Archaeal Ether Lipids

1.  Basic Chemistry

The Archaea represent one of the three primary kingdoms or domains of living organisms. The alternative name 'archaebacteria' is considered by some to be redundant. Archaea are single celled, lacking a nuclear membrane and having a low deoxyribonucleic acid content. They include thermophiles, halophiles and acidophiles, collectively termed 'extremophiles'. Some believe that they resemble the dominant organisms in the primeval biosphere. Many of the species are methanogenic, including those found in low-temperature environments. Four archaeal phyla are recognized, the Euryarchaeota, Crenarchaeota, Thaumarcheota and Korarcheota. These 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 environmental conditions also, and indeed it is now suggested that these may constitute up to 20% of the oceanic biomass.

The lipids of these organism 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 Eucarya (eukaryotes).

 

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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.

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 one to four cyclopentane rings in each chain, while the crenarchaeota may have an additional cyclohexane ring in the alkyl chains. There appear to be increasing proportions of cyclopentane rings in lipids from thermoacidophilic species with increasing environmental temperatures. Yet other lipids have 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 hyperthermophilic organisms of the Crenarchaeota.

 

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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.

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 Aeropyrum pernix has C25 archaeols only. In contrast, the genera Pyrobaculum, Thermoplasma and Sulfolobus utilize caldarchaeols (C40), including those with cyclopentane ring structures.

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As an alternative to the simple glycerol component, calditol from Sulfolobus solfaricus contains 2-hydroxymethyl-1,2,3,4,5-pentahydroxy-cyclopentane linked via an ether bond to the glycerol moiety at position sn-1. In other species, the alkyl groups are linked to tetritol.

While these core lipids are often found in the free state in organisms, more often they are completed by having a variety of polar head groups. These exist as 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. The glycolipids comprise mainly glucosyl and gentiobiosyl (β-D-glucosyl-(1→6)-β-D-glucosyl) units linked to the core alkylglycerols. 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.

 

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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. Streptococcus species contain dimeric lipids, termed 'glucopyronosy- and kojibiosyl-cardiolipins', while Halobacterium salinarum contains similar lipids with sulfo-tri- and diglycosyl-diethers esterified to the phosphate group of phosphatidic acid. However, the term ‘glycocardiolipin’ is misleading, as a glucose unit takes the place of the central glycerol of cardiolipin.

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. However, 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, which 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 recognisable by-products. 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 recognised 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 89.0% of the total phospholipid side chains. None of these lipids appear to have been adequately characterised as yet.

 

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. 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 multilamellar) and membrane packing densities, a property of potential value as carriers of therapeutic agents or as adjuvants of 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 interglycosyl 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.

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. Acidophiles are able to withstand a pH of zero and below. 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.

In mitochondria and bacteria, there is a tight association between cardiolipin and cytochrome c oxidase, and this is also true for the cardiolipin analogue in Archaea, suggesting that this is a truly universal lipid–protein interaction.

 

3.  Biochemistry

The unique chirality of the glycerol molecule in these 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.

 

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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 a by G1P-dehydrogenase, which is completely different from the well-known G3P dehydrogenase. 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. Comparisons of other enzymes in the biosynthesis of lipids in also relevant to this debate, which I prefer to leave to the experts (see the reading list below).

The isoprenoid chains are synthesised by a mechanism that appears to be very similar to the classical mevalonic acid pathway (see our web page on cholesterol biosynthesis). 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.

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. 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.

 

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Cyclopentane rings are presumably formed by internal cyclization of the biphytanyl chains involving the coupling of a methyl group with another carbon atom by mechanisms that have yet to be revealed. Molecular biology and gene studies have found a number of archaeal proteins with sequence similarities to members of the cytidine diphosphate (CDP)-alcohol phosphatidyltransferase family, suggesting that the biosynthesis mechanisms for the archeol serine, glycerol and inositol phospholipids resemble those for the bacterial analogues. Similarly, archaetidylethanolamine is probably synthesised by decarboxylation of archaetidylserine as in the bacterial equivalent. In the same way, archaeol glycolipids are synthesised by the transfer of glucose or gentiobiose from UDP-glucose or UDP-gentiobiose, respectively, to archaeol.

The formation of tetra-ether lipids is one of the most intriguing problems in lipid biochemistry. It is presumed that it must involve carbon-carbon bond formation between the two methyl termini of phytanyl or their precursor (unsaturated) chains. Such a reaction would be unprecedented in biochemistry, and it will be fascinating to learn how the story unfolds with further research.

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, with 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.

 

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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.

