Reconstruction of the archaeal isoprenoid ether lipid biosynthesis pathway in Escherichia coli through digeranylgeranylglyceryl phosphate

The membrane lipids of archaea are characterized by unique isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether (archaeol) and sn-2,3-dibiphytanyldiglycerol tetraether (caldarchaeol). The biosynthetic pathway for the tetraether lipid entails unprecedented head-to-head coupling of isoprenoid intermediates by an unknown mechanism involving unidentified enzymes. To investigate the isoprenoid ether lipid biosynthesis pathway of the hyperthermophilic archaeon, Archaeoglobus fulgidus, its lipid synthesis machinery was reconstructed in an engineered Escherichia coli strain in an effort to demonstrate, for the first time, efficient isoprenoid ether lipid biosynthesis for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP). The biosynthesis of DGGGP was verified using an LC/MS/MS technique and was accomplished by cloning and expressing the native E. coli gene for isopentenyl diphosphate (IPP) isomerase (idi), along with the A. fulgidus genes for G1P dehydrogenase (egsA) and GGPP synthase (gps), under the control of the lac promoter. The A. fulgidus genes for GGGP synthase (GGGPS) and DGGGP synthase (DGGGPS), under the control of the araBAD promoter, were then introduced and expressed to enable DGGGP biosynthesis in vivo. This investigation established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway for DGGGP biosynthesis and provides a platform useful for identification of subsequent, currently unknown, steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head coupling reaction yielding unsaturated caldarchaeol.     

Tetraether type polar lipids increase after logarithmic growth phase of Methanobacterium thermoautotrophicum in compensation for the decrease of diether lipids

Abstract The ratios of tetraether to diether type lipids in the total lipid during cell growth in batch cultures of Methanobacterium thermoautotrophicum ΔH (DSM 1053) were examined. The proportion of tetraether type lipids to the total lipid was about 80% during the log phase, and at the onset of the transient phase it began to rise up to about 93%. It was kept almost constant at that level throughout the stationary phase. The polar lipid composition changed with the age of the cell culture. The proportions of all the diether type polar lipids were lower and the levels of all tetraether type polar lipids were higher in the stationary phase than in the log phase. On the other hand, the composition of polar head groups, irrespective of the core lipids, was nearly constant in both growth phases measured so far despite the change in core lipid composition.     

Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.     

Structures of archaebacterial membrane lipids

Structural data on archaebacterial lipids is presented with emphasis on the ether lipids of the methanogens. These ether lipids normally account for 80–95% of the membrane lipids with the remaining 5–20% of neutral squalenes and other isoprenoids. Genus-specific combinations of various lipid core structures found in methanogens include diether-tetraether, diether-hydroxydiether, or diether-macrocyclic diether-tetraether lipid moieties. Some species have only the standard diether core lipid, but none are known with predominantly tetraether lipids as found in certain sulfur-dependent archaebacteria. The relative proportions of these lipid cores are known to vary in relation to growth conditions inMethanococcus jannaschii andMethanobacterium thermoautotrophicum. Polar headgroups in glycosidic or phosphodiester linkage to thesn-1 orsn-1 carbons of glycerol consist of polyols, carbohydrates, and amino compounds. The available structural data indicate a close similarity among the polar lipids synthesized within the species of the same genus. Detection of lipid molecular ions by mass spectrometry of total polar lipid extracts is a promising technique to provide valuable comparative data. Since these lipid structures are stable within the extreme environments that many archaebacteria inhabit, there may be specific applications for their use in biotechnology.     

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395 m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98°C (maximum growth temperature 102°C), but capable of prolonged survival at 105°C. Optimum growth was at pH 7 (range 5–8) and NaCl concentration 2.4% (range 1%–5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn²⁺-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA–DNA hybridization (<63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp. nov. Received: December 10, 1997 / Accepted: February 4, 1998     

Structural complexity in isoprenoid glycerol dialkyl glycerol tetraether lipid cores of Sulfolobus and other archaea revealed by liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry of membrane lipid cores from Sulfolobus species reveals isomeric forms of ring-containing isoprenoid glycerol dialkyl glycerol tetraether components not previously recognised via the use of NMR and liquid chromatography-mass spectrometry techniques. Equivalent isomerism was confirmed for the components in other hyperthermophilic genera and in sediments which contain the lipids of mesophilic archaea. The recognition of the isomeric structures in distinct archaeal clades suggests that profiles of tetraether lipids reported previously may have oversimplified the true lipid complexity in archaeal cultures and natural environments. Accordingly, the extent of variation in tetraether structures revealed by the work should direct more informative interpretations of lipid profiles in the future. Moreover, the results emphasise that tandem mass spectrometry provides a unique capability for assigning the structures of intact tetraether lipid cores for co-eluting species during chromatographic separation.     

