Glycolipid Fatty Acid Research Articles

Metabolic insights into high-altitude adaptation of Himalayan ‘Horsetails’ [Equisetum diffusum D. Don] with special reference to the fatty acid dynamicity

Equisetum diffusum D. Don, a perennial herb of Equisetaceae, is considered as native to the Himalayan mountain range. This plant has prolific distribution along different altitudes of Eastern Himalayan region. Generally, mountain flora, like E. diffusum, experience a combination of contrasting abiotic stresses and may represent a unique natural model for investigating physiological resilience to address climate change. So, the present study has been framed to compare the metabolic responses in E. diffusum growing at different elevation belts with an aim to understand the underlying basis for their high-altitude adjustment ability. The biochemical profiles of this plant, collected from three different altitudinal transects of Darjeeling hills, were compared. The fatty acid profiles of neutral lipids, membrane phospholipids and glycolipids demonstrated palmitic acid and oleic acid as the predominant fatty acids. The major outcome was the substantial increase of polyunsaturated fatty acids (PUFAs) and unsaturation index in glycolipids of the samples collected from higher altitudes. Major proportion of changes in PUFA content was contributed by arachidonic acid. Therefore, the plants homeostatically control the membrane fluidity by enhancing the unsaturation of glycolipid fatty acids. Moreover, significantly higher antioxidant enzyme activities in higher altitude samples reflect the involvement of these enzymes in coping with the oxidative stress condition and membrane damage. The stress responsive effects may be further augmented by enhanced content of secondary metabolites. Therefore, a combination of biochemical responses along with the changes in membrane plasticity can be predicted as the metabolic adjustment strategies for high-altitude adaptation of E. diffusum.

Beyond PLFA: Concurrent extraction of neutral and glycolipid fatty acids provides new insights into soil microbial communities

The analysis of phospholipid fatty acids (PLFAs) is one of the most common methods used to quantify the abundance, and analyse the community structure, of soil microbes. The PLFA extraction method can yield two additional lipid fractions—neutral lipids and glycolipids—which potentially hold additional, valuable information on soil microbial communities. Yet its quantitative sensitivity on complete neutral lipid (NLFA) and glycolipid fatty acid (GLFA) profiles has never been validated. In this study we tested (i) if the high-throughput PLFA method can be expanded to concurrently extract complete NLFA and GLFA profiles, as well as sterols, (ii) whether taxonomic specificities of signature fatty acids are retained across the three lipid fractions in pure culture strains, and (iii) whether NLFAs and GLFAs allow soil-specific fingerprinting to the same extent as PLFA analysis. By adjusting the polarity of chloroform with 2% ethanol for solid phase extraction, pure lipid standards were fully fractionated into neutral lipids, glycolipids, and phospholipids. Sterols eluted in the neutral lipid fraction, and a betaine lipid co-eluted with phospholipids. We found consistent taxonomic specificities of fatty acid markers across the three lipid fractions by analysing pure culture extracts representative of soil microbes. Fatty acid profiles from soil extracts, however, showed stronger differences between PLFAs, NLFAs, and GLFAs than between soil types. This indicates that PLFAs and NLFAs signify different community properties (biomass vs. carbon storage, putatively), and that GLFAs are sensitive markers for community traits which behave differently than PLFAs. Although we consistently found high abundances of characteristic sterols in fungal extracts, the PLFA extraction method only yielded miniscule amounts of ergosterol from soil extracts. We argue that concomitant measurement of fatty acid profiles from all three lipid fractions is a low-effort and potentially information-rich addition to the PLFA method, and discuss its applicability for soil microbial community analyses.

Variability in carbon uptake and (re)cycling in Antarctic cryptoendolithic microbial ecosystems demonstrated through radiocarbon analysis of organic biomarkers.

