Archaeal Membrane Research Articles

Reappraisal of archaeal C20-C25 diether lipid (extended archaeol) origin and use as a biomarker of hypersalinity

The diether core membrane lipid sesterterpanyl-phytanyl-glycerol (so-called extended archaeol and often abbreviated C20-C25) is considered as a hallmark of Haloarchaea, a clade of archaea thriving under extreme high salinities. We here report about extended archaeol occurrence in different saline aquatic settings with salinity ranging from ca. 50 psu (5 % NaCl w/v) to saturation (ca. 350 psu). This demonstrates that this lipid is not restricted to extreme saline environments but suggests a minimum salinity threshold of ca. 50 psu above which C20-C25 is most commonly produced. The proportion of C20-C25 relative to that of archaeol (C20-C20) did not appear linearly dependent on the salinity of the site and was potentially also influenced by pH and temperature, preventing its direct use as a quantitative salinity proxy based on the present data set. An extensive literature review of archaeal membrane lipid compositions further highlighted that taxonomy also contributes to the distribution of this lipid in the environment and identifies Natrialbales (one of the three orders of Haloarchaea) as the main source. Statistical analysis showed that, among Haloarchaea, C20-C25 producers display pH and salinity growth optima slightly higher than non-producers and are distributed within two distinct groups, one composed mostly of neutrophiles and one of alkaliphiles. In contrast, the presence of C20-C25 was not correlated to the optimal growth temperature of the strains. This suggests that two confounding parameters, i.e., taxonomy and adaptation to changes in salinity and/or pH, contribute to the distribution of C20-C25 within Haloarchaea.

Crossing the lipid divide

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Location and Conformational Ensemble of Menaquinone and Menaquinol, and Protein-Lipid Modulations in Archaeal Membranes.

Halobacteria, a type of archaea in high salt environments, have phytanyl ether phospholipid membranes containing up to 50% menaquinone. It is not understood why a high concentration of menaquinone is required and how it influences membrane properties. In this study, menaquinone-8 headgroup and torsion parameters of isoprenoid tail are optimized in the CHARMM36 force field. Molecular dynamics simulations of archaeal bilayers containing 0 to 50% menaquinone characterize the distribution of menaquinone-8 and menaquinol-8, as well as their effects on mechanical properties and permeability. Menaquinone-8 segregates to the membrane midplane above concentrations of 10%, favoring an extended conformation in a fluid state. Menaquinone-8 increases the bilayer thickness but does not significantly alter the area compressibility modulus and lipid chain ordering. Counterintuitively, menaquinone-8 increases water permeability because it lowers the free energy barrier in the midplane. The thickness increase due to menaquinone-8 may help halobacteria ameliorate hyper-osmotic pressure by increasing the membrane bending constant. Simulations of the archaeal membranes with archaerhodopsin-3 show that the local membrane surface adjusts to accommodate the thick membranes. Overall, this study delineates the biophysical landscape of 50% menaquinone in the archaeal bilayer, demonstrates the mixing of menaquinone and menaquinol, and provides atomistic details about menaquinone configurations.

How Can Immuno suppression After Organ Transplantation be Made More Effective and Safer? – A Review on Liposomal Formulations with Consideration of Archaeal Tetraetherlipid

Immune-suppressive agents such as methylprednisolone and cyclosporine exert tremendous side effects, because of high dosage and long-term application required for immune suppression after organ transplantation. Major side effects of methylprednisolone include bleeding of the gastro-intestinal tract, hypertension, and osteoporosis, whereas cyclosporine is nephrotoxic. Liposomes are phospholipid particles that allow delivery of drugs preferentially to the reticuloendothelial system. They can be prepared from phospholipids such as lecithin from soybean or egg yolk, other specific or modified lipids or from membrane-spanning tetraether lipid (TEL), which can be extracted and purified from archaeal cell membranes. One advantage in the use of liposomal application is reduced toxicity of many drugs. We report on various liposomal preparations of cyclosporine, methylprednisolone (L-MPL) and its palmitate derivative (L-MPLP). It has been documented that liposomal cyclosporine A (L-CsA), 1.75 mg/kg/day for seven days has potential for use as an immune-suppressive agent in rats with increased efficacy and decreased nephrotoxicity compared to commercially available forms of intravenous CsA. Liposomal methylprednisolone (L-MPL) 2 mg/kg, intravenously (IV), twice a week shows significantly prolonged cardiac allograft survival in rats and tissue-selective sequestration of the drug in comparison with the same dosage regimen of methylprednisolone in solution, administered daily. We report on organ distribution of L-MPLP in rats after intraperitoneal (IP) administration. Conclusion: Liposomal preparations of immunosuppressants have significantly higher immune-suppressive potential and lower toxicity than non-liposomal preparations. Bipolar TEL can be extracted, fractionated and purified from archaea to form stable liposomes which are extremely resistant, even to gastric fluid. Hence, TEL liposomes allow (besides IV and IP) for oral administration of immuno suppressants after organ transplantation with pharmacological and toxicological advantages as common liposomal phospholipid bilayer preparations.

