Tetraether Lipids Research Articles

Hidden molecular clues in marine sediments revealed by untargeted mass spectrometry imaging

Mass spectrometry imaging (MSI) in sedimentary archives can produce records of molecular proxies at μm-scale resolution. For example, in annually varved sediments of the Santa Barbara Basin, such a fine resolution allows deciphering sub-annual distributions of archaeal tetraether lipids, haptophyte-derived alkenones, and sterols. Herein, we reported the establishment of an untargeted data processing workflow aimed at dissecting the MSI datasets and extracting information beyond that obtained by targeted analysis of known molecular proxies. The combination of MSI and the untargeted workflow not only increases the spatial resolution for molecular stratigraphy but also dramatically broadens the number and diversity of molecular signals evaluated, enabling us to discover unique molecular signatures imprinted by various biogeochemical processes. We applied the proposed workflow to two MSI datasets that were both measured on the uppermost ∼10 cm of the Santa Barbara Basin sediments while covering different mass ranges. Two matrices of 18625×293 and 18963×323 (number of spectra × number of peaks) were, respectively, extracted after peak alignment using bin-wise kernel density estimation and subsequent peak picking by peak prominence filtering combined with geochemical context-based filtering. Feature extraction by non-negative matrix factorization revealed in total 15 stable molecular clusters with distinct spatial distributions in the sediments. Each cluster typically comprised several to dozens of compounds, with the majority of compounds in each cluster likely belonging to similar chemical taxonomies. Some of these clusters can be linked to specific biogeochemical processes. For example, chlorin-like compounds are possibly related to diatom production, alkenones are related to coccolithophorid production, and steranes and long-chain fatty acids likely represent terrigenous input. Supervised learning from these data mining results further extracted molecular signatures with proxy potential that appear to be linked to specific environmental conditions inferred from historical oceanographic data. However, generalizability to other sedimentary settings will require further investigation.

Structural insights into a spindle-shaped archaeal virus with a sevenfold symmetrical tail

Archaeal viruses with a spindle-shaped virion are abundant and widespread in extremely diverse environments. However, efforts to obtain the high-resolution structure of a spindle-shaped virus have been unsuccessful. Here, we present the structure of SSV19, a spindle-shaped virus infecting the hyperthermophilic archaeon Sulfolobus sp. E11-6. Our near-atomic structure reveals an unusual sevenfold symmetrical virus tail consisting of the tailspike, nozzle, and adaptor proteins. The spindle-shaped capsid shell is formed by seven left-handed helical strands, constructed of the hydrophobic major capsid protein, emanating from the highly glycosylated tail assembly. Sliding between adjacent strands is responsible for the variation of a virion in size. Ultrathin sections of the SSV19-infected cells show that SSV19 virions adsorb to the host cell membrane through the tail after penetrating the S-layer. The tailspike harbors a putative endo-mannanase domain, which shares structural similarity to a Bacteroides thetaiotaomicro endo-mannanase. Molecules of glycerol dibiphytanyl glycerol tetraether lipid were observed in hydrophobic clefts between the tail and the capsid shell. The nozzle protein resembles the stem and clip domains of the portals of herpesviruses and bacteriophages, implying an evolutionary relationship among the archaeal, bacterial, and eukaryotic viruses.

Discovery, structure and mechanism of a tetraether lipid synthase

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1–5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon–carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon–carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3–Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7–10.

Vesicular and Planar Membranes of Archaea Lipids: Unusual Physical Properties and Biomedical Applications.

