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Di- and Tetra-Alkyl Ether Lipids of the Archaea

The Archaea represent one of the three primary kingdoms or domains of living organisms. They are single celled with a low deoxyribonucleic acid content, and they lack a nuclear membrane. They include thermophiles, halophiles and acidophiles, collectively termed 'extremophiles', which some believe resemble the dominant organisms in the primeval biosphere; many of the species are methanogenic, including those found in low temperature environments. Four main archaeal phyla are recognized, the Euryarchaeota, Crenarchaeota, Thaumarchaeota and Korarchaeota, but this is changing as more DNA data becomes available. As the name suggests, extremophiles exist in extreme habitats, including hot springs and waters with high salt, alkali or acid conditions, but more recently it has become apparent that many such organisms exist in mild environments also. It is now suggested that they may constitute up to 20% of the oceanic biomass, where they are significant contributors to global biogeochemical pathways including the methane and nitrogen cycles.

The lipids of Archaea are very different from those of Bacteria, although there are also many parallels, and the differing structures and biosynthetic machinery constitute part of a continuing debate as to how and when the two groups diverged during evolution. In particular, they contain isoprenoid moieties linked to glycerol with unique stereochemistry. In addition to an interest because of their position in the current biosphere, these lipids are more resistant to degradation than many other biomolecules, and they are often preserved in the geologic record. Thus, they are markers that contribute to knowledge of earth history and geological processes.

1.  Basic Chemistry

The lipids of the Archaea are now known to contain many unique and characteristic polar lipids based on 2,3-dialkyl-sn-glycerol backbones, i.e. the stereochemistry is the opposite of that found in the two other primary kingdoms, Bacteria (eubacteria) and Eukarya (eukaryotes) (see our web page on phosphatidic acid, for example). The alkyl groups are isoprenoid in nature, and the simplest molecules of this type are derivatives of 2,3-diphytanyl-O-sn-glycerol (archaeol), i.e. with two C20 isoprenoid units (occasionally C25) attached to positions sn-2 and sn-3 of glycerol by ether linkages (sometimes designated C40 ether lipids). Many other isoprenoid groups are found linked in this way, including macrocyclic diethers. Generally, the alkyl chains are saturated, but forms with double bonds in various positions have been found in a few species of Archaea.

Structures - archaeol and caldarchaeol

In addition, tetraethers with strikingly different molecular architecture have been discovered as core lipids of the Archaea. These molecules have one or two polar head groups, which need not be the same (see below), with the 2,3-sn-glycerol moieties linked by two C40 alkyl components that are also isoprenoid in nature (C80 ether lipids). Thus, caldarchaeol (so-called because it is the predominant form in thermophilic Archaea) has two C40 isoprenoid units linked from position 2 to position 3’ and from position 3 to position 2’ (anti-parallel chains), while in isocardarcheol, they are linked from position 2 to position 2’ and from position 3 to position 3’ (parallel chains) of the two sn-glycerol moieties. While both forms can exist in the same species, the anti-parallel forms tend to predominate. The acronym GDGT is often used to denote such glycerol dialkyl glycerol tetraether lipids.

Some lipids of this type have both methyl branches and one to four cyclopentane rings in each chain, up to eight in total. It has been suggested that a nomenclature GDGT-x be employed, where x is the number of cyclopentane rings in the molecule. As the environmental temperatures increase and the pH decreases, the proportions of cyclopentane rings in lipids from thermoacidophilic species tend to increase. A further complication is found in the Thaumarchaeota, which can have an additional cyclohexane ring in the alkyl chains. Yet other lipids of this type have carbon-carbon links between the chains, forming an ‘H’-shape. For many years, there was thought to be a relatively limited number of forms in living organisms, but improved methods of analysis are showing the great complexity that exists in nature. Needless to say, the stereochemistry at the ring structures adds further complications. As an example, the structure illustrated forms part of the tetra-ether core of crenarchaeol from hyper-thermophilic organisms of the Thaumarchaeota.

Part structure of crenarchaeol

Yet other hydroxy-GDGT lipids have found with hydroxyl groups in position 3 of one of the alkyl chains, while structural analogues occur in marine sediments with only one glycerol moiety and with the alkyl groups terminating in hydroxyl groups. Finally, unsaturated diether lipids are found in some archaea that grow at very low temperatures (-20 to 10°C). The nature and relative amounts of the various lipid structures in archaeal membrane vary widely, depending on the specific organism and upon the environmental conditions. For example, the Halobacteriales only possess bilayer (mainly C20) membrane lipids, while the hyperthermophilic archaeon Aeropyrum pernix has C25 archaeols only. In contrast, the genera Pyrobaculum, Thermoplasma and Sulfolobus utilize caldarchaeols (C40), including those with cyclopentane ring structures.