Cryptoendolithic lichens and cyanobacteria living in porous sandstone in the high-elevation McMurdo Dry Valleys are purported to be among the slowest growing organisms on Earth with cycles of death and regrowth on the order of 103 -104 years. Here, organic biomarker and radiocarbon analysis were used to better constrain ages and carbon sources of cryptoendoliths in University Valley (UV; 1,800m.a.s.l) and neighboring Farnell Valley (FV; 1,700m.a.s.l). Δ14 C was measured for membrane component phospholipid fatty acids (PLFA) and glycolipid fatty acids, as well as for total organic carbon (TOC). PLFA concentrations indicated viable cells comprised a minor (<0.5%) component of TOC. TOC Δ14 C values ranged from -272‰ to -185‰ equivalent to calibrated ages of 1,100-2,550years old. These ages may be the result of fractional preservation of biogenic carbon and/or sudden large-scale community death and extended period(s) of inactivity prior to slow recolonization and incorporation of 14 C-depleted fossil material. PLFA Δ14 C values were generally more modern than the corresponding TOC and varied widely between sites; the FV PLFA Δ14 C value (+40‰) was consistent with modern atmospheric CO2 , while UV values ranged from -199‰ to -79‰ (calibrated ages of 1,665-610years). The observed variability in PLFA Δ14 C depletions is hypothesized to reflect variations in the extent of fixation of modern atmospheric CO2 and the preservation and recycling of older organic carbon by the community in various stages of sandstone recolonization. PLFA profiles and microbial community compositions as determined by molecular genetic characterizations and microscopy differed between the two valleys (e.g., predominance of biomarker 18:2 [>50%] in FV compared to UV), representing microbial communities that may reflect distinct stages of sandstone recolonization and/or environmental conditions. It is thus proposed that Dry Valley cryptoendolithic microbial communities are faster growing than previously estimated.

Body Mass Parameters, Lipid Profiles and Protein Contents of Zebrafish Embryos and Effects of 2,4-Dinitrophenol Exposure.

Morphology and physiology of fish embryos undergo dramatic changes during their development until the onset of feeding, supplied only by endogenous yolk reserves. For obtaining an insight how these restructuring processes are reflected by body mass related parameters, dry weights (dw), contents of the elements carbon and nitrogen and lipid and protein levels were quantified in different stages within the first four days of embryo development of the zebrafish (Danio rerio). The data show age dependent changes in tissue composition. Dry weights decreased significantly from 79μgdw/egg at 0hours post fertilization (hpf) to 61 μgdw/egg after 96 hpf. The amounts of total carbon fluctuated between 460 mg g-1 and 540 mg g-1 dw, nitrogen was at about 100 mg g-1 dw and total fatty acids were between 48–73 mg g-1 dw. In contrast to these parameters that remained relatively constant, the protein content, which was 240 mg g-1 at 0 hpf, showed an overall increase of about 40%. Comparisons of intact eggs and dechorionated embryos at stages prior to hatching (24, 30, 48 hpf) showed that the differences seen for dry weight and for carbon and nitrogen contents became smaller at more advanced stages, consistent with transition of material from the chorion to embryo tissue. Further, we determined the effect of 2,4-dinitrophenol at a subacutely toxic concentration (14 μM, LC10) as a model chemical challenge on the examined body mass related parameters. The compound caused significant decreases in phospholipid and glycolipid fatty acid contents along with a decrease in the phospholipid fatty acid unsaturation index. No major changes were observed for the other examined parameters. Lipidomic studies as performed here may thus be useful for determining subacute effects of lipophilic organic compounds on lipid metabolism and on cellular membranes of zebrafish embryos.

Production and early preservation of lipid biomarkers in iron hot springs.

The bicarbonate-buffered anoxic vent waters at Chocolate Pots hot springs in Yellowstone National Park are 51-54°C, pH 5.5-6.0, and are very high in dissolved Fe(II) at 5.8-5.9 mg/L. The aqueous Fe(II) is oxidized by a combination of biotic and abiotic mechanisms and precipitated as primary siliceous nanophase iron oxyhydroxides (ferrihydrite). Four distinct prokaryotic photosynthetic microbial mat types grow on top of these iron deposits. Lipids were used to characterize the community composition of the microbial mats, link source organisms to geologically significant biomarkers, and investigate how iron mineralization degrades the lipid signature of the community. The phospholipid and glycolipid fatty acid profiles of the highest-temperature mats indicate that they are dominated by cyanobacteria and green nonsulfur filamentous anoxygenic phototrophs (FAPs). Diagnostic lipid biomarkers of the cyanobacteria include midchain branched mono- and dimethylalkanes and, most notably, 2-methylbacteriohopanepolyol. Diagnostic lipid biomarkers of the FAPs (Chloroflexus and Roseiflexus spp.) include wax esters and a long-chain tri-unsaturated alkene. Surprisingly, the lipid biomarkers resisted the earliest stages of microbial degradation and diagenesis to survive in the iron oxides beneath the mats. Understanding the potential of particular sedimentary environments to capture and preserve fossil biosignatures is of vital importance in the selection of the best landing sites for future astrobiological missions to Mars. This study explores the nature of organic degradation processes in moderately thermal Fe(II)-rich groundwater springs--environmental conditions that have been previously identified as highly relevant for Mars exploration.