 

4.  Structurally Related Lipids from Anaerobic Bacteria

In recent years an unusual group of branched glycerol dialkyl glycerol tetraether lipids has been discovered in peat bogs and soils. In general, they consist of octacosane (C28) alkyl units with either 13,16-dimethyl- or 5,13,16-trimethyl substituents, and forms occur with 4 to 6 methyl groups attached to the n-alkyl chains and/or with 0 to 2 cyclopentyl moieties in the alkyl chain. In addition to being nonisoprenoid in nature, they differ from the lipids of the Archaea in that they have a 1,2-di-O-alkyl-sn-glycerol rather than a 2,3-di-O-alkyl-sn-glycerol configuration typical of the latter.

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It is believed that these branched membrane lipids are produced by anaerobic soil bacteria, probably of the genus Acidobacteria. The nature of the intact lipids from which they are derived has also to be fully elucidated, although some components have been identified with glucuronosyl or glucosyl units attached to the glycerol ether backbone.

 

5.  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.

 

Suggested Reading

  • Chong, P.L.-G. Archaebacterial bipolar tetraether lipids: Physico-chemical and membrane properties. Chem. Phys. Lipids, 163, 253-265 (2010) (DOI: 10.1016/j.chemphyslip.2009.12.006).
  • Corcelli, A. The cardiolipin analogues of Archaea. Biochim. Biophys. Acta, 1788, 2101-2106 (2009) (DOI: doi:10.1016/j.bbamem.2009.05.010).
  • Gattinger, A., Schloter, M. and Munch, J.C. Phospholipid etherlipid and phospholipid fatty acid fingerprints in selected euryarchaeotal monocultures for taxonomic profiling. FEMS Microbiol. Letts., 213, 133-139 (2002) (DOI: 10.1111/j.1574-6968.2002.tb11297.x).
  • Knappy, C.S., Barilla, D., de Blaquiere, J.P.A., Morgan, H.W., Nunn, C.E.M., Suleman, M., Tan, C.H.W. and Keely, B.J. Structural complexity in isoprenoid glycerol dialkyl glycerol tetraether lipid cores of Sulfolobus and other archaea revealed by liquid chromatography-tandem mass spectrometry. Chem. Phys. Lipids, 165, 648-655 (2012) (DOI: 10.1016/j.chemphyslip.2012.06.009).
  • Koga, Y. and Morii, H. Special methods for the analysis of ether lipid structure and metabolism in archaea. Anal. Biochem., 348, 1-14 (2006) (DOI: 10.1016/j.ab.2005.04.004).
  • Liu, X.L., Leider, A., Gillespie, A., Groger, J., Versteegh, G.J.M. and Hinrichs, K.U. Identification of polar lipid precursors of the ubiquitous branched GDGT orphan lipids in a peat bog in Northern Germany. Org. Geochem., 41, 653-660 (2010) (DOI: 10.1016/j.orggeochem.2010.04.004).
  • Lombard, J., López-García, P. and Moreira, D. Phylogenomic investigation of phospholipid synthesis in Archaea. Archaea, 630910 (2012) (DOI: 10.1155/2012/630910).
  • Matsumi, R., Atomi, H., Driessen, A.J.M. and van der Oost, J. Isoprenoid biosynthesis in Archaea - Biochemical and evolutionary implications. Res. Microbiol., 162, 39-52 (2011) (DOI: 10.1016/j.resmic.2010.10.003).
  • Morii, H., Kiyonari, K., Ishino, Y. and Koga, Y. A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in methanoarchaeon Methanothermobacter thermautotrophicus cells. J. Biol. Chem., 284, 30766-30774 (2009) (DOI: 10.1074/jbc.M109.034652).
  • Peretó, J., López-García, P. and Moreira, D. Ancestral lipid biosynthesis and early membrane evolution. Trends. Biochem. Sci., 29, 469-477 (2004) (DOI: 10.1016/j.tibs.2004.07.002).
  • Schouten, S., Hopmans, E.C. and Damsté, J.S.S. The organic geochemistry of glycerol dialkyl glycerol tetraether lipids: A review. Org. Geochem., 54 19-61 (2013) (DOI: 10.1016/j.orggeochem.2012.09.006).
  • Zhu, C., Lipp, J.S., Wormer, L., Becker, K.W., Schroder, J. and Hinrichs, K.U. Comprehensive glycerol ether lipid fingerprints through a novel reversed phase liquid chromatography-mass spectrometry protocol. Org. Geochem., 65, 53-62 (2013) (DOI: 10.1016/j.orggeochem.2013.09.012).

 

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Updated: April 15, 2014