Liposomes and planar membranes made of archaea or archaea-like lipids exhibit many unusual physical properties compared to model membranes composed of conventional diester lipids. Here, we review several recent findings in this research area, which include (1) thermosensitive archaeosomes with the capability to drastically change the membrane surface charge, (2) MthK channel’s capability to insert into tightly packed tetraether black lipid membranes and exhibit channel activity with surprisingly high calcium sensitivity, and (3) the intercalation of apolar squalane into the midplane space of diether bilayers to impede proton permeation. We also review the usage of tetraether archaeosomes as nanocarriers of therapeutics and vaccine adjuvants, as well as the biomedical applications of planar archaea lipid membranes. The discussion on archaeosomal therapeutics is focused on partially purified tetraether lipid fractions such as the polar lipid fraction E (PLFE) and glyceryl caldityl tetraether (GCTE), which are the main components of PLFE with the sugar and phosphate removed.

Influence of water conditions on peat brGDGTs: A modern investigation and its paleoclimatic implications

Peat brGDGTs (branched glycerol dialkyl glycerol tetraether lipids) have been proposed as an indicator of past temperature changes. However, recent Chinese peat brGDGTs-based Holocene temperature ranges, of >10 °C, are implausible as they are greater than the corresponding ranges obtained using other proxies. This indicates the possible role of additional factor(s) affecting peat brGDGTs, other than temperature. Here, we report the results of a comparative study of surface peat samples, from dry sites (without water cover when sampling) and water sites (with water cover when sampling), from three peatlands with contrasting environmental backgrounds in China. The results clearly demonstrate the influence of water conditions on the molecular distribution of peat brGDGTs. Specifically, the relevant methylation index (MBT′/MBT′5ME, which is proposed as a temperature indicator) of brGDGTs from the water sites is generally lower than that from the dry sites, which may be caused by the decoupling of water/air temperatures and/or different brGDGTs-producing bacterial communities controlled by the water conditions. Considering the long-term process of hydroseral succession from a water body (an analogue of a water site) to a peat bog (an analogue of a dry site), we estimate that around half of the range of the peat brGDGTs-based Holocene temperature record could result from long-term changes in hydrological conditions. If this effect is removed from the peat core MBT′5ME record, the residual range of the peat brGDGTs-based Holocene temperature record from the Narenxia peatland in the Altai Mountains, in arid inland China, is consistent with that of the quantitative pollen-based Holocene temperature record from the same peatland, highlighting the paleoclimatic significance of our findings.

Influence of bottom seawater oxygen on archaeal tetraether lipids in sediments: Implications for archaeal lipid-based proxies

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are important archaeal membrane lipids that widely occur in marine water columns and sediments. Based on its sensitivity to temperature, the paleotemperature index TEX86 was developed and extensively studied. However, GDGTs in sediments are not only affected by their external sources, but also by internal biochemical processes. The effect of bottom water redox conditions on GDGT composition and TEX86 in the surface sediments remains unclear. In this study, field investigations in the eastern China marginal seas were conducted to trace the responses of sedimentary GDGTs to bottom dissolved oxygen (DO). Our results showed that the TEX86 was independent of the DO level. However, the decrease in DO was accompanied by an increase in GDGT Ring Index (RI) and relative abundance of crenarchaeol (Cren) in contrast to the decrease pattern for GDGT-0, suggesting that DO has important effects on the composition of GDGTs but shows limited impact on TEX86 in the study area. These changes were probably driven by archaeal adaptation, shift of community structure and degradation of GDGTs. In addition, strong relationships between DO and RI and between DO and GDGT abundances (i.e., %GDGT-0, GDGT-0/Cren) indicate that these parameters can be used as potential redox proxies for bottom water, especially in relatively stable stratified waters. Further studies coupled with archaeal community are needed to cover more areas to constrain their applicability and driving mechanisms.