Greenhouse gas production and lipid biomarker distribution in Yedoma and Alas thermokarst lake sediments in Eastern Siberia.

Permafrost thaw leads to thermokarst lake formation and talik growth tens of meters deep, enabling microbial decomposition of formerly frozen organic matter (OM). We analyzed two 17-m-long thermokarst lake sediment cores taken in Central Yakutia, Russia. One core was from an Alas lake in a Holocene thermokarst basin that underwent multiple lake generations, and the second core from a young Yedoma upland lake (formed ~70years ago) whose sediments have thawed for the first time since deposition. This comparison provides a glance into OM fate in thawing Yedoma deposits. We analyzed total organic carbon (TOC) and dissolved organic carbon (DOC) content, n-alkane concentrations, and bacterial and archaeal membrane markers. Furthermore, we conducted 1-year-long incubations (4°C, dark) and measured anaerobic carbon dioxide (CO2 ) and methane (CH4 ) production. The sediments from both cores contained little TOC (0.7±0.4 wt%), but DOC values were relatively high, with the highest values in the frozen Yedoma lake sediments (1620mgL-1 ). Cumulative greenhouse gas (GHG) production after 1year was highest in the Yedoma lake sediments (226±212µgCO2 -Cg-1 dw, 28±36µg CH4 -Cg-1 dw) and 3 and 1.5 times lower in the Alas lake sediments, respectively (75±76µgCO2 -Cg-1 dw, 19±29µg CH4 -Cg-1 dw). The highest CO2 production in the frozen Yedoma lake sediments likely results from decomposition of readily bioavailable OM, while highest CH4 production in the non-frozen top sediments of this core suggests that methanogenic communities established upon thaw. The lower GHG production in the non-frozen Alas lake sediments resulted from advanced OM decomposition during Holocene talik development. Furthermore, we found that drivers of CO2 and CH4 production differ following thaw. Our results suggest that GHG production from TOC-poor mineral deposits, which are widespread throughout the Arctic, can be substantial. Therefore, our novel data are relevant for vast ice-rich permafrost deposits vulnerable to thermokarst formation.

A promiscuous archaeal cardiolipin synthase enables construction of diverse natural and unnatural phospholipids

Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain–based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif–containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is nonselective to the stereochemistry of the glycerol backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase that could be of interest for bio-catalytic purposes.

Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis.

Lipid membranes are essential cellular elements as they provide cellular integrity and selective permeability under a broad range of environmental settings upon cell growth. In particular, Archaea are commonly recognized for their tolerance to extreme conditions, which is now widely accepted to stem from the unique structure of their lipids. While enhancing the stability of the archaeal cell membrane, the exceptional properties of archaeal lipids also hinder their extraction using regular procedures initially developed for bacterial and eukaryotic lipids. The protocol described here circumvents these issues by directly hydrolyzing the polar head group(s) of archaeal lipids and extracting the resulting core lipids. Although leading to a loss of information on the nature of polar heads, this procedure allows the quantitative extraction of core lipids for most types of archaeal cells in an efficient, reproducible, and rapid manner.

Subseafloor Archaea reflect 139 kyrs of paleodepositional changes in the northern Red Sea.