Arctic gypsum endoliths: a biogeochemical characterization of a viable and active microbial community

Abstract. Extreme environmental conditions such as those found in the polar regions on Earth are thought to test the limits of life. Microorganisms living in these environments often seek protection from environmental stresses such as high UV exposure, desiccation and rapid temperature fluctuations, with one protective habitat found within rocks. Such endolithic microbial communities, which often consist of bacteria, fungi, algae and lichens, are small-scale ecosystems comprised of both producers and consumers. However, the harsh environmental conditions experienced by polar endolithic communities are thought to limit microbial diversity and therefore the rate at which they cycle carbon. In this study, we characterized the microbial community diversity, turnover rate and microbe–mineral interactions of a gypsum-based endolithic community in the polar desert of the Canadian high Arctic. 16S/18S/23S rRNA pyrotag sequencing demonstrated the presence of a diverse community of phototrophic and heterotrophic bacteria, archaea, algae and fungi. Stable carbon isotope analysis of the viable microbial membranes, as phospholipid fatty acids and glycolipid fatty acids, confirmed the diversity observed by molecular techniques and indicated that present-day atmospheric carbon is assimilated into the microbial community biomass. Uptake of radiocarbon from atmospheric nuclear weapons testing during the 1960s into microbial lipids was used as a pulse label to determine that the microbial community turns over carbon on the order of 10 yr, equivalent to 4.4 g C m−2 yr−1 gross primary productivity. Scanning electron microscopy (SEM) micrographs indicated that mechanical weathering of gypsum by freeze–thaw cycles leads to increased porosity, which ultimately increases the habitability of the rock. In addition, while bacteria were adhered to these mineral surfaces, chemical analysis by micro-X-ray fluorescence (μ-XRF) spectroscopy suggests little evidence for microbial alteration of minerals, which contrasts with other endolithic habitats. While it is possible that these communities turn over carbon quickly and leave little evidence of microbe–mineral interaction, an alternative hypothesis is that the soluble and friable nature of gypsum and harsh conditions lead to elevated erosion rates, limiting microbial residence times in this habitat. Regardless, this endolithic community represents a microbial system that does not rely on a nutrient pool from the host gypsum cap rock, instead receiving these elements from allochthonous debris to maintain a more diverse and active community than might have been predicted in the polar desert of the Canadian high Arctic.

Radiocarbon Evidence of Active Endolithic Microbial Communities in the Hyperarid Core of the Atacama Desert

The hyperarid core of the Atacama Desert is one of the driest and most inhospitable places on Earth, where life is most commonly found in the interior of rocks (i.e., endolithic habitats). Due to the extreme dryness, microbial activity in these habitats is expected to be low; however, the rate of carbon cycling within these microbial communities remains unknown. We address this issue by characterizing the isotopic composition ((13)C and (14)C) of phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) in colonized rocks from four different sites inside the hyperarid core. δ(13)C results suggest that autotrophy and/or quantitative conversion of organic matter to CO2 are the dominant processes occurring with the rock. Most Δ(14)C signatures of PLFA and GLFA were consistent with modern atmospheric CO2, indicating that endoliths are using atmospheric carbon as a primary carbon source and are also cycling carbon quickly. However, at one site the PLFA contained (14)C from atmospheric nuclear weapons testing that occurred during the 1950s and 1960s, indicating a decadal rate of carbon cycling. At the driest site (Yungay), based on the relative abundance and (14)C content of GLFA and PLFA, there was evidence of possible preservation. Hence, in low-moisture conditions, glycolipids may persist while phospholipids are preferentially hydrolyzed.