Intact Polar and Core Tetraether Lipids in Sediments from the Haiyang 4 Cold‐seep of the Northern South China Sea and their Implications

AbstractA number of cold seeps have been discovered in the northern South China Sea (SCS) including the Haiyang 4 cold‐seep area where Core 973‐5 was collected. Intact polar lipids (IPLs) and core lipids (CLs) were analyzed separately in sediments from Core 973‐5. The most abundant lipid biomarkers were isoprenoidal GDGTs (isoGDGTs), with Crenarchaeol and GDGT‐0 predominating. IPL‐isoGDGTs and CL‐isoGDGTs were mainly derived from Thaumarchaeota. IPL‐isoGDGTs were mainly produced and retained in situ thus containing most of the in situ microbiological information. Branched GDGTs were predominantly derived from generated in marine production, and mixed with some terrestrial inputs. All IPLs groups presented a high value in the sulfate‐methane transition zone (SMTZ). Furthermore, IPL and CL‐MI, IPL‐R0/4 showed the highest values within the SMTZ, while IPL and CL‐R4/i had the lowest values at the SMTZ, suggesting that the contribution of Methanophila and methanogenic to GDGTs increased, while the contribution of ammonia‐oxidizing Archaea to GDGTs decreased at the SMTZ.

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.

The distribution of intact polar lipid-derived branched tetraethers along a freshwater-seawater pH gradient in coastal East China Sea

In view of the application of microbial branched tetraether lipids (i.e., brGDGTs) in terrestrial pH reconstructions, their potential as seawater pH proxy is investigated from the Yangtze River Estuary (YRE) to the shelf region in the East China Sea (ECS). BrGDGTs occurring as ‘fossil’ core and in ‘living’ intact polar lipids (CL-brGDGTs and IPL-brGDGTs, respectively) in surface sediments are separately analyzed. The results show that riverine IPL-brGDGTs are rapidly lost at the transition from the YRE to the marine settings and sedimentary IPL-brGDGTs are predominantly produced in situ in brackish water environments. Among environmental parameters, surface water pH is a determinant factor controlling the cyclization of IPL-brGDGTs, with higher cyclization at higher pH. Hereby, two IPL-brGDGTs-based seawater pH calibrations are established in the brackish seawater environment in this study: surface pH = 7.28 + 1.08 × #Ringstetra (n = 18, r2 = 0.87, RMSE = 0.05, p < 0.01) and surface pH = 7.13 + 3.82 × f(Ic) + 4.29 × f(IIb') + 4.21 × f(IIIa) (n = 18, r2 = 0.90, RMSE = 0.04, p < 0.01), which are different from those established in terrestrial environments. This is the first work demonstrating that in situ produced microbial brGDGTs can trace seawater pH change in marine environment, which makes it promising to apply brGDGTs for seawater pH reconstructions in marine settings where the terrestrial inputs are negligible.

Self-assembly and phase transition properties of pure archaeal tetraether lipids

Lipid membrane compartments are universal features of life, but different organisms evolved diverse lipids to meet specific physiological and ecological requirements. Glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. GDGT lipids are characterized by multiple branching, ether linkages, cycloalkane rings, and often span the entire length of the membranes. These lipids are the primary constituents of the cell membranes of many Archaea, some of which are extremophilic organisms.

Characteristics of isoprenoid and branched tetraether lipids in the deep ocean: implications from surface sediments in the seamount area of the Western Pacific Ocean

The isoprenoid glycerol dialkyl glycerol tetraethers (isoGDGTs) and branched glycerol dialkyl glycerol tetraethers (brGDGTs) in marine sediments have been widely used for paleoceanographic reconstructions. However, their distributions in the deep ocean (> 4000 m) have not been well characterized. This study analyzed GDGTs in surface sediments from the seamount area of Marcus-Wake and Magellan, western Pacific Ocean (4431-5756 m). The results were further compared with published data of GDGTs in the South China Sea (SCS), equatorial Pacific Ocean, and Mariana Trench to reveal the provenance of GDGTs in the deep ocean and their suitability for paleoceanographic reconstruction. The ratios of GDGT-0/crenarchaeol (0.59–1.38) and GDGT-2/3 (10.65–14.78) indicate the contribution from benthic Thaumarchaeota in the seamount area. The isoGDGTs of the surface sediments from the seamount and the Mariana Trench regions were clustered for a redundancy analysis. They are characterized by the similar relative abundance of each isoGDGT, i.e. higher fractional abundance of GDGT-0 (26–49%) than those in the SCS (12–23%) and the equatorial Pacific Ocean (22–28%). This suggests isoGDGTs in the deep ocean (> 4000 m) are for both planktonic and benthic Thaumarchaeota and the benthic Thaumarchaeota likely produce excessive GDGT-0 in the seamount area. For brGDGTs, the higher IIIa/IIa ratios in the seamount area (1.67–4.83) than the marine-derived values (0.92), indicating in-situ production of brGDGTs. The discrepancy (0.7 to 5.0 °C) between sea surface temperature, deduced by TetraEther indeX (TEX86), and the observed sea surface temperature is likely due to less input of isoGDGTs from the pelagic Thaumarchaeota but more from the benthic Thaumarchaeota.