The major lipid cores of the archaeon <Emphasis Type="Italic">Ignisphaera aggregans</Emphasis>: implications for the phylogeny and biosynthesis of glycerol monoalkyl glycerol tetraether isoprenoid lipids

The lipid cores from Ignisphaera aggregans, a hyperthermophilic Crenarchaeon recently isolated from New Zealand hot springs, have been profiled by liquid chromatography–tandem mass spectrometry. The distribution revealed includes relatively high proportions of monoalkyl (also known as H-shaped) tetraether cores which have previously been implicated as kingdom-specific biomarkers for the Euryarchaeota. Such high expression of monoalkyl tetraether lipids is unusual in the archaeal domain and may indicate that formation of these components is an adaptive mechanism that allows I. aggregans to regulate membrane behaviour at high temperatures. The observed dialkyl tetraether and monoalkyl tetraether lipid distributions are similar but not fully concordant, showing differences in the average number of incorporated rings. The similarity supports a biosynthetic route to the ring-containing dialkyl and monoalkyl tetraether lipids via a dialkyl tetraether core containing zero rings, or a closely related structural relative, as an intermediate. Currently, however, the precise nature of the biosynthetic route to these lipids cannot be deduced.     

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.     

Effects of temperature and growth phase on lipid and biochemical composition of Isochrysis galbana TK1

The lipid and biochemical composition of the haptophyte Isochrysis galbana TK1 was examined. Cultures were grown at 15 °C and 30 °C, and harvested in the exponential and early stationary growth phases. Carbohydrate and protein content varied at the two culture temperatures and growth phases. The highest protein content was found at the exponential growth phase at 15 °C, and the highest carbohydrate content was found at the stationary phase at the same culture temperature. Lipid accumulated in the stationary growth phase and its content was higher at 30 °C than at 15 °C regardless of the growth phase. The neutral lipids were the major class of lipid found in all the cultures. The stationary phase culture had a higher proportion of neutral lipids than the exponential phase culture and the proportion decreased slightly when culture temperature was increased from 15 °C to 30 °C. Phospholipid levels remained constant at the two temperatures, but slightly decreased in the stationary phase. Glycolipids in the exponentially growing cells were higher than those from stationary growth phase and increased with temperature. Polyunsaturated fatty acids (PUFAs) predominated in glycolipids and phospholipids. Cells grown at 15 °C contained higher proportion of 18:3 (n–3) and 22:6 (n–3) with a corresponding decrease in 18:2 (n–6), monounsaturated and saturated fatty acids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Monolayers of ether lipids from archaebacteria

The surface behavior of six different ether lipids from archaebacteria, based on condensation of glycerol or more complex polyols with two isoprenoid alcohols at 20 or 40 carbon atoms, was investigated in monolayers at the air-water interface.The compounds with no complex polar group (GD, GDGT, GDNT) form monolayers showing a reversible collapse at surface pressure as low as 22 dynes/cm. This collapse pressure decrease with temperature in such a way that the film tension remains constant. In condensed films, these molecules do not assume a completely upright position.Lipids with complex polar ends (HL, GLB, PLII) form films more stable to compression. Forcearea characteristics and surface moment values of HL monolayers are similar to those of analogous ester lipids with fatty acid chains. Monolayers of the two bipolar lipids, GLB and PLII, at room temperature present a more condensed state, probably due to the lateral cohesion between long alkyl chains, but a lower collapse pressure.For all bipolar lipids, the area expansion induced by temperature increase is larger than that of monopolar ones.Abbreviations GD Glycerol diether (2,3-di-O-phytanyl-sn-glycerol - GDGT Glycerol-dialkyl-glycerol tetraether - GDNT Glycerol-dialkyl-nonitol tetraether - GLB Glycolipid B - PLII Phospholipid II - HL Total lipid extract from Halobacterium halobium     

Proportions of diether, macrocyclic diether, and tetraether lipids in Methanococcus jannaschii grown at different temperatures.