Differential response of fatty acid composition in the different lipid classes from particulate matter in a high arctic fjord (Kongsfjorden, Svalbard)

Lipid classes and the respective fatty acid composition of natural particulate matter were studied on a seasonal basis in the Arctic fjord Kongsfjorden (Svalbard) during the early summer of 2006 and the spring, summer of 2007. Polar lipids were the major lipid class most of the times in 2006 and at all times in 2007. Among neutral lipids triglycerides were dominant. Polar lipids were divided into glycolipids (chloroplast membranes) and phospholipids (live cell membranes). Glycolipids were further divided into monogalactosyldiglycerides (MGDG), the major glycolipid, followed by digalactosyldiglycerides (DGDG) and Sulfoquinovosyldiglycerides (SQDG). In 2007, changes in both polar lipid constituents showed similar increasing trend from May to mid June but subsequently showed opposite trends from July to September. The seasonal pattern of particulate glycolipids was one of the low concentrations of MGDG in June followed by an increase between late June and September. SQDG exhibited a similar trend while DGDG displayed an opposing trend. In 2007, fatty acid composition of phospholipids, glycolipids and neutral lipids was dominated by saturated acids at all times followed by mono unsaturated acids and polyunsaturated acids with docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and 18:5n-3 as major contributors. Glycolipid fatty acid pattern differed from that of phospholipids, showing a more important contribution of 18:5n-3. Neutral lipid composition differed from the other two classes by a larger contribution of 16:1n-5 and percentages of EPA, DHA, 18:5 and 18:4n-3 lower than in the two structural classes. Factorial correspondence analysis (FCA) of the fatty acid composition of all classes illustrated a seasonal transition in species composition and/or physiological states for the phospholipids and the different processes of chloroplast membrane adaptation in relation to taxonomic changes for the glycolipids. Neutral lipid changes were more complex because of the combined influence of growth rates, nutrient limitations and community shifts. The differential response of the different lipid classes is discussed in relation with the complex interactions between community structure, environmental adaptation and metabolic processes.

Distributions of phospholipid and glycolipid fatty acids in two strains of different functional Erythrobacter sp. isolated from South China Sea

The comparison of the fatty acids between aerobic anoxygenic phototrophic bacteria (AAPB) and their phylogenetic relatives has been a fascinating but yet enigmatic topic, enhancing our understanding of physiological variations between these evolutionarily related microorganisms. Two strains of marine bacteria, both phylogenetically falling into Erythrobacter sp., were isolated from the South China Sea, and demonstrated, respectively, to be an aerobic anoxygenic phototrophic bacteria (AAPB) (JL475) which is capable of anoxygenic photosynthesis via BChl a, and an obligate heterotroph (JL316) with a lack of BChl a, on the basis of phylogenetic analysis and pure culture cultivation. Phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) of the two strains were extracted and analyzed by gas chromatographymass spectrometry. The PLFA in JL475 AAPB are characterized by C18:1 C18:2ω7,13 and C18:1, with the C18:2ω7,13 being a specific compound for AAPB and in particular for Erythrobacter longus and some of its phylogenetically closely related relatives. The JL316 strain is characterized in PLFA by the presence of C18:1, C16:1 and C16:0, and in particular C17:1. GLFA do not show any discrimination between the two strains. Four α,ω-dicarboxylic acids, including 1,8-octanedioic acid, 1,9-nonanedioic acid, 1,10-decanedioic acid and 1,11-undecanedioic acid, are present only in JL316 GLFA, presumably derived from metabolic products. C14-C16 2-hydroxy fatty acids were found in the two strains, probably assuming a similar function of their LPS in outer membranes.

Biomarker analysis of microbial diversity in sediments of a saline groundwater seep of Salt Basin, Nebraska

Lipids extracted from sediments in a saline seep in the Salt Basin of Lancaster County, Nebraska included alkanes, alkenes, alkanols, phytol, C 27–30 sterols, C 30–32 hopanoids, tetrahymanol, glycolipid and phospholipid fatty acids, and lipopolysaccharide hydroxyl fatty acids. Biomarker profiles suggest that the brine seeps of Salt Basin support a microbial ecosystem adapted to a relatively highly saline and sulfidic environment. The phospholipid fatty acid (PLFA) and lipopolysaccharide hydroxyl fatty acid profiles are consistent with the presence of large numbers of sulfate-reducing bacteria (SRB) in black, sulfidic muds surrounding the seeps. In the context of field and laboratory observations, the presence of large amounts of glycolipid fatty acids is attributed to large populations of photosynthetic microorganisms (cyanobacteria, phytoplankton, and purple sulfur bacteria) that likely play important roles in the local cycling of carbon and sulfur. The sterol profile and the detection of polyunsaturated alkenes (C 21:6, C 21:7, C 30:4, and C 30:5) implicates microalgae as important contributors of organic matter at the site. Comparatively high concentrations of phytol (58.2 μg g −1 dry wt sediment) record the activity of photosynthetic organisms in the system. The δ 13C of phytol (−37.1‰) is compatible with a dominance of microalgae, cyanobacteria, or higher plants and a lesser contribution from phototrophic sulfur bacteria. The presence of various intermediate degradation products of phytol (phytenes and phytadienes) indicates that SRB likely mediate the chemical reduction of phytol in the anaerobic zone. The presence of C 30–32 hopanols can be attributed to cyanobacteria and methanotrophs in oxic regions of the water column, whereas bacterivorous ciliates and phototrophic sulfur bacteria living at the chemocline are likely sources of tetrahymanol. The carbon isotopic composition of individual fatty acids and neutral lipids helps to identify source organisms. These microorganisms and others constitute a unique and integrated ecosystem prescribed by the geochemistry of the Salt Basin.