Stability Monomolecular Properties of Archaeal Tetraether Lipids Layers onto Solid Substrates

This paper reports the recent findings related to the stability properties of tetraether lipid layers. Organizations moleculars of chemical structure modified of Langmuir-Blodgett layers of archael tetraether lipids from the archebacterium Sulfolobus acidocaldarius on the wafer silicon substrates are investigated stable and organized. The behavior of Langmuir-Blodgett layers of chemical structure modified of archaeal tetraether lipids on the wafer silicon substrates is characterization using Differential Scanning Calorimetry (DSC) and Atomic Force Microscopy (AFM). The thermodynamics behavior and stability of Langmuir-Blodgett layers of archael tetraether lipids on the wafer silicon substrates are shown. Stability of the lipid membranes is of great importance to a number of biomedical applications, including intravenous drug delivery, biomaterials, and biosensors.

Metabolic and ecological controls on the stable carbon isotopic composition of archaeal (isoGDGT and BDGT) and bacterial (brGDGT) lipids in wetlands and lignites

The glycerol dialkyl glycerol tetraether (GDGT) lipids of archaea and bacteria have been extensively studied in both modern and ancient wetlands, but their specific biological sources and biogeochemical significance in such settings remain unclear. The stable carbon isotopic composition of GDGTs, and associated tetraether lipids such as butanetriol - DGTs (BDGTs), can help reveal aspects of the carbon metabolism of their source organism. Here we present an in-depth compound-specific analyses of archaeal (isoGDGTs and BDGT-0) and bacterial (brGDGTs) tetraether lipid δ13C values from the Florida Everglades wetland, supplemented by 16S rRNA gene profiling of the archaeal community. We interpret this alongside GDGT δ13C analyses from three additional freshwater wetland sediments, and two ancient wetland deposits (lignites) from the Miocene. Our data suggests that isoGDGT-0 δ13C values, which range from –22.5‰ to −36.5‰ across our wetland sites, could to some degree reflect the relative importance of the acetotrophic and CO2-reducing hydrogenotrophic methanogenic pathways, though definitively delineating between the two is challenging due to (a) potential incorporation of dissolved inorganic carbon (DIC) by CO2 reducers, and (b) the diluting effect of GDGTs derived from seemingly heterotrophic/fermentative Bathyarchaeia, the most abundant archaeal class in the Everglades. In the Everglades, which harbours abundant Methanomassiliicoccales, BDGT-0 is significantly more 13C-depleted than all other lipids, with a δ13C signature at certain depths (∼−40.0 to −45.0‰) that is consistent with an important source from these (likely) methylotrophic methanogens. This adds to a growing body of evidence on the underappreciated role of methylotrophic methanogenesis, largely driven by Methanomassiliicoccales, in the freshwater methane cycle. However, slightly more 13C-enriched values (∼−35.0‰) at certain depths in the same site provide evidence for additional, possibly heterotrophic sources under certain conditions, and BDGT-0 δ13C values in the Miocene lignite (≈ δ13C of TOC) are consistent with a solely heterotrophic source. Where available for measurement, δ13C values of isoGDGTs-1 to -3, averaging –32.7 ± 2.3‰, were significantly more 13C-depleted than isoGDGT-0, suggesting a different mixture of sources, likely including an autotrophic/mixotrophic component derived from archaea belonging to the Crenarchaeota, Nitrososphaeria (formerly Thaumarchaeota) and/or MBG-D/DHVEG-1. Finally, brGDGT δ13C values are generally consistent with their proposed bacterial heterotrophic origin and biosynthetic effects, averaging out at around 4‰ depleted relative to total organic carbon (TOC). Importantly, we find no significant variation between the δ13C values of different brGDGTs across our wetland types. This highlights that spatial and temporal changes in commonly applied brGDGT-based proxies likely do not co-occur with changes in the heterotrophic lifestyle of their producer(s), discounting a possible confounding factor in brGDGT palaeothermometry. Taken together, our results yield insights into wetland archaeal communities and the lipids they produce, and underline the applicability of tetraether δ13C analyses in probing aspects of microbial carbon cycling in modern and ancient wetland sediments.

