Composition of the lipids of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> and their origin from its host <Emphasis Type="Italic">Ignicoccus</Emphasis> sp. strain KIN4/I

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent ¹³C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.     

Effect of growth temperature on ether lipid biochemistry in Archaeoglobus fulgidus

The archaea are distinguished by their unique isoprenoid ether lipids, which typically consist of the sn-2,3-diphytanylglycerol diether or sn-2,3-dibiphytanyldiglycerol tetraether core modified with a variety of polar headgroups. However, many hyperthermophilic archaea also synthesize tetraether lipids with up to four pentacyclic rings per 40-carbon chain, presumably to improve membrane thermal stability at temperatures up to∼110 °C. This study aimed to correlate the ratio of tetraether to diether core lipid, as well as the presence of pentacyclic groups in tetraether lipids, with growth temperature for the hyperthermophilic archaeon, Archaeoglobus fulgidus. Analysis of the membrane core lipids of A. fulgidus using APCI–MS analysis revealed that the tetraether-to-diether lipid ratio increases from 0.3 ± 0.1 for cultures grown at 70°C to 0.9 ± 0.1 for cultures grown at 89°C. Thin-layer chromatography (TLC) followed by APCI–MS analysis provided evidence for no more than one pentacycle in the hydrocarbon chains of tetraether lipid from cultures grown at 70°C and up to 2 pentacycles in the tetraether lipid from cultures grown at higher temperatures. Analysis of the polar lipid extract using TLC and negative-ion ESI–MS suggested the presence of diether and tetraether phospholipids with inositol, glycosyl, and ethanolamine headgroup chemistry.     

Novel alk-1-enyl ether lipids isolated from Clostridium acetobutylicum

Alkenyl ether analogues of phosphatidylglycerol (plasmenylglycerol), bisphosphatidylglycerol (cardiolipin) (plasmenylglycerolphosphatidic acid), monoglycosyldiglyceride and diglycosyldiglyceride were isolated from the polar lipids of Clostridium acetobutylicum and characterized by chemical analyses and degradation. The position of the alkenyl ether bond (at C-1) and of the acyl ester bond (at C-2) as well as the configuration at C-2 of the phospholipids are the same as of the alkenyl ether phospholipids known so far. The alkenyl ether analogue of monoglycosyldiglyceride contains a galactosyl residue, that of diglycosyldiglyceride a glucosyl-galactosyl residue, glucosyl forming the terminal unit.     

Dominance of mixed ether/ester,intact polar membrane lipids in five species of the order Rubrobacterales: Another group of bacteria not obeying the “lipid divide”

The composition of the core lipids and intact polar lipids (IPLs) of five Rubrobacter species was examined. Methylated (ω-4) fatty acids (FAs) characterized the core lipids of Rubrobacter radiotolerans, R. xylanophilus and R. bracarensis. In contrast, R. calidifluminis and R. naiadicus lacked ω-4 methyl FAs but instead contained abundant (i.e., 34–41 % of the core lipids) ω-cyclohexyl FAs not reported before in the order Rubrobacterales. Their genomes contained an almost complete operon encoding proteins enabling production of cyclohexane carboxylic acid CoA thioester, which acts as a building block for ω-cyclohexyl FAs in other bacteria. Hence, the most plausible explanation for the biosynthesis of these cyclic FAs in R. calidifluminis and R. naiadicus is a recent acquisition of this operon. All strains contained 1-O-alkyl glycerol ether lipids in abundance (up to 46 % of the core lipids), in line with the dominance (>90 %) of mixed ether/ester IPLs with a variety of polar headgroups. The IPL head group distribution of R. calidifluminis and R. naiadicus differed, e.g. they lacked a novel IPL tentatively assigned as phosphothreoninol. The genomes of all five Rubrobacter species contained a putative operon encoding the synthesis of the 1-O-alkyl glycerol phosphate, the presumed building block of mixed ether/ester IPLs, which shows some resemblance with an operon enabling ether lipid production in various other aerobic bacteria but requires more study. The uncommon dominance of mixed ether/ester IPLs in Rubrobacter species exemplifies our recent growing awareness that the lipid divide between archaea and bacteria/eukaryotes is not as clear cut as previously thought.     

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, <Emphasis Type="Italic">Cherax destructor</Emphasis> and <Emphasis Type="Italic">Engaeus sericatus</Emphasis> (Astacidae,Parastacidae)

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81–82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-β-1,4-glucanase, β-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-β-1,4-glucanase and β-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-β-d-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.     

Changes in the Lipid Composition of Mucor hiemalis Sporangiospores Related to the Age of the Spore-Forming Culture

Analysis of sporangiospore lipids of the fungus Mucor hiemalis F-1156 showed that alterations occur in the content of fatty acids and individual classes of lipids during long-term cultivation (for about 20 days). The changes in the lipid composition related to the age of the spore-forming mycelium suggest an important role of sporangiospore lipids in spore germination and in further development of the spherical cells formed in this processes. The M. hiemalis F-1156 sporangiospores with a lipid pool exhausted during long-term cultivation can give rise to both mycelial and yeastlike growth.     

