Diether and tetraether lipids are fundamental components of the archaeal cell membrane. Archaea adjust the degree of tetraether lipid cyclization in order to maintain functional membranes and cellular homeostasis when confronted with pH and/or thermal stress. Thus, the ability to adjust tetraether lipid composition likely represents a critical phenotypic trait that enabled archaeal diversification into environments characterized by extremes in pH and/or temperature. Here we assess the relationship between geochemical variation, core- and polar-isoprenoid glycerol dibiphytanyl glycerol tetraether (C-iGDGT and P-iGDGT, respectively) lipid composition, and archaeal 16S rRNA gene diversity and abundance in 27 geothermal springs in Yellowstone National Park, Wyoming. The composition and abundance of C-iGDGT and P-iGDGT lipids recovered from geothermal ecosystems were distinct from surrounding soils, indicating that they are synthesized endogenously. With the exception of GDGT-0 (no cyclopentyl rings), the abundances of individual C-iGDGT and P-iGDGT lipids were significantly correlated. The abundance of a number of individual tetraether lipids varied positively with the relative abundance of individual 16S rRNA gene sequences, most notably crenarchaeol in both the core and polar GDGT fraction and sequences closely affiliated with Candidatus Nitrosocaldus yellowstonii. This finding supports the proposal that crenarchaeol is a biomarker for nitrifying archaea. Variation in the degree of cyclization of C- and P-iGDGT lipids recovered from geothermal mats and sediments could best be explained by variation in spring pH, with lipids from acidic environments tending to have, on average, more internal cyclic rings than those from higher pH ecosystems. Likewise, variation in the phylogenetic composition of archaeal 16S rRNA genes could best be explained by spring pH. In turn, the phylogenetic similarity of archaeal 16S rRNA genes was significantly correlated with the similarity in the composition of C- and P-iGDGT lipids. Taken together, these data suggest that the ability to adjust the composition of GDGT lipid membranes played a central role in the diversification of archaea into or out of environments characterized by extremes of low pH and high temperature.
tetraether lipids; Nitrosocaldus; amoA; nitrification; crenarchaeol; community ecology; phylogenetic ecology
A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria.
archaea; ether lipids; isoprenoids; biosynthesis; lipid divide
The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition.
All cells are surrounded by a lipid membrane that protects the cell, controls the movement of molecules into and out of the cell, and passes messages about environmental conditions to the cell. This membrane is made of two layers of molecules called lipids, with various proteins embedded in it. There are many different types of lipid molecules that together help to keep the membrane flexible. Moreover, lipid molecules of particular types can also come together to form ‘rafts’ that help the membrane to carry out its various roles.
Given the complexity of the cell membrane, cell biologists often use simpler model membranes and computer simulations to explore how the different types of lipid molecules are organized within the membrane. According to the ‘picket fence’ model the cell membrane is divided into small compartments as a result of its interaction with the dense network of actin fibers that acts as a skeleton inside the cell.
Recent computer simulations have predicted that these interactions can influence the distribution of lipids and proteins within the membrane. In particular, they can prevent the drastic re-arrangement of lipids into regions of high and low viscosity at low temperature. This temperature dependent re-arrangement of the membrane is known as lipid phase separation.
Honigmann et al. have now used computer simulations and two advanced techniques—super-resolution optical STED microscopy and fluorescence correlation spectroscopy—to explore the properties of a model membrane in the presence of a dense network of actin fibers in fine detail. The results show that, in agreement with the simulation predictions of the ‘picket fence’ model, the actin fibers bound to the membrane prevent lipid phase separation happening at low temperatures. Moreover, the actin fibers also help to organize the distribution of lipids and proteins within the membrane at physiological temperatures. Honigmann et al. also suggest that the actin fibers cause the membrane to curve in a way that can reinforce the influence of the ‘picket fence’.
The results show that the ‘raft’ and ‘picket fence’ models are connected, and that a cell can control the properties of its membrane by controlling the interactions between the membrane and the actin fibers that make up the skeleton of the cell.
membrane organization; cortical actin; lipid phase separation; STED microscopy; fluorescence correlation spectroscopy; pinning sites; None
Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme.