Ambient ultraviolet radiation in the Arctic reduces root biomass and alters microbial community composition but has no effects on microbial biomass

AbstractWe assessed the effects of ambient solar ultraviolet (UV) radiation on below‐ground parameters in an arctic heath in north‐eastern Greenland. We hypothesized that the current UV fluxes would reduce root biomass and mycorrhizal colonization and that these changes would lead to lower soil microbial biomass and altered microbial community composition. These hypotheses were tested on cored soil samples from a UV reduction experiment with three filter treatments (Mylar, 60% UV‐B reduction; Lexan, up to 90% UV‐B reduction+UV‐A reduction; UV transparent Teflon, filter control) and an open control treatment in two study sites after 3 years' manipulation. Reduction of both UV‐A and UV‐B radiation caused over 30% increase in the root biomass of Vaccinium uliginosum, which was the dominant plant species. UV reduction had contrasting effects on ericoid mycorrhizal colonization of V. uliginosum roots in the two sites, while it had no clear effects on fungal (ergosterol) or microbial biomass (measured both with fumigation–extraction and quantitative lipid biomarker analysis) in soil. However, principal component analysis of lipid biomarkers (phospholipid and glycolipid fatty acid profiles) showed that microbial community composition was altered by UV reduction. Although the UV responses were slight considering the large dose difference between the treatments (from near‐ambient to up to 90% UV‐B reduction), we cannot rule out the possibility that the recovery of ozone layer would change the below‐ground functioning of arctic ecosystems.

Lipid biomarkers and carbon-isotopes of modern travertine deposits (Yellowstone National Park, USA): Implications for biogeochemical dynamics in hot-spring systems

Lipid biomarkers and 13C fractionation patterns were used to understand the dynamics of carbon cycling during microbial metabolisms in different environments of travertine precipitation (called facies) at Spring AT-1 on Angel Terrace in the Mammoth Hot Springs complex of Yellowstone National Park, USA. Microbial mats that encrust travertine deposits were collected for analyses of lipid biomarkers and carbon isotopes along the continuous drainage outflow system of Spring AT-1. The spring water exhibits a continuous temperature drop from 71°C in the vent at top to 24°C in the distal slope at bottom. Phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) exhibit distinctly different compositions in each of the facies, which are consistent with partitioning of the bacterial 16S rRNA gene sequences in the Spring AT-1 travertine facies (Fouke et al., 2003). The δ 13C composition of total biomass within the microbial mats decreases from −16.1‰ in the vent to −23.5‰ in the distal slope. However, lower values occur in the pond (−26.0‰) and the proximal slope (−28.0‰) between the vent and the distal slope. Isotopic compositions of PLFA and GLFA have variations similar to those of total biomass. The average δ 13C values of PLFA are −12.4 ± 5.2‰ (n = 10 individual fatty acids, same below) in the vent, −33.0 ± 3.1‰ (n = 11) in the pond, −33.7 ± 3.8‰ (n = 16) in the proximal slope, and −22.4 ± 3.4‰ (n = 10) in the distal slope; the average δ 13C values of GLFA are −19.6 ± 3.0‰ (n = 3) in the vent, −30.4 ± 4.7‰ (n = 8) in the pond, −36.9 ± 2.8‰ (n = 12) in the proximal slope, and −27.9 ± 3.1‰ (n = 13) in the distal slope. In particular, fatty acids in the vent are enriched in 13C relative to the total biomass, which is consistent with the notion that the biosynthetic pathways of the extant microbial community in the vent may be dominated by Aquificales using the reversed tricarboxylic acid cycle. Fractionations between fatty acids and total biomass in the pond, the proximal slope and the distal slope suggest the involvement of other biosynthetic pathways for CO 2 fixation by extant microbial populations. The results indicate that lipid biomarkers provide valuable information on the changing diversity and activity of microbial communities in different depositional environments. Carbon-isotope fractionations, on the other hand, can provide insight into the operating biosynthetic pathways associated with different organisms in the changing environment. This integrated approach may serve as a powerful tool for identifying functional metabolism within a community and identify shifts in microbial community structure in modern hot-spring systems.