Growth of Methanococcus jannaschii over a wide temperature range (47 to 75 degrees C) is correlated with an ability to alter dramatically the proportions of three ether lipid cores. These lipids shifted from predominantly diether (2,3-di-O-phytanyl-sn-glycerol) at the lower growth temperatures to macrocyclic diether and tetraether at near optimal growth temperatures. Lipid head groups varied as well, especially with respect to an increase in phosphate at the higher temperatures.     

Extraction and composition of polar lipids from the archaebacterium, Methanobacterium thermoautotrophicum: effective extraction of tetraether lipids by an acidified solvent

The usual Bligh and Dyer method could extract only a small part of the lipids of Methanobacterium thermoautotrophicum. When the water in the solvent was replaced by 5% trichloroacetic acid, the lipid recovery reached the maximum level, which was 6 times higher than that by the former method. The use of HCl (2 M) or disruption of cells was also effective but prolonged extraction with the HCl-containing solvent caused degradation of some phosphoglycolipids. Twenty-three spots of polar lipids were detected on a thin-layer chromatogram of the total lipid. These were 10 phospholipids (18%), 6 aminophospholipids (17%), 3 aminophosphoglycolipids (15%), 2 phosphoglycolipids (31%), and 2 glycolipids (19%). The predominant polar lipids were a highly polar phosphoglycolipid (PGL1, 30%) and a glycolipid (GL1a, 16%). The other major lipids included an aminophospholipid (PNL1a, 9%), and an aminophosphoglycolipid (PNGL1, 7%). The complete structure determination of PNL1a, GL1a, and PNGL1 is described in the accompanying paper. Acetolysis of the total lipids followed by acid methanolysis was required for the complete cleavage of polar head groups, releasing core residues of diphytanyl glycerol diether (C20 diether) and dibiphytanyl diglycerol tetraether (C40 tetraether). A densitometric assay of a thin-layer chromatogram showed that the ratio of C20 diether and C40 tetraether was 1:14. GLC analysis of alkyl chlorides prepared from the total lipid by BCl3 treatment showed that phytanyl (C20), biphytanyl (C40), and unidentified alkyl chains accounted for 10, 83, and 7 mol% of the total alkyl chains, respectively. Strong acid hydrolysis of the macromolecular residue obtained after lipid extraction gave a significant amount of C40 tetraether, which had probably been bound covalently to other substances in the cells.     

Tetraether membrane lipids of <Emphasis Type="Italic">Candidatus “Aciduliprofundum boonei”</Emphasis>, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.     

The effect of fluoride on the growth and fatty acid composition of Sphagnum fimbriatum at two temperatures

The fatly acid composition of different lipid fractions (neutral, glyco- and phospholipids) was studied in Sphagnum fimbriatum Wils, gametophytes grown in aseptic cultures at two temperatures (15°C and 25°C). The effect of a growth-retarding concentration (0.1 mM) of KF was also investigated. Fifteen-day treatment with KF affected the fatty acid composition more strongly at the higher than at the lower temperature. The proportion of palmitic acid (16:0) increased but the proportion of linoleic (18:2) decreased in all the lipid fractions, and that of linolenic (18:3) acid decreased in the fractions containing glyco- and neutral lipids. This indicates that the fluoride ions inhibit lengthening of the fatty acid chain. Compared with gametophytes grown at 25°C, material cultivated at 15°C had a much higher proportion of a highly unsaturated fatty acid, linolenic acid (18:3), in all the lipid fractions, but a lower proportion of oleic acid (18:1) in the neutral and phospholipids, and a lower proportion of linoleic (18:2) acid in all three fractions.     