Formula of calditolAs an alternative to the simple glycerol component, calditol is present in a subset of thermoacidophilic archaea of the Sulfolobales order within the Crenarchaeota phylum, such as Sulfolobus solfaricus, and contains the nine-carbon nonitol with a five-membered ring, i.e. 2-hydroxymethyl-1,2,3,4,5-pentahydroxy-cyclopentane, linked to a glycerol moiety at position sn-1 with positions sn-2 and 3 attached to isoprene units to form a glycerol-dialkyl-calditol-tetraether. A radical S-adenosylmethionine protein required for calditol synthesis has been identified in S. acidocaldarius, and deletion of this renders the organism sensitive to low pH. Calditol is therefore essential to protect the archaeal cells from acidic stress. As a further alternative to glycerol in some other species, the alkyl groups are linked to tetritol (1,2,3,4-butanetetrol).

While these core lipids are sometimes found in the free state in organisms, more often they are completed by having a variety of polar head groups. These exist both as phospho- and glycolipids (and as a combination of both), and as sulfated forms of these. Most of the polar head groups of phospholipids are similar to those of organisms of the other primary kingdoms and include ethanolamine, L-serine, glycerol, myo-inositol, and even choline in phosphodiester-linkage, while the glycolipids comprise mainly glucosyl and gentiobiosyl (β‑D‑glucosyl-(1→6)-β-D-glucosyl) units linked to the core alkylglycerols. In the Halobacteriaceae, the basic diglycosyl residue is α-D-mannosyl(1-2)-α‑D-glucose. However, in some species of Archaea there are some unique polar groups, such as di- and trimethylaminopentanetetrols, glucosaminyl-myo-inositol and glucosyl-myo-inositol. New structures are still being discovered.

The main lipids in Archaea

The simplest lipids of this type are based upon the archaeol backbone, so that archaetidic acid is the monophosphate ester of archeol and is the equivalent of phosphatidic acid from eukaryotes, while archaetidylethanolamine is analogous to phosphatidylethanolamine, for example. Indeed, most of the conventional phospholipids have archaeal equivalents, including an analogue of cardiolipin. Similarly, gentiobiosyl archaeol could be considered as an analogue of the diglycosyldiacylglycerols found in higher plants. Other such lipids, which may lack conventional equivalents, include archaetidylglycerosulfate, archaetidylglycerophosphate methyl ester and triglycosyl archaeol lipids. It appears that aminolipids and glycolipids containing pH-sensitive β-D-galactofuranosyl units are common in the methanogens, but they are absent from thermophiles. Halobacterium salinarum and related species contain similar lipids with sulfated tri-, di- and mono-glycosyl-diethers esterified to the phosphate group of phosphatidic acid, while Halobellus sp. contain comparable molecular forms together with phosphatidylglycerosulfate.

The lipids based on the caldarchaeol and other tetra-ether cores are much more complicated. Sometimes only one of the glycerol moieties is attached to a polar moiety, so caldarchaetidic acid is the monophosphate ester of caldarchaeol, but more often both glycerols are linked to polar moieties, and these are always different, e.g. glycosyl caldarchaetidylserine contains a glycosyl moiety at one end of the molecule and a serine phosphate at the other. Thus, the extensively studied species Methanobacterium thermoautotrophicum contains four phospholipids (archaetidic acid, archaetidylethanolamine, archaetidylserine, archaetidyl-myo-inositol) and one glycolipid (gentiobiosyl archaeol) based on archaeol, together with four phospholipids (caldarchaetidic acid, caldarchaetidylethanolamine, caldarchaetidylserine, caldarchaetidyl-myo-inositol), one glycolipid (gentiobiosyl caldarchaeol) and three mixed glyco-phospholipids (gentiobiosyl caldarchaetidylethanolamine, gentiobiosyl caldarchaetidylserine, gentiobiosyl caldarchaetidyl-myo-inositol) based on caldarchaeol.