Isoprenoidal glycerol dialkyl glycerol tetraethers (iGDGTs) are core membrane lipids of many archaea that enhance the integrity of cytoplasmic membranes in extreme environments. We examined the iGDGT profiles and corresponding aqueous geochemistry in 40 hot spring sediment and microbial mat samples from the U.S. Great Basin with temperatures ranging from 31 to 95°C and pH ranging from 6.8 to 10.7. The absolute abundance of iGDGTs correlated negatively with pH and positively with temperature. High lipid concentrations, distinct lipid profiles, and a strong relationship between polar and core lipids in hot spring samples suggested in situ production of most iGDGTs rather than contamination from local soils. Two-way cluster analysis and non-metric multidimensional scaling (NMS) of polar iGDGTs indicated that the relative abundance of individual lipids was most strongly related to temperature (r2 = 0.546), with moderate correlations with pH (r2 = 0.359), nitrite (r2 = 0.286), oxygen (r2 = 0.259), and nitrate (r2 = 0.215). Relative abundance profiles of individual polar iGDGTs indicated potential temperature optima for iGDGT-0 (≤70°C), iGDGT-3 (≥55°C), and iGDGT-4 (≥60°C). These relationships likely reflect both physiological adaptations and community-level population shifts in response to temperature differences, such as a shift from cooler samples with more abundant methanogens to higher-temperature samples with more abundant Crenarchaeota. Crenarchaeol was widely distributed across the temperature gradient, which is consistent with other reports of abundant crenarchaeol in Great Basin hot springs and suggests a wide distribution for thermophilic ammonia-oxidizing archaea (AOA).
archaea; iGDGTs; hot springs; Great Basin; lipids
The temperature limits of growth of a number of yeast species were examined, and on this basis the organisms were classified into different thermal categories. The following species were examined: Leucosporidium frigidum and Leucosporidium nivalis, psychrophilic, temperature limits of growth, -2 to 20 degrees C; Canadian lipolytica mesophilic, temperature limits of growth, 5 to 35 degrees Candida parapsilosis and Saccharomyces telluris, thermotolerant, temperature limits of growth, 8 to 42 degrees C; Torulopsis bovina and Candida slooffi, thermophilic, temperature limits of growth, 25 to 45 degrees C and 28 to 45 degrees C, respectively. The membrane lipid and cytochrome composition of mitochrondrial fractions isolated from these yeasts were compared. There was a direct correlation between the growth temperature and the degree of membrane of lipid unsaturation; the lower the temperature, the greater the degree of lipid unsaturation. The membrane lipid composition of the thermophilic yeasts were distinguished by the high percentage (30 to 40%) of saturated fatty acid, as compared with the mesophilic and psychrophilic yeasts. The latter contained approximately 90% unsaturated fatty acid, 55% of which was linolenic acid, C alpha-18:3. Changes in phospholipid composition in relation to temperature were also noted. The respiratory-deficient thermophile, C. slooffi, was characterized by the absence of cardiolipin (sensitivity 0.1 mug of phosphorus) and cytochrome aa3. The absence of conventional mitochondrial structures in this thermophilic microorganism is tentatively suggested although low concentrations of cytochromes b, c, and c1 were detected by low-temperature spectroscopy. On the other hand, the respiratory-competent thermophile, T. bovina, was characterized by a high cardiolipin (25% of the total phospholipid) and cytochrome aa3 content (1 nmol/mg of mitochrondrial protein). Low-temperature spectra showed the presence of one b-type cytochrome in the thermophilic yeasts, two b-type cytochromes in the mesophilic yeasts, and three b-type cytochromes in the psychrophilic yeasts. It was concluded that a knowledge of the properties of the biological membrane is fundamental to an understanding of the ability of a microorganism to grow and reproduce in different temperature environments.
Novel cardiolipins from Archaea were detected by screening the intact polar lipid (IPL) composition of microbial communities associated with methane seepage in deep-sea sediments from the Pakistan margin by high-performance liquid chromatography electrospray ionization mass spectrometry. A series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol, BPG) represented 0.5% to 5% of total archaeal IPLs. These molecules are similar to the recently described cardiolipin analogues with four phytanyl chains from extreme halophilic archaea. It is worth noting that cardiolipin analogues from the seep archaeal communities are composed of four isoprenoidal chains, which may contain differences in chain length (20 and 25 carbon atoms) and degrees of unsaturation and the presence of a hydroxyl group. Two novel diether lipids, structurally related to the BPGs, are described and interpreted as degradation products of archaeal cardiolipin analogues. Since archaeal communities in seep sediments are dominated by anaerobic methanotrophs, our observations have implications for characterizing structural components of archaeal membranes, in which BPGs are presumed to contribute to modulation of cell permeability properties. Whether BPGs facilitate interspecies interaction in syntrophic methanotrophic consortia remains to be tested.