Characterization of multiple-substrate utilization by anthracene-degrading Mycobacterium frederiksbergense LB501T.

Stable carbon isotope analysis of biomass and analyses of phospholipid fatty acids (PLFA), glycolipid fatty acids (GLFA), and mycolic acids were used to characterize mixed-substrate utilization by Mycobacterium frederiksbergense LB501T under various substrate regimens. The distinct (13)C contents of anthracene and glucose as representatives of typical hydrophobic pollutants and naturally occurring organic compounds, respectively, were monitored during formation into biomass and used to quantify the relative contributions of the two carbon sources to biomass formation. Moreover, the influence of mixed-substrate utilization on PLFA, GLFA, and mycolic acid profiles and cell surface hydrophobicity was investigated. Results revealed that M. frederiksbergense LB501T degrades anthracene and forms biomass from it even in the presence of more readily available dissolved glucose. The relative ratios of straight-chain saturated PLFA to the corresponding unsaturated PLFA and the total fraction of saturated cyclopropyl-branched PLFA of M. frederiksbergense LB501T depended on the carbon source and the various rates of addition of mixed substrates, whereas no such trend was observed with GLFA. Higher proportions of anthracene in the carbon source mixture led to higher cell surface hydrophobicities and more-hydrophobic mycolic acids, which in turn appeared to be valuable indicators for substrate utilization by M. frederiksbergense LB501T. The capability of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria to utilize readily available substrates besides the poorly available PAHs favors the buildup of PAH-degrading biomass. Feeding of supplementary carbon substrates may therefore promote bioremediation, provided that it sustains the pollutant-degrading population rather than other members of the microbial community.

Influence of the growth substrate on ester-linked phospho- and glycolipid fatty acids of PAH-degrading Mycobacterium sp. LB501T.

The influences of poorly water-soluble anthracene on ester-linked phospholipid fatty acid (PLFA) and glycolipid fatty acid (GLFA) profiles of Mycobacterium sp. LB501T were studied. Bacteria were cultivated on either anthracene or glucose (one culture with successively amended small doses of this substrate and one with excess concentrations) to distinguish between influences of the chemical structure and the bioavailability of the growth substrate. Results revealed that GLFA and PLFA profiles of M. sp. LB501T depended on the availability and the structure of the carbon source. Fatty acid profiles obtained with anthracene differed from those obtained with excess glucose. They were interpreted as a specific adaptation to this poorly bioavailable polycyclic aromatic hydrocarbon (PAH). In contrast, profiles obtained with low glucose concentrations showed clear signs of starvation stress. Stable carbon isotopic ratios (delta13C) of GLFA and PLFA of M. sp. LB501T were analysed to characterize the 13C-fractionation during the biosynthesis of individual fatty acids and to evaluate their value as markers for substrate usage. Although the delta13C values of PLFA and GLFA showed differential isotope fractionation during anthracene- and glucose-degradation, they were sufficiently distinct to be used as signatures of bacterial substrate usage.