The management of metabolic energy storage during the life cycle of mayflies: a comparative field investigation of the collector-gatherer <Emphasis Type="Italic">Ephemera danica</Emphasis> and the scraper <Emphasis Type="Italic">Rhithrogena semicolorata</Emphasis>

The concentration and seasonal dynamics of the major energy storage components, triglycerides and glycogen, were measured in two species of mayfly (Rhithrogena semicolorata and Ephemera danica) with contrasting life cycle strategies living in a small mountain stream. E. danica is a burrowing, semivoltine collector-gatherer; R. semicolorata is univoltine and scrapes periphyton from stones. This is the first publication which focuses on the role of metabolic energy sources during the larval life span of two mayfly species until the larvae emerge. Although triglycerides are the major energy reserve in both species (>84% of total energy storage) throughout the whole larval development their seasonal dynamic differed considerably. In R. semicolorata the triglyceride concentration declined during the last weeks prior to emergence in both sexes. The same pattern was found in female larvae of E. danica, but not in male E. danica. It is suggested that females use triglycerides in the last larval stages for egg maturation, which is completed in the last larval instar. In male E. danica the triglyceride concentrations remained high until emergence, presumably due to their high energy demands as adults for their swarming flights. Glycogen concentrations did not show such a difference between species and sexes. Its significance as a storage substrate for energy is rather low; however, concentrations decreased in both species and sexes prior to emergence.     

Phylogenomic analysis of lipid biosynthetic genes of Archaea shed light on the ‘lipid divide’

The lipid membrane is one of the most characteristic traits distinguishing the three domains of life. Membrane lipids of Bacteria and Eukarya are composed of fatty acids linked to glycerol‐3‐phosphate (G3P) via ester bonds, while those of Archaea possess isoprene‐based alkyl chains linked by ether linkages to glycerol‐1‐phosphate (G1P), resulting in the opposite stereochemistry of the glycerol phosphate backbone. This ‘lipid divide’ has raised questions on the evolution of microbial life since eukaryotes are thought to have evolved from the Archaea, requiring a radical change in membrane composition. Here, we searched for homologs of enzymes involved in membrane lipid and fatty acid synthesis in a wide variety of archaeal genomes and performed phylogenomic analyses. We found that two uncultured archaeal groups, i.e. marine euryarchaeota group II/III and ‘Lokiarchaeota’, recently discovered descendants of the archaeal ancestor leading to eukaryotes, lack the gene to synthesize G1P and, consequently, the capacity to synthesize archaeal membrane lipids. However, our analyses reveal their genetic capacity to synthesize G3P‐based ‘chimeric lipids’ with either two ether‐bound isoprenoidal chains or with an ester‐bound fatty acid instead of an ether‐bound isoprenoid. These archaea may reflect the ‘archaea‐to‐eukaryote’ membrane transition stage which have led to the current ‘lipid divide’.     

Energy content,storage substances,and construction and maintenance costs of Mediterranean deciduous leaves

Summary At monthly intervals water content, crude fibre, total and protein nitrogen, sugars, starch, total lipids, ash content and calorific total energy were measured throughout the lifespan of the leaves of the deciduous mediterranean shrubs Pistacia terebinthus L. and Cotinus coggygria Scop. From these data the construction costs and maintenance costs, as well as the construction costs of non-storage compounds and energy expenditure values were calculated. The latter values were also calculated for the evergreen stemmed shrub Ephedra distachya for reasons of comparison with an evergreen mediterranean species. The water status in the deciduous leaves is stable for a long time during the drought period until the beginning of senescence in August/September. In Cotinus an early and considerable increase of storage compounds is found, whilst in Pistacia terebinthus the accumulation is more uniform until August. The N-content is rather low compared with other deciduous leaves, the calorific energy is in the lower range of the values reported for similar species. The construction costs of the leaves of both deciduous species are significantly lower than those calculated by Williams et al. (1987) for two drought deciduous chaparral species but are in agreement with the data reported by other authors on deciduous leaves. Contrary to the findings of Williams et al. they are lower than those of evergreen species; this is also true when the construction cost of the non-storage compounds alone is considered. The values found for Ephedra are similar to the maintenance costs do not show a significant variation in the deciduous leaves. They are higher than those known for evergreen leaves but somewhat lower than the values calculated for deciduous chaparral leaves.     