The vertical distribution of subseafloor archaeal communities is thought to be primarily controlled by in situ conditions in sediments such as the availability of electron acceptors and donors, although sharp community shifts have also been observed at lithological boundaries suggesting that at least a subset of vertically stratified Archaea form a long-term genetic record of coinciding environmental conditions that occurred at the time of sediment deposition. To substantiate this possibility, we performed a highly resolved 16S rRNA gene survey of vertically stratified archaeal communities paired with paleo-oceanographic proxies in a sedimentary record from the northern Red Sea spanning the last glacial-interglacial cycle (i.e., marine isotope stages 1-6; MIS1-6). Our results show a strong significant correlation between subseafloor archaeal communities and drastic paleodepositional changes associated with glacial low vs. interglacial high stands (ANOSIM; R=.73; p= .001) and only a moderately strong correlation with lithological changes. Bathyarchaeota, Lokiarchaeota, MBGA, and DHVEG-1 were the most abundant identified archaeal groups. Whether they represented ancient cell lines from the time of deposition or migrated to the specific sedimentary horizons after deposition remains speculative. However, we show that the majority of sedimentary archaeal tetraether membrane lipids were of allochthonous origin and not produced in situ. Slow post-burial growth under energy-limited conditions would explain why the downcore distribution of these dominant archaeal groups still indirectly reflect changes in the paleodepositional environment that prevailed during the analyzed marine isotope stages. In addition, archaea seeded from the overlying water column such as Thaumarchaeota and group II and III Euryarchaeota, which were likely not have been able to subsist after burial, were identified from a lower abundance of preserved sedimentary DNA signatures, and represented direct markers of paleoenvironmental changes in the Red Sea spanning the last six marine isotope stages.

Diverse communities of Bacteria and Archaea flourished in Palaeoarchaean (3.5–3.3 Ga) microbial mats

AbstractLimited taxonomic classification is possible for Archaean microbial mats and this is a fundamental limitation in constraining early ecosystems. Applying Fourier transform infrared spectroscopy (FTIR), a powerful tool for identifying vibrational motions attributable to specific functional groups, we characterized fossilized biopolymers in 3.5–3.3 Ga microbial mats from the Barberton greenstone belt (South Africa). Microbial mats from four Palaeoarchaean horizons exhibit significant differences in taxonomically informative aliphatic contents, despite high aromaticity. This reflects precursor biological heterogeneity since all horizons show equally exceptional preservation and underwent similar grades of metamorphism. Low methylene to end‐methyl (CH2/CH3) absorbance ratios in mats from the 3.472 Ga Middle Marker horizon signify short, highly branchedn‐alkanes interpreted as isoprenoid chains forming archaeal membranes. Mats from the 3.45 Ga Hooggenoeg Chert H5c, 3.334 Ga Footbridge Chert, and 3.33 Ga Josefsdal Chert exhibit higherCH2/CH3ratios suggesting mostly longer, unbranched fatty acids from bacterial lipid precursors. Absorbance ratios of end‐methyl to methylene (CH3/CH2) in Hooggenoeg, Josefsdal and Footbridge mats yield a range of values (0.20–0.80) suggesting mixed bacterial and archaeal architect communities based on comparison with modern examples. Higher (0.78–1.25)CH3/CH2ratios in the Middle Marker mats identify Archaea. This exceptional preservation reflects early, rapid silicification preventing the alteration of biogeochemical signals inherited from biomass. Since silicification commenced during the lifetime of the microbial mat,FTIRsignals estimate the affinities of the architect community and may be used in the reconstruction of Archaean ecosystems. Together, these results show that Bacteria and Archaea flourished together in Earth's earliest ecosystems.

Effects of temperature and pH on archaeal membrane lipid distributions in freshwater wetlands

Freshwater wetlands harbour diverse archaeal communities and associated membrane lipid assemblages, but the effect of environmental factors (e.g. pH and temperature) on the distribution of these lipids is relatively poorly constrained. Here we explore the effects of temperature and pH on archaeal core-lipid and intact polar lipid (IPL) derived core lipid distributions in a range of wetlands. We focus, not only on the commonly studied isoprenoidal glycerol dialkyl glycerol tetraethers (isoGDGTs), but also widen our analyses to include more recently identified, but relatively widespread, archaeal lipids such as isoGDGT isomers, methylated isoGDGTs (Me-GDGTs), and butanetriol and pentanetriol tetraethers (BDGTs and PDGTs). Based on multivariate analysis and a globally distributed set of wetlands, we find that the degree of isoGDGT cyclisation does increase along with temperature and pH in wetlands. However, and unlike in some other settings, this relationship is obscured in simple scatterplots due to the incorporation of isoGDGTs from highly diverse archaeal sources with multiple ring-temperature or ring-pH relationships. We further show that the relative abundance of early eluting to later eluting isoGDGT isomers increases with pH, representing a previously unknown and seemingly widespread archaeal membrane homeostasis mechanism or taxonomic signal. The distribution and abundance of crenarchaeol, a marker for Thaumarchaeota, demonstrates that in wetlands these Archaea, likely involved in ammonia oxidation, are restricted primarily to the generally drier soil/sediment surface and typically are more abundant in circumneutral pH settings. We identify Me-GDGTs and Me-isoGMGTs (homologs of isoGDGTs and isoGMGTs, but with additional methylation on the biphytanyl chain) as ubiquitous in wetlands, but variation in their abundance and distribution suggests changing source communities and/or membrane adaptation. The high relative abundance of BDGTs and PDGTs in the perennially anoxic part of the peat profile (catotelm), as well as their elevated abundance in a circumneutral pH wetland, is consistent with an important input from their only known culture source, belonging to the methanogenic Methanomassiliicoccales. Our results underline the diversity of archaeal membrane lipids preserved in wetlands and provide a baseline for the use of archaeal lipid distributions in wetlands as tracers of recent or ancient climate and biogeochemistry.