28 Lipid composition of members of the algal class chlorarachniophyceae

The algal class Chlorarachniophyceae is comprised of a small group of unicellular eukaryotic algae that are often characterized by an unusual amoeboid morphology. This morphology is hypothesized to be the result of a secondary endosymbiosis in which a green alga was engulfed as prey by a nonphotosynthetic amoeba or amoebaflagellate. Whereas much is known about the phylogenetic relationships of individual chlorarachniophytes to one another, and to possible ancestral host organisms in the genera Cercomonas and Heteromita, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, seven organisms were examined for their fatty acid and sterol composition. These included Bigelowiella natans, Chlorarachnion globusum, Chlorarachnion reptans, Gymnochlora stellata, Lotharella amoeboformis, Lotharella globosa, and Lotharella sp. Fatty acids associated with chloroplast‐associated glycolipids, cytoplasmic membrane‐associated phospholipids, and storage triglycerides were characterized. Glycolipid fatty acids were found to be of limited composition, containing principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0), which ranged in relative percentage from 67–90% and 10–29%, respectively, in these seven organisms. Triglyceride‐associated fatty acids were found to be similar. Phospholipid fatty acid composition was more variable. The principal phospholipid fatty acids, 16:0 (25–32%) and a compound tentatively identified as docosapentaenoic acid [22:5(n‐3)] (26–35%), were found along with a number of C18 and C20 fatty acids. All organisms contained two sterols as free sterols. These were tentatively identified as 24‐ethylcholesta‐5,22E‐dien‐3b‐ol (stigmasterol; 70–95%) and 24‐methylcholesta‐5,22E‐dien‐3b‐ol (brassicasterol; 5–30%).

Aglycone modulation of glycolipid receptor function

The aglycone has been largely ignored in consideration of glycoconjugate function. Evidence is reviewed which suggests that the role of the lipid in glycolipid carbohydrate function may be particularly significant. The lipid moiety can promote or reduce carbohydrate exposure of membrane glycolipids. Theoretical calculation has indicated that the plane of the plasma membrane can restrict the permitted conformations of a given glycolipid oligosaccharide. Thus the lipid moiety may influence the relative conformation of such carbohydrate sequences. Evidence of ceramide regulation of glycolipid function can be found in studies of enzyme substrate specificity, antiglycolipid recognition and bacterial/host cell interactions. Studies of verotoxin binding to its glycolipid receptor globotriaosyl ceramide indicate that modulation of receptor function by glycolipid fatty acid content plays an important role in in vitro binding assays, cell cytotoxicity and intracellular routing.

Glycosphingolipid headgroup orientation in fluid phospholipid/cholesterol membranes: similarity for a range of glycolipid fatty acids

Galactosyl ceramide (GalCer) was labeled for nuclear magnetic resonance (NMR) spectroscopy by replacement of a hydrogen atom at C6 of the galactose residue with deuterium. Wideline 2H NMR of [d1]GalCer permitted consideration of a mechanism traditionally entertained for cell surface recognition site modulation: that the nature of the fatty acid attached to the sphingosine backbone of glycosphingolipids (GSLs) importantly influences carbohydrate headgroup orientation. Comparison was made among various glycolipid fatty acids by altering hydroxylation, saturation, and chain length. Studies were carried out in unsonicated bilayer membranes mimicking several important characteristics of cell plasma membranes: fluidity, low GSL content, predominant [sn-2]monounsaturated phosphatidylcholine (PC) (1-palmitoyl-2-oleoyl PC), and the presence of cholesterol. Spectroscopy was performed on samples over a range of temperatures, which included the physiological. 2H NMR spectra of [d1]GalCer having 18-carbon saturated fatty acid (stearic acid), cis-9-unsaturated fatty acid (oleic acid), D- and L-stereoisomers of alpha-OH stearic acid, or 24-carbon saturated fatty acid (lignoceric acid) were importantly similar. This argues that for GSLs dispersed as minor components in fluid membranes, variation of the glycolipid fatty acid does not provide as much potential for direct conformational modulation of the carbohydrate portion as has sometimes been assumed. However, there was some evidence of motional differences among the species studied. The 2H NMR spectra that were obtained proved to be more complex than was anticipated. Their features could be approximated by assuming a combination of axially symmetric and axially asymmetric glycolipid motions. Presuming the appropriateness of such a analysis, at a magnetic field of 3.54 T (23.215 MHz), the experimental spectra suggested predominantly asymmetric motional contributions. At the higher field of 11.7 T (76.7 MHz, equivalent to a proton frequency of 500 MHz), spectra indicated dominance by axially symmetric rotational modes. There was also evidence of some bilayer orientation in the stronger magnetic field. The unusual observation of spectral differences between the two magnetic field strengths may involve a diamagnetic response to high field on the part of some liposome physical characteristics.