Different archaeal species can contain distinctive variants on the basic structures, and these are proving useful for taxonomic purposes and for studies of microbial ecology. Also, because of their saturated nature and the relatively stable ether bonds, residues of archaeal ether lipids can survive well in rocks and sediments, and can serve as markers for the Archaea in general and even for particular organisms over geological time spans. However, slow degradation does occur via hydrolysis, oxidation and other reactions with formation of by-products that are still recognizable as of Archaeal origin. Archaeal lipids are an important element of research in organic geochemistry as they are found in most environments, including soil, peat, marine and lacustrine water columns and sediments, hot springs and stalagmites. They have even been claimed as ‘most ubiquitous lipid on Earth’.

It is not always recognized that Archaeal species can contain appreciable amounts of lipids containing conventional fatty acids. Most of these are not linked by ester bonds, so may be in unesterified form or as amide-linked components of amino-lipids, such as ornithine lipids, sphingolipids or proteolipids. However, in Methanothermus fervidus, esterified fatty acids amounted to 89% of the total phospholipid side chains. None of these lipids appear to have been adequately characterized as yet. Although sterols and hopanoids are not present, Archaea contain polyterpenes, i.e. carotenoids, polyprenols, quinones and apolar polyisoprenoids, which may have related functions in regulating the physical properties of membranes.

Anaerobic soil bacteria of the genus Acidobacteria produce tetralkylglycerol ethers that are superficially similar to those of the Archaea, but are derived from 1,2-di-O-alkyl-sn-glycerol. They may also contain membrane-spanning fatty acids such as iso-diabolic acid (13,16-dimethyl-octacosanedioic acid).

2.  Archaeal Lipids in Membranes

Diether phospholipids resemble the more conventional diacyl phospholipids from eukaryotes in many aspects of their physical properties, and in particular they have an ability to form bilayer membrane structures. On the other hand, tetra-ether polar lipids can span the membranes of the organisms to form in effect a membrane monolayer, which is believed to stiffen the membrane at high growth temperatures. Physical chemical methods, such as freeze fracturing, are not able to separate the two leaves of the bilayer, for example. In aqueous solution, the bipolar ether lipids especially form remarkably stable liposomes of different sizes (uni- and multi-lamellar) and membrane packing densities, a property of potential value as carriers of therapeutic agents or as adjuvants for drugs and vaccines.

The complex archaeal lipids are distributed asymmetrically in membranes. A study of the distribution of lipids between the inner and outer leaflets of the membrane of Methanobacterium thermoautotrophicum has demonstrated that a high proportion of the gentiobiose units of both the di- and tetra-ether lipids are exposed on the outer aspect of the cells, where inter-glycosyl hydrogen bonding may assist in stabilizing the membrane structure. Similarly, much of the gentiobiose unit of gentiobiosyl caldarchaetidylethanolamine is on the outer surface with the phosphoethanolamine unit inside, although most of the archaetidylethanolamine (diether) is in the outer leaflet of the membrane bilayer. The phosphoserine and phosphoinositol residues of both diether and tetraether polar lipids are mainly oriented towards the cytoplasmic surface of the membrane, as is expected in Eukaryotes.

Scottish thistleAt the growth temperature of any organism, membranes are in a liquid-crystalline state in which a high degree of lipid movement is possible to enable membrane proteins to function properly and to maintain the barrier function. Most bacteria achieve this by changing the chain-length and degree of unsaturation and/or branching of the fatty acid chains. In archaea, the plasma membrane is in a liquid–crystalline state between a monolayer of dibiphytanyldiglycerol tetraether lipids and a bilayer of diphytanylglycerol di-ether lipids. While the phytanyl chains of archaeal membranes maintain this phase over a wide range of growth temperatures, the lipid compositions do change in response to extreme temperature variations. Ether lipids are much more stable to chemical attack via oxidation or acid/base treatment than acyl lipids, and there is increasing evidence that they have a major role in archaeal membranes in enabling the organisms to tolerate extremes of temperature, salt concentrations and pH. The addition of cyclic structures such as five-membered rings to the trans-membrane portion of the lipids appears to be an adaptation to high temperatures, conferring enhanced membrane packing and reduced fluidity properties, and strengthens the hydrogen bonding at the membrane surface. The presence of a covalent bond between the alkyl chains in H-shaped tetraethers may reinforce the strength of the membrane. For example, halophiles can thrive at salinities greater than 20-25%, while the optimal growth temperature for many thermophiles is 80°C, and some have survived a temperature as high as 120°C. Some acidophiles are able to withstand a pH of zero. It has been demonstrated that the tetra-ether membrane monolayers especially have a limited permeability for protons even at the higher growth temperatures that have been observed. It appears that Archaea adjust the composition of their membrane lipids to maintain their proton permeability within a narrow range. Membranes containing tetra-ether lipids are also able to withstand high concentrations of metal ions and pH gradients that approach 5 pH units.