Intact Ether Lipids in Trench Sediments Related to Archaeal Community and Environmental Conditions in the Deepest Ocean

AbstractArchaea play an important role in marine biogeochemical cycle; however, their phylogenetic distribution and lipid composition in the hadal zone (6–11 km water depth) are poorly known. Here, we analyzed archaeal membrane lipids and 16S rRNA gene sequences in sediments from Mariana Trench (MT), Massau Trench (MS), and New Britain Trench (NBT), varying from 1,560 to 10,840 m depth. Forty‐two intact polar lipids (IPLs) were identified, including glycerol dialkyl glycerol tetraethers (GDGTs), OH‐GDGTs, glycerol dialkyl diethers (GDDs), and archaeol (AR) with polar headgroups of monohexose (1G), dihexose (2G), trihexose (3G), and hexose‐phosphohexose (HPH). Compositional and spatial distribution patterns of archaeal lipids suggest benthic Thaumarchaeota as a major source for IPLs, consistent with the predominance of Thaumarchaeota genes (>80%). The redundancy analysis (RDA) based on lipid and 16S rRNA data separates samples into three groups: extremely deep water (MT), significant terrestrial influence (NBT 1, 4, and 6), and predominant marine influence (MS, NBT 2, 3, 7, and 10). 1G‐GDDs and 1G‐AR positively correlate with water depth, likely reflecting the adaptation of benthic archaea to elevated hydrostatic pressure or variation of archaeal community in trench sediments. Bathyarchaeota are more abundant in sediments receiving terrestrial input; this pattern was attributed to their capability of utilizing terrestrial organic matter as an energy source. Our study highlights important environmental influences (e.g., pressure and organic matter quality and quantity) on benthic archaeal community and archaeal IPL compositions, which should be considered when IPLs and core lipids are applied as chemotaxonomic markers and paleo‐proxies.

Lipid Phase Separation Induced by the Apolar Polyisoprenoid Squalane Demonstrates Its Role in Membrane Domain Formation in Archaeal Membranes.

Archaea synthesize methyl-branched, ether phospholipids, which confer the archaeal membrane exceptional physicochemical properties. A novel membrane organization was proposed recently to explain the thermal and high pressure tolerance of the polyextremophilic archaeon Thermococcus barophilus. According to this theoretical model, apolar molecules could populate the midplane of the bilayer and could alter the physicochemical properties of the membrane, among which is the possibility to form membrane domains. We tested this hypothesis using neutron diffraction on a model archaeal membrane composed of two archaeal diether lipids with phosphocholine and phosphoethanolamine headgroups in the presence of the apolar polyisoprenoid squalane. We show that squalane is inserted in the midplane at a maximal concentration between 5 and 10 mol % and that squalane can modify the lateral organization of the membrane and induces the coexistence of separate phases. The lateral reorganization is temperature- and squalane concentration-dependent and could be due to the release of lipid chain frustration and the induction of a negative curvature in the lipids.

Archaeal Membrane Lipid-Based Paleothermometry for Applications in Polar Oceans

Archaeal Membrane Lipid-Based Paleothermometry for Applications in Polar Oceans

Structural studies of geranylgeranylglyceryl phosphate synthase, a prenyltransferase found in thermophilic Euryarchaeota.

