new species of the genus Pedobacter. For this reason, we propose the name Pedobacter nototheniae sp. nov. with the type strain 36B243T (= LMG 30634T = CCM 8855T = CIP 111622T).

Haloparvum sedimenti gen. nov., sp. nov., a member of the family Haloferacaceae.

Two extremely halophilic archaeal strains, DYS4T and Y2, were isolated from rock salt of the Jiangcheng Salt Mine, Yunnan province, China. Cells of the two strains were non-motile, pleomorphic rods and Gram-stain-negative. The cells produced light red-pigmented colonies. Strains DYS4T and Y2 required 2.6-3.4 M NaCl, pH 7.5- 8.0 and 42 ºC in aerobic conditions for optimal growth. Mg2+ was required for growth. The major polar lipids of both strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl glucosyl diether. An unidentified minor glycolipid spot was present for strains DYS4T and Y2, which differentiates them from the closely related species of the genera Halorubrum and Halopenitus. The lipid core of the glycolipid was sn-2,3-diphytanylglycerol (C20C20). The sequence similarity of the 16S rRNA gene demonstrated that the closest relatives of strains DYS4T and Y2 were Halorubrum aidingense 31-hongT (94.1 % and 93.6 % 16S rRNA gene sequence similarity to DYS4T and Y2, respectively) and Halopenitus salinus SKJ47T (93.4% and 93.1%). Phylogenetic analysis of the 16S rRNA gene and the rpoB' gene revealed that strains DYS4T and Y2 formed an independent lineage closely related to the genera Halorubrum and Halopenitus. The DNA G+C contents of strains DYS4T and Y2 were 68.2 and 67.0 mol%, respectively. The DNA-DNA relatedness value between strains DYS4T and Y2 was 90.0 ± 0.5%, while that between strain DYS4T and other closest relatives was less than 26 % (19 ± 0.7 % for Halorubrum aidingense 31-hongT and 25 ± 0.3% for Halopenitus salinus SKJ47T). The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains DYS4T and Y2 (=CGMCC 1.15000=JCM 30892) represent a novel species of a new genus within the family Haloferacaceae, for which the name Haloparvum sedimenti gen. nov., sp. nov. is proposed. The type strain of the type species is DYS4T (=CGMCC 1.14998T=JCM 30891T).

Halosiccatus urmianus gen. nov., sp. nov., a haloarchaeon from a salt lake.

A novel, orange-pigmented, halophilic archaeon, strain DC8T, was isolated from Urmia salt lake in north-west Iran. The cells of strain DC8T were non-motile and pleomorphic, from small rods to triangular or disc shaped. The novel strain needed at least 2.5 M NaCl and 0.02 M MgCl2 for growth. Optimal growth was achieved at 4.0 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.5 and 45 °C, respectively, and it was able to grow over a pH range of 7.0 to 8.5 and a temperature range of 25 to 55 °C. Analysis of the 16S rRNA gene sequence showed that strain DC8T was a member of the family Halobacteriaceae; however, its similarity was as low as 90.1 %, 89.3 % and 89.1 % to the most closely related haloarchaeal taxa, including type species of members of the genera Halosimplex, Halobaculum and Halomicrobium, respectively. The G+C content of its DNA was 68.1 mol%. Polar lipid analyses revealed that strain DC8T contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and phosphatidic acid. One unknown phospholipid, two major glycolipids and one minor glycolipid were also detected. The only quinone present was MK-8 (II-H2). The physiological, biochemical and phylogenetic differences between strain DC8T and other extremely halophilic archaeal genera with validly published names supported that this strain represents a novel species of a new genus within the family Halobacteriaceae, for which the name Halosiccatus urmianus gen. nov., sp. nov. is proposed. The type strain is strain DC8T ( = IBRC-M 10911T = CECT 8793T).

Examination of the structures of several glycerolipids from marine macroalgae by NMR and GC-MS.

Several classes of glycerolipids were isolated from the total lipids of the algae Saccharina cichorioides, Eualaria fistulosa, Fucus evanescens, Sargassum pallidum, Silvetia babingtonii (Ochrophyta, Phaeophyceae), Tichocarpus crinitus, and Neorhodomela larix (Rhodophyta, Florideophyceae). The structures of these lipids were examined by nuclear magnetic resonance (NMR) spectroscopy, including 1D ((1) H and (13) C) and 2D (COSY, HSQC and HMBC) experiments. All of the investigated algae included common galactolipids and sulfonoglycolipids as the major glycolipids. Minor glycolipids isolated from S. cichorioides, T. crinitus, and N. laris were identified as lyso-galactolipids with a polar group consisted of the galactose. Comparison of the (1) H NMR data of minor nonpolar lipids isolated from the extracts of the brown algae S. pallidum and F. evanescens with the (1) H NMR data of other lipids allowed them to be identified as diacylglycerols. The structures of betaine lipids isolated from brown algae were confirmed by NMR for the first time. The fatty acid compositions of the isolated lipids were determined by gas chromatography-mass spectrometry.