Distributions and sources of isoprenoidal GDGTs in Lake Lugano and other central European (peri-)alpine lakes: Lessons for their use as paleotemperature proxies

Isoprenoidal glycerol dialkyl glycerol tetraether (isoGDGT) lipids occur ubiquitously in freshwater and marine environments. Since their distribution varies with temperature, sedimentary isoGDGTs have been used as proxies for the reconstruction of past continental climate for almost two decades. Yet, their application in lacustrine sediments is still not well constrained because the niches of isoGDGT-producing microorganisms in lakes are often ill-defined. Here, we study the distribution of isoGDGTs and their hydroxy derivatives (OH-isoGDGTs) in the water column of the deep (288 m), meromictic northern basin of Lake Lugano (Switzerland) using quantitative analysis of various pools of isoGDGTs and the stable carbon isotopic composition of isoGDGT-derived biphytanes. We provide strong evidence for archaeal water column sources of the isoGDGTs, based on comparison of lipid data with microbial diversity determined by 16S rRNA next generation sequencing. We find highest concentrations (i.e., 40 ng L−1) of crenarchaeol, the isoGDGTs specific for thaumarchaea, in suspended particle matter (SPM) from deeper (30–100 m) waters below the thermocline. This correlates well with thaumarchaeal 16S rRNA gene abundances, comprised by a single thaumarchaeote of the order Nitrosopumilales. The concentrations of OH-isoGDGT with 0–2 cyclopentane rings follow this profile, suggesting an identical archaeal source. In the deeper anoxic waters, the archaeal community changes substantially and was comprised of various members of the Bathyarchaeota, Diapherotrites, Euryarchaeota, and Woesearchaeota. This change is accompanied by a changing distribution of isoGDGTs with a high contribution of GDGT-0 with a polar head group, and a more negative δ13C value of the acyclic biphytane derived thereof. Comparison of the isoGDGT composition (distribution and δ13C) in the surface sediment with that of the sinking particle flux studied over 1 year at three depths indicates substantial downward transport of isoGDGTs to the sediments from the waters between the thermocline and the anoxic hypolimnion, but not from the deeper (>100 m) waters. The relatively low value for the isoGDGT-based TEX86 value (ca. 0.40) in the surface sediment is similar to that of the in-situ produced isoGDGTs in the waters below the thermocline, and is consistent with the year-around low water temperatures (<6 °C). We also analyzed the isoGDGT composition in surface sediments of 36 additional (peri-)alpine lakes in Central Europe; their isoGDGT concentration and distribution show a clear-cut difference based on lake size. In relatively large and deep lakes, like the Lake Lugano North Basin, the crenarchaeol concentration was much higher than in small-sized and shallow lakes, because Nitrosopumilales spp. have seemingly no niche in shallower waters (0–∼30 m depth), and production, and hence preservation in the sedimentary record, of crenarchaeol (and other thaumarchaeal isoGDGTs) is much lower than in large lakes. We argue that in the mid-latitudinal large and deep (peri-)alpine lakes Nitrosopumilales spp. generally thrive in cold waters below the thermocline, where temperature is largely not affected by fluctuations in mean annual air temperature. Therefore, the TEX86 paleothermometer is relative insensitive to atmospheric temperature changes in these lakes. In the small-sized and shallow lakes and ponds crenarchaeol can still be present but is probably mainly derived from Nitrososphaerales spp. (either from in-situ production or soil erosion). These thaumarchaea are characterized by a higher relative content of the crenarchaeol isomer than the Nitrosopumilales spp. at the same temperature, which may compromise accurate paleotemperature estimations using the TEX86 for small/shallow lakes.