Biochemical Bases for Controlled-atmosphere Effects on Reducing Chilling Injury of `Hass' Avocado Fruit

California-grown `Hass' avocado fruit were stored at 5C, in air or a controlled atmosphere (CA) of 2% oxygen and 5% carbon dioxide. Fruit were evaluated at 0, 2, 4, 6, 8, 10, and 12 weeks, both immediately upon removal from storage and after ripening at 20C. Severe chilling injury (flesh browning) developed in the airstored fruit after 6 weeks, while only moderate symptoms were observed in CA-stored avocado fruit after 12 weeks. Lipid peroxidation breakdown products increased during storage and ripening in both air and CA treatments. Sterols, steryl esters, steryl glycosides, glycolipids, and phospholipids were analyzed. Quantity of acylated steryl glycoside in ripe fruit changed from 34 nmoles initially, to 51 or 27 nmoles after 6 weeks at 5C in air or CA, respectively. Glycolipid fatty acid unsaturation in air-stored fruit decreased with the development of chilling injury. Fatty acid unsaturation in phospholipids (phosphatidylinositol, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) of air-stored avocados decreased with the development of chilling injury. CA storage delayed the development of chilling injury and the loss of fatty acid unsaturation.

Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects

Deuterium wide line NMR spectroscopy was used to study cholesterol effects on the ceramide portions of two glycosphingolipids (GSLs) distributed as minor components in fluid membranes. The common existence of very long fatty acids on GSLs was taken into account by including one glycolipid species with fatty acid chain length matching that of the host matrix, and one longer by 6 carbons. N-stearoyl and N-lignoceroyl galactosyl ceramide with perdeuterated fatty acid (18:0[d35] GalCer and 24:0[d47] GalCer) were prepared by partial synthesis. They were dispersed in bilayer membranes having the 18-carbon-fatty-acid phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), as major component. Glycolipid fatty acid chain behavior and arrangement were analyzed using order profiles derived from their 2H-NMR spectra. Cholesterol effects on order parameter profiles for 18:0[d35] GalCer, with chain length equal to that of the host matrix, followed the pattern known for acyl chains of phospholipids. The presence of sterol led to restriction of trans/gauche isomerization along the length of the chain, with the largest absolute increase in order parameters being toward the surface, but somewhat greater relative effect just below the "plateau" region. In cholesterol-containing membranes, order parameter profiles for the long chain species, 24:0[d47] GalCer, showed a characteristic secondary "plateau" associated with carbon atoms C14 to C23, a feature also present in SOPC bilayers without cholesterol and in pure hydrated 24:0[d47] GalCer. Cholesterol-induced ordering effects on the long chain glycolipid were similar to those described for the shorter chain species, but were minimal at the methyl terminus. Within a given membrane,SCD profiles for 1 8:O[d3] GalCer and 24:0[d47] GalCer were quantitatively similar to a membrane depth of C13 to C14. SCD values at C16 and C17 were about 15% and 28% higher, respectively, for the long chain GSL than for its short chain analogue inSOPC/cholesterol (compared to 21 and 31%, respectively, in membranes without cholesterol). Nitroxide spin labels attached rigidly to C16 of the long chain glycolipid gave EPR order parameters that were twice as high as for the same spin label at C16 on the shorter chain glycolipid in both matrices. It would appear that the above factors impose a tendency for the "extra" portion of the 24-carbon chain to cross the bilayer midplane where it may interact with terminal portions of acyl chains in the opposing monolayer; however, steric constraints, and probably collision events associated with lateral diffusion, induce wide orientation fluctuations in the segment involved.

Production of sophorose lipid by Candida (Torulopsis) apicola grown on glucose

The production of extracellular sophorose lipid by Candida apicola IMET 43747 was studied after growth on glucose as sole carbon source. In the presence of citrate and after 120 h of cultivation, the microcrystalline lactonic sophorose lipid started to appear in the medium. The lipid was shown to accumulate in the cell wall and only a minor part was liberated into the medium. By means of NMR studies it was shown that the cell wall-bound and the liberated lactonic sophorose lipid were identical and had a hydroxy Δ 8-hexadecenoic acid backbone. Biotransformation of d-[1- 13C]glucose revealed that in glucose-grown cells glucose was degraded to trioses which were rearranged and flow backward via the gluconeogenic pathway into the glycolipid as documented by scrambling of the 13C label from the C-1 position into the d-[6- 13C]glucose of sophorose. A further part of the trioses was used for de novo synthesis of the glycolipid fatty acid moiety via acetyl-CoA. By use of a mixed substrate, i.e., glucose and hexadecane, part of the glucose added was directly incorporated into the carbohydrate moiety of the lipid. These results are discussed in terms of the physiological significance of the sophorose lipid to act as extracellular carbon storage material.