It has been proposed that magnesium ions can bridge the negative charges of adjacent anionic phospholipids to act in part as a surrogate for cardiolipin, a molecule that is known to control the curvature of membranes. However, there is a tight association between cardiolipin and cytochrome c oxidase in mitochondria and bacteria, and this is also true for the cardiolipin analogue in Archaea, suggesting that this is a truly universal lipid-protein interaction. Remarkably high concentrations of menaquinones are present in membranes of some extremophiles such as the haloarchaea, where it has been suggested that they act as ion permeability barriers and as a powerful shield against oxidative stress, in addition to their functions as electron and proton transporters.

3.  Biosynthesis

The unique chirality of the glycerol molecule in Archaeal lipids is a consequence of the specificity of the enzyme that reduces dihydroxy acetone phosphate, i.e. the product is sn-glycerol-1-phosphate rather than sn-glycerol-3-phosphate, as in bacteria and eukaryotes.

In eukaryotes, there are a number of routes to the generation of sn-glycerol-3-phosphate, including via glycolysis, in addition to via D-glyceraldehyde-3-phosphate as illustrated (see our web page on ether lipids). Dihydroxyacetone phosphate (DHAP) is a key intermediate, which in Archaea is converted to sn-glycerol-1-phosphate by an NADH-dependent reduction by G1P-dehydrogenase, which is completely different from the well-known G3P-dehydrogenase. Both use adenine nucleotides (NADH or NADPH), but G1P-dehydrogenase uses Zn2+ at its active site and transfers the pro-R hydrogen of NADH while G3P-dehydrogenase transfers the pro-S hydrogen.

Synthesis of sn-glycerol-1-phosphate

The evolutionary significance of this is a matter of debate, with various experts ranking the deviation in these pathways at different points in the divergence of Archaea and Bacteria from a primitive ancestral cell. However, two recently discovered archaeal groups, i.e. marine euryarchaeota group II/III and 'Lokiarchaeota', which are believed to be descendants of the archaeal ancestor leading to eukaryotes, lack the gene for synthesis of sn-glycerol-1-phosphate, and cannot synthesise archaeal membrane lipids, although they are able to produce 'chimeric' lipids with ether-bound isoprenoidal chains or with one ester-bound fatty acid replacing an ether-bound isoprenoid. These archaeal species may reflect a transition stage between archaea and eukaryotes. A preliminary communication suggests that some bacterial phyla encode a complete pathway for archaeal membrane lipid biosynthesis in addition to that for conventional bacterial lipids.

The isoprenoid chains in archaea are synthesised by a mechanism that appears to be similar if not identical to the classical mevalonic acid pathway (see our web page on cholesterol biosynthesis) at least in the first steps, and it involves the universal five-carbon subunits isopentenyl pyrophosphate and dimethylallyl pyrophosphate. For example, the key enzyme HMG-CoA reductase from Sulfolobus solfataricus showed more than 40% similarity to eucaryal homologues. However, some of the later steps in isoprenoid biosynthesis are different from those in the classical mevalonic acid pathway, suggesting a divergence in archaeal metabolism from both bacteria and eukaryotes at a very early stage in their evolution from a common ancestor. There are also appreciable differences among the archaeal phyla. The isoprenoid building blocks undergo sequential condensation reactions leading first to the formation of geranyl diphosphate (C10), then farnesyl (C15), geranylgeranyl (C20), farnesylgeranyl (C25) diphosphates, and so forth, reactions catalysed by enzymes of a family of synthases common to all three domains of life.

Ether bonds are formed by coupling the terpenoid chains as geranylgeranyl units, first to position 3 of sn-glycerol-1-phosphate and then to position 2 to form sn-2,3-digeranylgeranylglycerol-1-phosphate by cytoplasmic prenyl transferases (geranylgeranylglyceryl diphosphate synthases).