Evidence for Enzymatic Backbone Methylation of the Main Membrane Lipids in the Archaeon Methanomassiliicoccus luminyensis.

Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers (BDGTs and PDGTs, respectively) are recently identified classes of archaeal membrane lipids that are prominent constituents in anoxic subseafloor sediments. These lipids are intriguing, as they possess unusual backbones with four or five carbon atoms instead of the canonical three-carbon glycerol backbone. In this study, we examined the biosynthesis of BDGTs and PDGTs by the methanogen Methanomassiliicoccus luminyensis, the only available isolate known to produce these compounds, via stable isotope labeling with [methyl-13C]methionine followed by mass spectrometry analysis. We show that their biosynthesis proceeds from transfer(s) of the terminal methyl group of methionine to the more common archaeal membrane lipids, i.e., glycerol dibiphytanyl glycerol tetraethers (GDGTs). As this methylation targets a methylene group, a radical mechanism involving a radical S-adenosylmethionine (SAM) enzyme is probable. Over the course of the incubation, the abundance of PDGTs relative to BDGTs, expressed as backbone methylation index, increased, implying that backbone methylation may be related to the growth shift to stationary conditions, possibly due to limited energy and/or substrate availability. The increase of the backbone methylation index with increasing sediment age in a sample set from the Mediterranean Sea adds support for such a relationship. IMPORTANCE Butanetriol and pentanetriol dibiphytanyl glycerol tetraethers are membrane lipids recently discovered in anoxic environments. These lipids differ from typical membrane-spanning tetraether lipids because they possess a non-glycerol backbone. The biosynthetic pathway and physiological role of these unique lipids are currently unknown. Here, we show that in the strain Methanomassiliicoccus luminyensis, these lipids are the result of methyl transfer(s) from an S-adenosyl methionine (SAM) intermediate. We observed a relative increase of the doubly methylated compound, pentanetriol dibiphytanyl glycerol tetraether, in the stationary phase of M. luminyensis as well as in the subseafloor of the Mediterranean Sea and thus introduced a backbone methylation index, which could be used to further explore microbial activity in natural settings.

Archaeal tetraether lipids and their biphytane carbon isotope composition in sediments along an estuarine biogeochemical gradient