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

Analysis of dietary phytochemicals needs to be applauded: Glycosylated plant sterols

AbstractIn this issue of European Journal of Lipid Science and Technology, Lampi and co‐workers [p. 656–669] report the content of glycosylated plant sterols (steryl glycosides and acylated steryl glycosides) as minor glycolipids found in common edible plants.See accompanying article http://dx.doi.org/10.1002/ejlt.201200033

Steryl glycosides and acylated steryl glycosides in plant foods reflect unique sterol patterns

AbstractGlycosylated plant sterols (steryl glycosides, SG and acylated steryl glycosides, ASG) are minor glycolipids found in a variety of plants at different levels and in different compositions. They have until now been far less studied than other sterol conjugates, though they possess at least the same potential health benefits as free or esterified plant sterols. Additionally, due to the more hydrophilic conjugated part, they may possess further beneficial properties in forming water‐soluble structures. We studied the content and sterol profiles of glycosylated sterols in various plant foods to be able to better understand their levels in human diets, as well as to identify sources with high levels and/or unique sterol profile for possible extraction for ingredients. Glycosylated sterols contributed up to almost 60% of total sterols in e.g. potatoes, indicating that they indeed are an important group of sterols in plants. The ratio of ASG and SG varied from 0.4 to 3.6, indicating significant differences in the proportions of these two conjugates in various plants. Furthermore, extremely large differences were observed in the sterol profiles not only between but also within different plants, which indicates that the sterol profile of the glycosylated sterols does not always reflect the total sterol composition in the same plant tissue.Practical applications: Glycosylated sterols have hydrophilic properties owing to the carbohydrate moiety of the conjugate. As a consequence, they could potentially be introduced to water‐based functional foods (unlike the hydrophobic free sterols and sterol esters in current applications). Various plant materials or side streams of food processes were shown to be good sources of these glycosylated plant sterols, and could be used as sources for extraction of glycosylated sterols for food ingredients. Furthermore, the detailed compositional information reported here will provide a basis to select also the desired sterol composition for the product. Finally, the data presented in this paper is invaluable for estimating the contribution of different foods as sources of glycosylated sterols in the diet.See commentary by Jiménez‐Escrig [p. 615–616]

The putative phosphatase All1758 is necessary for normal growth, cell size and synthesis of the minor heterocyst-specific glycolipid in the cyanobacterium Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 differentiates nitrogen-fixing heterocysts arranged in a periodic pattern when deprived of a fixed source of nitrogen. In a genetic screen for mutations that prevent diazotrophic growth, open reading frame all1758, which encodes a putative serine/threonine phosphatase, was identified. Mutation of all1758 resulted in a number of seemingly disparate phenotypes that included a delay in the morphological differentiation of heterocysts, reduced cell size, and lethality under certain conditions. The mutant was incapable of fixing nitrogen under either oxic or anoxic conditions, and lacked the minor heterocyst-specific glycolipid. Pattern formation, as indicated by the timing and pattern of expression from the promoters of hetR and patS fused transcriptionally to the gene for green fluorescent protein (GFP), was unaffected by mutation of all1758, suggesting that its role in the formation of heterocysts is limited to morphological differentiation. Transcription of all1758 was constitutive with respect to both cell type and conditions of growth, but required a functional copy of all1758. The reduced cell size of the all1758 mutant and the location of all1758 between the cell division genes ftsX and ftsY may be indicative of a role for all1758 in cell division. Taken together, these results suggest that the protein encoded by all1758 may represent a link between cell growth, division and regulation of the morphological differentiation of heterocysts.