Life cycle and adaptive strategies of three non-annual,ruderalCardueae: Onopordum nervosum,Carthamus arborescens,andCirsium scabrum

The life cycle and autoecology of three Mediterranean thorny species,Onopordum nervosum (Iberian endemic),Cirsium scabrum (Western Mediterranean) andCarthamus arborescens (Iberian-North African), of potential use in agriculture as bioenergetic, forage or oil producing plants were studied. These threeCardueae spp. are good examples of ways in which Mediterranean plants adapt their typical annual cycle to avoid summer droughts.O. nervosum is a perennial, monocarpic, heliophilous species which flowers in early summer and grows even under the extreme xeric conditions of the Mediterranean summers. It is a basiphilous plant usually found in sandy-loamy and sandy-clayey-loamy soils with a marked geographical variability in its seed germination.C. scabrum is a perennial, monocarpic species which has a long life cycle, flowering in summer and growing only during the wet months. It is not well-adapted to droughts and is usually restricted to acid soils.C. arborescens is a perennial, polycarpic species which flowers in spring and dries up in summer. It is usually found in basic loamy-sandy or sandy-loamy soils poor in organic matter.     

The prevalence of <Emphasis Type="Italic">cis</Emphasis>-9-hexadecenoic acid is a specific feature of the fatty acid profile of zygomycetes from the order <Emphasis Type="Italic">Kickxellales</Emphasis>

The fatty acid profiles of zygomycetes from the family Kickxellaceae of the order Kickxellales were studied with reference to the species Kicksella alabastrina of the key genus Kicksella of the family and the species Linderina pennispora. When synthesized de novo, the lipids of these species show the prevalence of cis-9-hexadecenoic acid. This trait is stable and does not depend on cultivation conditions and can, therefore, be considered as a specific chemotaxonomic characteristic of fungi from the order Kickxellales. The fatty acid profiles of the two fungi under study are similar to that of sea buckthorn oil.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 99–103.Original Russian Text Copyright © 2005 by Konova, Kochkina, Galanina.     

Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Pathway,inhibition and regulation of methyl <Emphasis Type="Italic">tertiary</Emphasis> butyl ether oxidation in a filamentous fungus, <Emphasis Type="Italic">Graphium</Emphasis> sp.

The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority (∼95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.     

Microsatellite DNA loci from the typical halophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (Brassicaceae)

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.     

Evolution of male sterility mechanisms in gynodioecious and dioecious species ofCirsium (Cynareae,Compositae)

The genusCirsium comprises both gynodioecious and dioecious species. The observation of microsporogenesis in female plants ofC. montanum, C. oleraceum, C. palustre andC. spinosissimum shows that the male sterility is due to a degeneration of the tapetum. This degeneration occurs more or less early according to the species and, in the light of these results, a scheme of evolution in the male sterility mechanism is proposed. Furthermore, the male sterility mechanism inC. montanum is very similar to that previously found in female plants of the dioecious speciesC. arvense. This fact enhances the possibility of evolution of the dioecy ofC. arvense from the gynodioecy found in other species. According to these results, a general scheme of evolution of sexes in the genusCirsium is proposed.     

Growth of three fungi on poultry fat or beef tallow

Aspergillus niger, Geotrichum candidum and Mucor miehei, when grown on a medium containing beef tallow or poultry fat for 5 days at 28°C, in Roux bottles, shake-flasks or fermenters, synthesized from 0.4 to 3.2 g lipids/I. The degree of fat utilization varied from 15 to 70%. The type of fat in the medium, the species of fungi and its cultivation method all influenced the fatty acid composition of fats remaining in the medium, as well as the intracellular lipids of the fungi.     

Cultivation of Sponge Larvae: Settlement,Survival, and Growth of Juveniles

The aim of this study was to culture sponge juveniles from larvae. Starting from larvae we expected to enhance the survival and growth, and to decrease the variation in these parameters during the sponge cultures. First, settlement success, morphological changes during metamorphosis, and survival of Dysidea avara, Ircinia oros, Hippospongia communis, under the same culture conditions, were compared. In a second step, we tested the effects of flow and food on survival and growth of juveniles from Dysidea avara and Crambe crambe. Finally, in a third experiment, we monitored survival and growth of juveniles of D. avara and C. crambe transplanted to the sea to compare laboratory and field results. The results altogether indicated that sponge culture from larvae is a promising method for sponge supply and that laboratory culture under controlled conditions is preferred over sea cultures in order to prevent biomass losses during these early life stages.     

Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng<Emphasis Type="Italic">rol</Emphasis> genes transferred from<Emphasis Type="Italic"> Agrobacterium</Emphasis> to<Emphasis Type="Italic"> Nicotiana</Emphasis>

The Ngrol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca. It is thought that bacterial infection resulted in the transfer of the Ngrol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not. Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes. The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species. This paper focuses on studies of the Ngrol genes in present-day plants and during the evolution of the genus Nicotiana. The functional sequences of several Ngrol genes may have been conserved after their ancient introduction from a bacterium to the plant. Resurrection of an ancestral function of one of the Ngrol genes, as examined by physiological and evolutionary analyses, is also described. The origin of the Ngrol genes is then considered, based on results of molecular phylogenetic analyses. The effects of the horizontal transfer of the Ngrol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.Seishiro Aoki is the recipient of the Botanical Society Award for Young Scientist, 2002.     

Archaeobacterial Ether Lipid Liposomes (Archaeosomes) as Novel Vaccine and Drug Delivery Systems

ABSTRACT:?

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeo-bacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.