The stable carbon isotopic composition of Thaumarchaeota-produced crenarchaeol (δ13Ccren) is thought to reflect the δ13C of dissolved inorganic carbon (DIC) and is valuable in carbon cycle research. Recently, an isotope flux-balance model was proposed that δ13Ccren is dependent on passive CO2 uptake and a slow rate of intracellular conversion to HCO3−, which has been validated in open ocean environments. However, further studies are required in environments with larger ranges of aqueous CO2 and diverse archaeal communities to examine whether the δ13Ccren value could still be predicted by the model. Here, we report δ13C values of biphytanes chemically released from the archaeal tetraether lipids (i.e., GDGTs) in sediments along a transect exhibiting shifts in the archaeal community and showing gradients of salinity, pH, and CO2 in the Pearl River Estuary, South China. Methanogenic and methanotrophic Euryarchaeota are present in the upper estuary, although not significant, as revealed by the elevated relative abundances of GDGT-0 and higher values of methane index in suspended particulate matter (SPM) and surface sediments, as well as the more 13C-depleted C40:0, C40:1, and C40:2 biphytanes than the C40:3 biphytane released from sedimentary GDGTs. Toward the lower estuary and coastal sea, GDGT distributions in SPM and sediments tend to indicate archaeal communities under normal marine conditions. However, there are still differences in δ13C values between C40:0 and C40:3 biphytanes by up to 2.4‰ in sediments, although C40:1, C40:2, and C40:3 were similar in δ13C, likely due to the existence of marine group II Euryarchaeota. Along such a physicochemical and biogeochemical gradient, the values of sedimentary δ13C40:3 increase monotonically with the increase of mean annual pH and the decrease of mean annual CO2. This phenomenon resembles the reported δ13CGDGT profiles, along with the variations in pH and CO2, in water column in the open ocean, and hence consistent in direction with the isotope flux-balance model for Thaumarchaeota lipids. However, our estimated apparent biosynthetic isotope effect (εAr) of crenarchaeol, ranging from 15.2‰ to 19.7‰, is mostly lower than those estimated in the open ocean. With the constraints of thaumarchaeal growth rates in such a dynamic estuarine environment as well as those in the open ocean, we suggest slight reductions in catalytic efficiency and intracellular kinetic isotope effect of intracellular CO2 hydration relative to previously employed values. Such modifications would slightly change the sensitivities of εAr to archaeal growth rate and ambient CO2 concentration but might be more suitable for variable hydrology and community environments.

The Polar Lipid Fraction E from Sulfolobus acidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents to Reduce the Leakage of the Antivascular Drug Combretastatin A4 Disodium Phosphate from Tetraether/Diester Hybrid Archaeosomes

Liposomes have many advantages as therapeutic capsules over free drugs such as small molecule drugs and nucleic acids. Cholesterol is commonly used as a membrane stabilizing agent in liposomal drugs (e.g., mRNA-lipid nanoparticle COVID-19 vaccines). However, due to the vulnerability of cholesterol to oxidation and the etiological role of cholesterol in many disorders, it is desirable to find an alternative means to stabilize liposomal membranes for drug delivery. In this study, we demonstrated that the polar lipid fraction E (PLFE), which contains exclusively bipolar tetraether macrocyclic lipids, isolated from the thermoacidophilic archaeon S. acidocaldarius can greatly stabilize the liposomal formulation of the anti-vascular drug, combretastatin A4 disodium phosphate (CA4P). Stability was assessed by determining the leakage rate constant k of entrapped CA4P fluorometrically. We found that, at 37 °C, PLFE decreases the k value monotonically from 1.54 × 10−2 s−1 for 100% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes to 3.4 × 10−5 s−1 for 100% PLFE archaeosomes, a change of k by two orders of magnitude. The changes in k of CA4P leakage are correlated well with the changes in liposomal CA4P’s cytotoxicity against MCF-7 breast cancer cells. We further showed that the reduction in spontaneous leakage of entrapped CA4P by PLFE can be attributed to the increased membrane surface charge and the increased membrane order and packing tightness in liposomes, as reflected by the zeta potential (−6.83 to −41.1 mV from 0 to 100 mol% PLFE) and diphenylhexatriene (DPH) fluorescence polarization (0.13 to 0.4 from 0 to 100 mol% PLFE) measurements. Moreover, we showed that PLFE slows down CA4P leakage more than cholesterol in POPC liposomes. These results together suggest that PLFE lipids can serve as an effective stabilizing agent for liposomal drugs and could potentially be useful for the optimization of liposomal CA4P for cancer treatment.