Membrane adaptation in the hyperthermophilic archaeon Pyrococcus furiosus relies upon a novel strategy involving glycerol monoalkyl glycerol tetraether lipids

Microbes preserve membrane functionality under fluctuating environmental conditions by modulating their membrane lipid composition. Although several studies have documented membrane adaptations in Archaea, the influence of most biotic and abiotic factors on archaeal lipid compositions remains underexplored. Here, we studied the influence of temperature, pH, salinity, the presence/absence of elemental sulfur, the carbon source and the genetic background on the lipid core composition of the hyperthermophilic neutrophilic marine archaeon Pyrococcus furiosus. Every growth parameter tested affected the lipid core composition to some extent, the carbon source and the genetic background having the greatest influence. Surprisingly, P. furiosus appeared to only marginally rely on the two major responses implemented by Archaea, i.e. the regulation of the ratio of diether to tetraether lipids and that of the number of cyclopentane rings in tetraethers. Instead, this species increased the ratio of glycerol monoalkyl glycerol tetraethers (GMGT, aka. H-shaped tetraethers) to glycerol dialkyl glycerol tetraethers in response to decreasing temperature and pH and increasing salinity, thus providing for the first time evidence of adaptive functions for GMGT. Besides P. furiosus, numerous other species synthesize significant proportions of GMGT, which suggests that this unprecedented adaptive strategy might be common in Archaea.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998     

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.     

Effect of weak constant magnetic field on the composition and content of lipids in radish seedlings at various temperatures

We studied the effects of weak permanent homogenous hirizontal magnetic field (PMF) (400 A/m) on the composition and content of lipids and composition of their fatty acids (FAs) in radish (Raphanus sativus L. var. radicula D.C., cv. Rosovo-krashyi s belym konchikom) seedlings at temperatures of 20 and 10°C. We compared lipid composition and content in seedlings at the phase of developed cotyledons (20°C, 5-day-old, and 10°C, 8-day-old seedlings) under low light and in darkness with the lipid composition and content in dry seeds. The seedlings grown in geomagnetic field (GMF) served as a control. In dry seeds, about 99% of total lipids comprised neutral lipids (NL) and only 1% were polar lipids (PL). Triacylglycerols predominated among NL comprising 93% of total seed lipids. During seed germination, NLs were consumed and PL were produced: the amount of glycolipids increased in control by 3.5–5 times and the amount of phospholipis, by 1.5–2 times.In the light at 20°C, PMF suppressed the formation of PL (by 18%), whereas in darkness, it stimulated it approximately by 80% as compared with control. In the light at 10°C, PMF slightly stimulated PL formation; in darkness, it did not almost affect their synthesis. In all treatments, PMF increased the ratio of phospholipids to sterols by 30–100%. Among FA, PMF exerted the strongest effect on the content of erucic acid: it increased in the light and in darkness at 20°C approximately by 25% and decreased at 10°C in the light by 13%. PMF behaved as a correction factor affecting lipid metabolism on the background of light and temperature action.     

Tetraether-linked membrane monolayers in <Emphasis Type="Italic">Ferroplasma</Emphasis> spp: a key to survival in acid

Ferroplasma acidarmanus thrives in hot, extremely low pH, metal-rich solutions associated with dissolving metal sulfide ore deposits. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin layer chromatography analyses of F. acidarmanus membranes indicate that tetraether lipids predominate, with at least three core lipid structures. NMR measurements indicate that the cytoplasmic pH of F. acidarmanus is ~5.6. The optimal growth pH is ~1.2, and the lowest growth pH is ~0.0. Thus, these organisms maintain pH gradients across their membranes that approach 5 pH units. Tetraether lipids were originally thought to be specifically associated with thermophiles but are now known to be widely distributed within the archaeal domain. Our data, in combination with recently published results for thermophilic and mesothermophilic acidophilic archaea, indicate that there may be a stronger association between tetraether lipids and tolerance to acid and/or large metal ion gradients.     

Structural and physicochemical properties of polar lipids from thermophilic archaea

The essential general features required for lipid membranes of extremophilic archaea to fulfill biological functions are that they are in the liquid crystalline phase and have extremely low permeability of solutes that is much less temperature sensitive due to a lack of lipid-phase transition and highly branched isoprenoid chains. Many accumulated data indicate that the organism’s response to extremely low pH is the opposite of that to high temperature. The high temperature adaptation does not require the tetraether lipids, while the adaptation of thermophiles to acidic environment requires the tetraether polar lipids. The presence of cyclopentane rings and the role of polar heads are not so straightforward regarding the correlations between fluidity and permeability of the lipid membrane. Due to the unique lipid structures and properties of archaeal lipids, they are a valuable resource in the development of novel biotechnological processes. This microreview focuses primarily on structural and physicochemical properties of polar lipids of (hyper)thermophilic archaea.