Archaeal plasma membranes appear to be extremely durable and almost impermeable to water and ions, in contrast to the membranes of Bacteria and Eucaryota. Additionally, they remain liquid within a temperature range of 0–100°C. These are the properties that have most likely determined the evolutionary fate of Archaea, and it may be possible for bionanotechnology to adopt these from nature. In this work, we use molecular dynamics simulations to assess at the atomistic level the structure and dynamics of a series of model archaeal membranes with lipids that have tetraether chemical nature and “branched” hydrophobic tails. We conclude that the branched structure defines dense packing and low water permeability of archaeal-like membranes, while at the same time ensuring a liquid-crystalline state, which is vital for living cells. This makes tetraether lipid systems promising in bionanotechnology and material science, namely for design of new and unique membrane nanosystems.
Branched glycerol dialkyl glycerol tetraethers (bGDGTs) are membrane-spanning lipids that likely stabilize membranes of some bacteria. Although bGDGTs have been reported previously in certain geothermal environments, it has been suggested that they may derive from surrounding soils since bGDGTs are known to be produced by soil bacteria. To test the hypothesis that bGDGTs can be produced by thermophiles in geothermal environments, we examined the distribution and abundance of bGDGTs, along with extensive geochemical data, in 40 sediment and mat samples collected from geothermal systems in the U.S. Great Basin (temperature: 31–95°C; pH: 6.8–10.7). bGDGTs were found in 38 out of 40 samples at concentrations up to 824 ng/g sample dry mass and comprised up to 99.5% of total GDGTs (branched plus isoprenoidal). The wide distribution of bGDGTs in hot springs, strong correlation between core and polar lipid abundances, distinctness of bGDGT profiles compared to nearby soils, and higher concentration of bGDGTs in hot springs compared to nearby soils provided evidence of in situ production, particularly for the minimally methylated bGDGTs I, Ib, and Ic. Polar bGDGTs were found almost exclusively in samples ≤70°C and the absolute abundance of polar bGDGTs correlated negatively with properties of chemically reduced, high temperature spring sources (temperature, H2S/HS−) and positively with properties of oxygenated, low temperature sites (O2, NO−3). Two-way cluster analysis and nonmetric multidimensional scaling based on relative abundance of polar bGDGTs supported these relationships and showed a negative relationship between the degree of methylation and temperature, suggesting a higher abundance for minimally methylated bGDGTs at high temperature. This study presents evidence of the widespread production of bGDGTs in mats and sediments of natural geothermal springs in the U.S. Great Basin, especially in oxygenated, low-temperature sites (≤70°C).
geothermal springs; membrane-spanning lipids; bGDGTs; thermophiles; Great Basin; lipids
Thermophilic organisms are able to live at high temperatures ranging from 50 to > 100°C. Their proteins must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive. Subtle differences between thermophilic and mesophilic molecules can be found when sequences or structures from homologous proteins are compared, but often these differences are family-specific and few general rules have been derived. The availability of complete genome sequences has now made it feasible to perform a large-scale comparison between mesophilic and thermophilic proteins, the latter of which primarily come from archaeal genomes although a few complete genomes of thermophilic eubacteria are also available.
We compared mesophilic proteins with their thermophilic counterparts of archaeal or eubacterial origins independently. This was based on the assumption that in these two kingdoms, different mechanisms may have been exploited for the adaptation of proteins at high temperatures. We derived the environment specific amino acid compositions of thermophilic proteins from 10 archaeal and seven eubacterial genomes, by aligning a large number of sequences from thermophilic proteins with their close mesophilic homologues of known three-dimensional (3D) structure. We further analysed environment specific substitutions, which lead from mesophilic proteins to either archaeal or eubacterial thermophilic proteins.