Archaea are uniquely adapted to thrive in harsh environments, and one of these adaptations involves the archaeal membrane lipids, which are characterized by their isoprenoid alkyl chains connected via ether linkages to glycerol 1-phosphate. The membrane lipids of the thermophilic and acidophilic euryarchaeota Thermoplasma volcanium are exclusively glycerol dibiphytanyl glycerol tetraethers. The first committed step in the biosynthetic pathway of these archaeal lipids is the formation of the ether linkage between glycerol 1-phosphate and geranylgeranyl diphosphate, and is catalyzed by the enzyme geranylgeranylglyceryl phosphate synthase (GGGPS). The 1.72 Å resolution crystal structure of GGGPS from T. volcanium (TvGGGPS) in complex with glycerol and sulfate is reported here. The crystal structure reveals TvGGGPS to be a dimer, which is consistent with the absence of the aromatic anchor residue in helix α5a that is required for hexamerization in other GGGPS homologs; the hexameric quaternary structure in GGGPS is thought to provide thermostability. A phylogenetic analysis of the Euryarchaeota and a parallel ancestral state reconstruction investigated the relationship between optimal growth temperature and the ancestral sequences. The presence of an aromatic anchor residue is not explained by temperature as an ecological parameter. An examination of the active site of the TvGGGPS dimer revealed that it may be able to accommodate longer isoprenoid substrates, supporting an alternative pathway of isoprenoid membrane-lipid synthesis.

Elasticity and phase behaviour of biomimetic membrane systems containing tetraether archaeal lipids

Archaeal single-cell microorganisms are characterised by particularly strong resistance to harsh environments at extreme temperature ranges, pH values, high pressure and salinity. The recognition of molecules rendering the archaeal membranes stable in such conditions represents an important step towards understanding the molecular mechanisms, which underlie the cellular survival, in order to effectively exploit them in biomedicine and bionanotechnology. Here we report on the bending elasticity and phase behaviour of model lipid membranes containing tetraether archaeal lipids. The bending elasticity modulus of membranes composed of palmitoyl-oleoyl phosphatidylcholine and glycerol dialkyl glycerol tetraether extract from the archaeon Thermoplasma acidophilum’s plasma membrane is measured by analysis of the thermal shape fluctuations of nearly spherical giant unilamellar vesicles (tens of micrometers in size). The bending rigidity is reported to non-monotonically depend on the archaeal lipids’ concentration in the bilayer. At 50 wt% of tetraether lipids in the membrane we measure around 20% decrease of the bending modulus compared to the studied single-component phosphocholine bilayer. The membrane resistance to bending increases at archaeal lipids’ content higher than 70 wt%. These findings support the hypothesis about a probable looping conformation at very low amounts of bipolar lipids in the membrane and prevailing spanning tetraether molecules at higher concentrations. Fluorescence microscopy reveals structural phase coexistence at low temperatures (6 °C) for 75 wt% of bolalipids in the bilayer. Laurdan spectroscopy measurements of large unilamellar vesicles (hundreds of nanometers in size) provide evidences for increased lipid ordering at the glycerol level induced by the presence of up to 90 wt% of bolalipid in the bilayer. At this archaeal lipid content we measure increased bending rigidity of the membrane. The fatty acyl chain mobility probed by DPH fluorescence spectroscopy is significantly reduced in the presence of bolalipids. The lipid ordering decreases with increasing the temperature to an extent depending on the bolalipid content in the membrane. The presence of bipolar lipids in the bilayer affects the lipid packing more strongly at the glycerol level compared to the hydrophobic core of the membrane. By elucidating the effect of tetraether lipids on the structural and mechanical properties of the bilayer, the reported results are expected to help in advancing on liposome-based pharmaceuticals and biomedical applications as well as for developments in sensorics and nanotechnology.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl− binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl−-releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl− uptake was faster than that of MF NpHR. These differences in the Cl−-releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl− release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl− uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl−-binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR′ state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl− pumping.

Lipidomics in archaeal membrane adaptation to environmental stresses and growth conditions: A review of culture-based physiological studies

Membrane lipids are thought to be a crucial part of the homeoviscous adaptation of archaea to extreme conditions. This article reviews the recent lipidomic studies of physiological membrane adaptations of archaea, assesses the biomolecular basis of an organic paleothermometer, TEX86, and contemplates the future directions of archaeal lipidomics. The studies of extremophilic archaea have revealed that at least three different molecular mechanisms are involved in membrane adaptation of archaea: (1) regulation of the number of cyclopentane rings of caldarchaeol, (2) alteration of the diether-to-tetraether lipid ratio, and (3) variation of the proportion of saturated and unsaturated lipids. However, most of the studies have focused on a limited number of archaeal ether-linked lipids, such as glycerol dialkyl glycerol tetraethers (GDGTs), which only represent a fraction of the entire lipidome. Environmental factors such as growth temperature and pH have been most frequently reported, but biotic factors, including growth phases, nutrition, and enzymatic activities affecting the membrane lipid composition are often overlooked. Membrane lipids of mesophilic ammonia-oxidizing marine Thaumarchaeota have been applied in the reconstruction of past sea surface temperatures. However, recent culture-based physiological studies have demonstrated that non-thermal biotic factors, including dissolved oxygen, ammonia oxidation rate and the growth rate, are the main drivers of GDGT cyclization in Nitrosopumilus maritimus . Moreover, other related strains or ecotypes exhibit a markedly different set of stress adaptations. A trend is now developing to examine the whole lipid profile (lipidome) for studies of archaeal physiology and biochemistry related to lipid biosynthesis (lipidomics) to gain a better understanding of the biological mechanisms underpinning the applications of membrane lipid-based proxies in biogeochemical or ecological research.