Biosynthesis of archeol lipids

The membrane protein cytidine diphosphate (CDP)-archaeol synthase catalyses the transfer of the nucleotide to its specific archaeal lipid substrate, leading to the formation of a CDP-activated precursor (CDP-archaeol) to which polar head groups are attached. Molecular biology and gene studies have found a number of archaeal proteins with sequence similarities to members of the (CDP)-alcohol phosphatidyltransferase family that add the polar head groups, suggesting that the biosynthesis mechanisms for the archeol serine and archeol glycerol phospholipids resemble those for the bacterial analogues other than for the inositol lipids (see below). Similarly, archaetidylethanolamine is probably synthesised by decarboxylation of archaetidylserine as in the bacterial equivalent. Archaeol glycolipids are synthesised by the transfer of glucose or gentiobiose from UDP-glucose or UDP-gentiobiose, respectively, to archaeol. The archaeal geranylgeranyl reductase is able to use the isoprenoid chains either in free form or bound to complex lipids, but hydrogenation of the double bond in position 2 is only possible when the isoprenoid chain is in bound form so the reaction is believed to be completed at this final stage. In species that produce di-C25 ether lipids, such as A. pernix, the prenyl transferases are mainly responsible for the specificity.

The formation of tetra-ether lipids is one of the most intriguing problems in lipid biochemistry. Although it was presumed for many years that it must involve carbon-carbon bond formation between the two methyl termini of isoprenoid chains, which the evidence suggests must be unsaturated, such a reaction would be unprecedented in biochemistry and alternative proposed mechanisms are under investigation. More recently, it has been suggested that head-to-head condensation of two C20 isoprenyl molecules, each containing an isopropylidene double bond might take place prior to attachment to the glycerol unit. Presumably, this reaction could be catalysed by an enzyme similar to the phytoene synthase involved in the biosynthesis of carotenoids. Two C40 chains with a diphosphate residue at each end could then be attached to the first glycerol by enzymes that are sufficiently flexible to accommodate a range of structural types, before the second glycerol is attached. Only then would the double bonds be hydrogenated.

Proposed intermediate in the fomation of glycerol dialkyl glycerol tetraethers

Cyclopentane rings are presumably formed by internal cyclization of the biphytanyl chains at an undefined stage in biosynthesis, probably involving the coupling of a methyl group with another carbon atom by mechanisms that have yet to be revealed. This would be highly unusual in that it involves non-activated carbon atoms, and an alternative hypothesis is that the rings are formed during chain elongation to form the C40 units with isopentenyl phosphate before attachment to glycerol.

Much remains to be learned of fatty acid biosynthesis in Archaea. It seems that acyl carrier protein is missing, but some aspects appear to resemble the biosynthetic mechanism in bacteria. Some of the enzymes for β-oxidation of fatty acids are also present in Archaea, and it has been suggested that these might operate in reverse in order to synthesise fatty acids.

Inositol lipids: The biosynthesis of the inositol lipids in Archaea and those few bacterial species that contain such lipids has proved of particular interest because the biosynthetic mechanism is very different from that in Eukaryotes, and this has evolutionary implications. Glucose-6-phosphate is converted to 1L-myo-inositol 1-phosphate (synonymous with inositol 3-phosphate) by an inositol phosphate synthase, and this is reacted with CDP-archaeol to form archaetidylinositol 3-phosphate by an archaetidylinositol phosphate synthase. This differs from the mechanism in Eukaryotes in that it is the 1-hydroxyl group of inositol 3-phosphate that is transferred rather than the 1-hydroxyl of free inositol. Finally, archaetidylinositol is produced via the action of a phosphatase.

Biosynthesis of archaetidylinositol

4.  Analysis

Structural analysis of the archaeal lipids is technically daunting. Chemical degradative methods were first used, but modern mass spectrometric procedures have now come to the fore, especially with HPLC in combination with electrospray and atmospheric-pressure chemical ionization. It is simpler from a technical standpoint to analyse the core lipids after removal of the polar moieties, when both GC-MS and HPLC-MS techniques can be used for analysis. Nuclear magnetic resonance (NMR) spectroscopy is then invaluable for determination of the stereochemistry of the various structural units. On the other hand, it is now possible to study the intact lipids by HPLC-MS methodology in order to determine the nature and proportions of the various head groups as well as of the alkyl moieties.

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Lipid listings Credits/disclaimer Updated: October 30th, 2019 Author: William W. Christie LipidWeb icon