Soil pH and aridity influence distributions of branched tetraether lipids in grassland soils along an aridity transect

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are membrane lipids of certain soil bacteria, and their relative distributions are used as a proxy for air temperature and soil pH. While temperature is recorded by the degree of methylation, soil pH is reflected by the amount of internal cyclization and the relative abundance of 6-methyl isomers. Since the exact producers of brGDGTs remain enigmatic, the mechanisms underlying their empirical relationships with temperature and soil pH, and thus the reliability of brGDGT-based paleorecords, are not well understood, especially in arid regions where mean annual precipitation (MAP) is less than 500 mm. Here, we evaluate the influence of soil pH and aridity on brGDGT distributions in grassland soils along an aridity transect (MAP = 173–415 mm) in Inner Mongolia. While the absolute and fractional abundance of 6-methyl brGDGTs increases with increasing soil pH and aridity, following the trend in the global surface soil calibration dataset, the degree of cyclization does not. This indicates that in arid regions, soil pH reconstructions based on the relative contribution of 6-methyl brGDGTs are likely more reliable than those based on the degree of cyclization. Furthermore, 5- and 6-methyl brGDGTs respond differently to aridity, supporting prior suggestions that the distribution of brGDGTs could be the result of changes in bacterial community composition instead of the direct physiological alteration of molecular structures by the source organisms. Analysis of the bacterial community composition in the same soil transect indicates that the relative abundance of Acidobacteria, the phylum hosting potential brGDGT source-organisms, shows a poor relationship with aridity. Instead, Verrucomicrobia (r2 = 0.70, p < 0.01), and its subclass Spartobacteria (r2 = 0.70, p < 0.01) in particular, show a significant negative correlation with aridity, resembling that of 5-methyl brGDGTs. Similarly, Actinobacteria are positively correlated with aridity (r2 = 0.59, p < 0.01), following the same trend as that of 6-methyl brGDGTs. The ability of certain cultures of Verrucomicrobia and Actinobacteria to produce iso-C15:0 fatty acids that could serve as building blocks for brGDGTs hints that Verrucomicrobia and Actinobacteria could possibly produce brGDGTs in arid soils.

Certain, but Not All, Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE–PLFE and PLFE–substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Planktonic Archaeal Ether Lipid Origins in Surface Waters of the North Pacific Subtropical Gyre.

Thaumarchaeota and Thermoplasmatota are the most abundant planktonic archaea in the sea. Thaumarchaeota contain tetraether lipids as their major membrane lipids, but the lipid composition of uncultured planktonic Thermoplasmatota representatives remains unknown. To address this knowledge gap, we quantified archaeal cells and ether lipids in open ocean depth profiles (0–200 m) of the North Pacific Subtropical Gyre. Planktonic archaeal community structure and ether lipid composition in the water column partitioned into two separate clusters: one above the deep chlorophyll maximum, the other within and below it. In surface waters, Thermoplasmatota densities ranged from 2.11 × 106 to 6.02 × 106 cells/L, while Thaumarchaeota were undetectable. As previously reported for Thaumarchaeota, potential homologs of archaeal tetraether ring synthases were present in planktonic Thermoplasmatota metagenomes. Despite the absence of Thaumarchaeota in surface waters, measurable amounts of intact polar ether lipids were found there. Based on cell abundance estimates, these surface water archaeal ether lipids contributed only 1.21 × 10–9 ng lipid/Thermoplasmatota cell, about three orders of magnitude less than that reported for Thaumarchaeota cells. While these data indicate that even if some tetraether and diether lipids may be derived from Thermoplasmatota, they would only comprise a small fraction of Thermoplasmatota total biomass. Therefore, while both MGI Thaumarchaeota and MGII/III Thermoplasmatota are potential biological sources of archaeal GDGTs, the Thaumarchaeota appear to be the major contributors of archaeal tetraether lipids in planktonic marine habitats. These results extend and confirm previous reports of planktonic archaeal lipid sources, and further emphasize the need for Thermoplasmatota cultivation, to better characterize the membrane lipid constituents of marine planktonic Thermoplasmatota, and more precisely define the sources and patterns of archaeal tetraether lipid distributions in marine plankton.