Halomicrobium zhouii sp. nov., a halophilic archaeon from a marine solar saltern

A halophilic archaeon, strain TBN51(T), was isolated from a marine solar saltern in Jiangsu, China. The colonies were red-pigmented and the cells were pleomorphic, motile and Gram-staining-negative. The strain was able to grow at 20-55 °C (optimum 42 °C), in the presence of 1.4-5.1 M NaCl (optimum 2.6 M), with 0-1.0 M MgCl(2) (optimum 0.05 M) and at pH 5.5-9.5 (optimum pH 7.0). Cells lysed in distilled water; the minimal NaCl concentration to prevent such lysis was 8 % (w/v). The major polar lipids of strain TBN51(T) were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and an unidentified glycolipid. The latter lipid and a minor glycolipid also detected in the novel strain were chromatographically identical to sulfated and non-sulfated mannosyl glucosyl diether, respectively. Analysis revealed that strain TBN51(T) had three dissimilar 16S rRNA genes. Phylogenetic analysis based on the sequences of these genes indicated that the novel strain was most closely related to Halomicrobium mukohataei JCM 9738(T) (89.2-94.8 % sequence similarity) and Halomicrobium katesii DSM 19301(T) (88.8-94.8 %). In similar comparisons of rpoB' gene sequences, strain TBN51(T) also appeared most closely related to Hmc. mukohataei JCM 9738(T) (88.5 % sequence similarity) and Hmc. katesii DSM 19301(T) (88.1 %). The genomic DNA G+C content of strain TBN51(T) was 69.1 mol%. The results of DNA-DNA hybridizations indicated that strain TBN51(T) represented a novel species since it showed relatedness values of only 23 % with Hmc. mukohataei JCM 9738(T) and 21 % with Hmc. katesii DSM 19301(T). It was concluded that strain TBN51(T) represents a novel species of the genus Halomicrobium, for which the name Halomicrobium zhouii sp. nov. is proposed. The type strain is TBN51(T) ( = CGMCC 1.10457(T) = JCM 17095(T)).

Halogranum gelatinilyticum sp. nov. and Halogranum amylolyticum sp. nov., isolated from a marine solar saltern, and emended description of the genus Halogranum

Two extremely halophilic archaeal strains, designated TNN44(T) and TNN58(T), were isolated from Tainan marine solar saltern near Lianyungang city, Jiangsu province, China. Cells of the two strains were pleomorphic and Gram-stain-negative; colonies were red-pigmented. Strains TNN44(T) and TNN58(T) were able to grow at 20-50 °C (optimum 37 °C for both), in the presence of 1.4-5.1 M NaCl (optimum 3.4-3.9 M NaCl) and at pH 5.5-9.0 (optimum pH 6.5-7.0); neither strain required Mg(2+) for growth. Cells lysed in distilled water. On the basis of 16S rRNA gene sequence analysis, strains TNN44(T) and TNN58(T) were related closely to Halogranum rubrum RO2-11(T) (96.2 and 97.2 % similarity, respectively). The polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate (trace), and one major glycolipid and one minor glycolipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively; other trace unidentified lipids were also detected. The DNA G+C content of strains TNN44(T) and TNN58(T) was 64.0 and 62.0 mol%, respectively. The level of DNA-DNA relatedness between strains TNN44(T) and TNN58(T) was 37.2 %, and these two strains showed a low level of DNA-DNA relatedness with Halogranum rubrum RO2-11(T) (40.6 and 44.4 %, respectively). Two novel species of the genus Halogranum are proposed to accommodate these two strains, Halogranum gelatinilyticum sp. nov. (type strain TNN44(T) = CGMCC 1.10119(T) = JCM 16426(T)) and Halogranum amylolyticum sp. nov. (type strain TNN58(T) = CGMCC 1.10121(T) = JCM 16428(T)).

Positive and negative tandem mass spectrometric fingerprints of lipids from the halophilic Archaea Haloarcula marismortui

Lipids from the extremely halophilic Archaea, Haloarcula marismortui, contain abundant phytanyl diether phospholipids, namely archaetidic acid (AA), archaetidylglycerol (AG), archaetidylglycerosulfate (AGS), with mainly archaetidylglycerophosphate methyl ester (AGP-Me). These were accompanied by a triglycosyl archaeol (TGA), lacking characteristic sulfate groups. Tandem-mass spectrometry was employed to provide fingerprints for identifying these known lipids, as well as small amounts of unsaturated phospholipids. These contained 3 and 6 double bonds in their archaeol moiety, suggested by negative tandem-MS of intact phospholipids, as indicated by differences between their pseudo-molecular ion and specific fragment ions designated as pi(2). The core ether lipids were confirmed by electrospray ionization mass spectrometry (ESI-MS) as 2,3-di-O-phytanyl-sn-glycerol (C20, C20), which gave rise to a precursor-ion at m/z 660 [M+Li](+), and its fragment ion at m/z 379 [M+Li](+), consistent with mono-O-phytanyl-glycerol. Furthermore, lithiated ions at m/z 654 (MS(1)), 379 (MS(2)) and m/z 648 (MS(1)), 373 (MS(2)), combined with (1)H/(13)C NMR chemical shifts at delta 5.31-121.6 (C2/2'-H2/2'), 5.08-124.9 (C6/6'-H6/6') and 5.10-126.0 (10/10'-H10/10') confirmed the presence of unsaturated homologs of archaeol. We carried out a comprehensive study on the lipids present in cells of H. marismortui. We used positive and negative ESI-MS with tandem-MS, which served as a fingerprint analysis for identifying the majority of component lipids.