Constraining the formation of authigenic carbonates in a seepage-affected cold-water coral mound by lipid biomarkers.

Cold-water coral (CWC) mounds are build-ups comprised of coral-dominated intervals alternating with a mixed carbonate-siliciclastic matrix. At some locations, CWC mounds are influenced by methane seepage, but the impact of methane on CWC mounds is poorly understood. To constrain the potential impact of methane on CWC mound growth, lipid biomarker investigations were combined with mineralogical and petrographic analyses to investigate the anaerobic oxidation of methane (AOM) and authigenic carbonate formation in sediment from a seep-affected CWC mound in the Gulf of Cadiz. The occurrence of AOM was confirmed by characteristic lipids found within a semi-lithified zone (SLZ) consisting of authigenic aragonite, high-magnesium calcite and calcium-excess dolomite. The formation of high-Mg calcite is attributed to AOM, acting as a lithifying agent. Aragonite is only a minor phase. Ca-excess dolomite in the SLZ and upper parts may be formed by organoclastic sulphate reduction, favouring precipitation by increased alkalinity. The AOM biomarkers in the SLZ include isoprenoid-based archaeal membrane lipids, such as abundant glycerol dibiphytanyl glycerol tetraethers (GDGTs) dominated by GDGT-2. The δ13 C values of GDGT-2, measured as ether-cleaved monocyclic biphytanes, are as low as -100‰ versus V-PDB. Further, bacterial dialkyl glycerol diethers with two anteiso-C15 alkyl chains and δ13 C values of -81‰ are interpreted as biomarkers of sulphate-reducing bacteria. The lipid biomarker signatures and mineralogical patterns suggest that anaerobic methane-oxidizing archaea of the ANME-1 group thrived in the subsurface at times of slow and diffusive methane seepage. Petrographic analyses revealed that the SLZ was exhumed at some point (e.g. signs of bioerosion of the semi-lithified sediment), providing a hard substrate for CWC larval settlement. In addition, this work reveals that AOM-induced semi-lithification likely played a role in mound stabilization. Lipid biomarker analysis proves to be a powerful tool to disentangle early diagenetic processes induced by microbial metabolisms.

Construction of an artificial biosynthetic pathway for hyperextended archaeal membrane lipids in the bacterium Escherichia coli.

Archaea produce unique membrane lipids, which possess two fully saturated isoprenoid chains linked to the glycerol moiety via ether bonds. The isoprenoid chain length of archaeal membrane lipids is believed to be important for some archaea to thrive in extreme environments because the hyperthermophilic archaeon Aeropyrum pernix and some halophilic archaea synthesize extended C25,C25-archaeal diether-type membrane lipids, which have isoprenoid chains that are longer than those of typical C20,C20-diether lipids. Natural archaeal diether lipids possessing longer C30 or C35 isoprenoid chains, however, have yet to be isolated. In the present study, we attempted to synthesize such hyperextended archaeal membrane lipids. We investigated the substrate preference of the enzyme sn-2,3-(digeranylfarnesyl)glycerol-1-phosphate synthase from A. pernix, which catalyzes the transfer of the second C25 isoprenoid chain to the glycerol moiety in the biosynthetic pathway of C25,C25-archaeal membrane lipids. The enzyme was shown to accept sn-3-hexaprenylglycerol-1-phosphate, which has a C30 isoprenoid chain, as a prenyl acceptor substrate to synthesize sn-2-geranylfarnesyl-3-hexaprenylglycerol-1-phosphate, a supposed precursor for hyperextended C25,C30-archaeal membrane lipids. Furthermore, we constructed an artificial biosynthetic pathway by introducing 4 archaeal genes and 1 gene from Bacillus subtilis in the cells of Escherichia coli, which enabled the E. coli strain to produce hyperextended C25,C30-archaeal membrane lipids, which have never been reported so far.