Glycolipids from the red alga Chondria armata (Kütz.) Okamura

Three distinct fractions containing polar glycolipids (PF(1-3)) were isolated from the chloroform soluble fraction of crude methanolic extract of red alga Chondria armata (Kütz.) Okamura on gel chromatography over Sephadex LH20. Their structure was elucidated by multidimensional nuclear magnetic resonance (NMR) techniques like 1H, 1H correlation spectroscopy (COSY), 1H, 1H total COSY (TOCSY), 1H, 13C heteronuclear multiple quantum coherence (HMQC), and 1H, 13C heteronuclear multiple bond correlation (HMBC) complemented by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. The coupling constant of the anomeric proton in 1H NMR spectrum and sign of rotation indicated an exclusive configuration of the sugar molecules in the glycerolipids. Major glycolipids were identified as (2R)-2-O-(5,8,11,14-eicosatetranoyl)-3-O-alpha-d-galactopyranosyl-sn-glycerol (GL2), its pentacetate (GL1), and (2R)-1-O-(palmitoyl)-2-O-(5,8,11, 14,17-eicosapentanoyl)-3-O-beta-d-galactopyranosyl-sn-glycerol (GL3). Each was methanolysed to give the same galactosylglycerol which on ESI-MS provided a pseudomolecular ion at m/z 309 representing deacylated glycolipid with the sodiated sugar moiety. Additionally, six minor glycolipids were also identified on the basis of ESI-MS. These include a 1,2-di-O-acyl-3-O-(acyl-6'-galactosyl)-glycerol (GL1a), sulfonoglycolipids 2-O-palmitoyl-3-O-(6'-sulfoquinovopyranosyl)-glycerol (GL2a) and its ethyl ether derivative (GL2b), 1-oleoyl-2-palmitoyl-3-O-galactosyl glycerol (GL3a), and 1,2-diacyl phosphatidyl glycerol (GL3b). GL1, GL1a, and GL2b are new to the literature. The novelty of the remaining identified compounds lies in the diversity of their fatty acid composition. Antimicrobial properties of these glycolipids against pathogens were evaluated. The yeast Candida albicans and the bacteria Klebsiella sp. were as sensitive as the standard Nystatin and antibiotic Streptomycin against PF3. Considerable activity was expressed by the same metabolite against the fungus Cryptococcus neoformans as compared to the control. Weak activity against the bacteria Shigella flexineri and Vibrio cholerae and the fungus Aspergillus fumigatus was also observed. Fraction PF2 was weakly active against some strains whereas all of them were resistant to its acetyl derivative PF1. Antimicrobial activity of glycolipids is being reported here for the first time.

Isolation and Comparative Analysis of Glycolipid Fractions in Bifidobacteria

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (Rf) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875T), Rhodococcus equi PCMT 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H2O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H2O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G1 and G2), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part.

Previously, we showed that quantitatively minor several glycolipids totaling only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97-112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779-86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.

The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood group antigen

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transfrmation and bovine i-active glycolipid ( NeuAcα2→3Gal β1→ 4GlcNac β1 → 3Gal β1 → 4GlcNac β1 → 3Glc β1 → 1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

Two Families of Murine Carbohydrate Ligands for E-Selectin

In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram binding experiments andin situliquid secondary ion mass spectrometry (LSIMS) with neoglycolipids derived from their endoglycoceramidase-released oligosaccharides, in conjunction with compositional and linkage analyses, one of the glycolipid ligands in kidney was identified as the Lex-active extended globo-glycolipid:Galβ1-4GlcNAcβ1-6GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-Cer;|||Fucα1,3||Ga1β1,3|.Neoglycolipids enriched for the ligand structures were obtained from oligosaccharides released by endo-β-galactosidase from the 32D c13 cells. By TLC-LSIMS and antibody binding, the main E-selectin binding determinant on these was identified as sialyl-Lea.