Our comparisons were based on homology-based structural predictions for a large number of thermophilic proteins. We demonstrated that thermal adaptation in the archaeal and eubacterial kingdoms is achieved in different ways. The main differences concern the usage of Gln, Ile and positively charged amino acids. In particular archaeal organisms appeared to have acquired thermostability by substituting non-charged polar amino acids (such as Gln) with Glu and Lys, and non-polar amino acids with Ile on the surface of proteins.
Since the reclassification of all life forms in three Domains (Archaea, Bacteria, Eukarya), the identity of their alleged forerunner (Last Universal Common Ancestor or LUCA) has been the subject of extensive controversies: progenote or already complex organism, prokaryote or protoeukaryote, thermophile or mesophile, product of a protracted progression from simple replicators to complex cells or born in the cradle of "catalytically closed" entities? We present a critical survey of the topic and suggest a scenario.
LUCA does not appear to have been a simple, primitive, hyperthermophilic prokaryote but rather a complex community of protoeukaryotes with a RNA genome, adapted to a broad range of moderate temperatures, genetically redundant, morphologically and metabolically diverse. LUCA's genetic redundancy predicts loss of paralogous gene copies in divergent lineages to be a significant source of phylogenetic anomalies, i.e. instances where a protein tree departs from the SSU-rRNA genealogy; consequently, horizontal gene transfer may not have the rampant character assumed by many. Examining membrane lipids suggest LUCA had sn1,2 ester fatty acid lipids from which Archaea emerged from the outset as thermophilic by "thermoreduction," with a new type of membrane, composed of sn2,3 ether isoprenoid lipids; this occurred without major enzymatic reconversion. Bacteria emerged by reductive evolution from LUCA and some lineages further acquired extreme thermophily by convergent evolution. This scenario is compatible with the hypothesis that the RNA to DNA transition resulted from different viral invasions as proposed by Forterre. Beyond the controversy opposing "replication first" to metabolism first", the predictive arguments of theories on "catalytic closure" or "compositional heredity" heavily weigh in favour of LUCA's ancestors having emerged as complex, self-replicating entities from which a genetic code arose under natural selection.
Life was born complex and the LUCA displayed that heritage. It had the "body "of a mesophilic eukaryote well before maturing by endosymbiosis into an organism adapted to an atmosphere rich in oxygen. Abundant indications suggest reductive evolution of this complex and heterogeneous entity towards the "prokaryotic" Domains Archaea and Bacteria. The word "prokaryote" should be abandoned because epistemologically unsound.
This article was reviewed by Anthony Poole, Patrick Forterre, and Nicolas Galtier.
Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using 2H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ9-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2′ and 16′ positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer.
Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-2H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime (2H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type 2H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.
Cannabinoid receptor agonist; Win55212-2; CP55940; THC; Lipid membrane phase transition; Drug membrane interaction; Lipid domain; Solid state NMR
Complete structures of nearly 40 ether polar lipids from seven species of methanogens have been elucidated during the past 10 years. Three kinds of variations of core lipids, macrocyclic archaeol and two hydroxyarchaeols, were identified, in addition to the usual archaeol and caldarchaeol (for the nomenclature of archaeal [archaebacterial] ether lipids, see the text). Polar head groups of methanogen phospholipids include ethanolamine, serine, inositol, N-acetylglucosamine, dimethyl- and trimethylaminopentanetetrol, and glucosaminylinositol. Glucose is the sole hexose moiety of glycolipids in most methanogens, and galactose and mannose have been found in a few species. Methanogen lipids are characterized by their diversity in phosphate-containing polar head groups and core lipids, which in turn can be used for chemotaxonomy of methanogens. This was shown by preliminary simplified analyses of lipid component residues. Core lipid analysis by high-pressure liquid chromatography provides a method of determining the methanogenic biomass in natural samples. There has been significant progress in the biosynthetic studies of methanogen lipids in recent years. In vivo incorporation experiments have led to delineation of the outline of the synthetic route of the diphytanylglycerol ether core. The mechanisms of biosynthesis of tetraether lipids and various polar lipids, and cell-free systems of either lipid synthesis, however, remain to be elucidated. The significance and the origin of archaeal ether lipids is discussed in terms of the lipid composition of bacteria living in a wide variety of environments, the oxygen requirement for biosynthesis of hydrocarbon chains, and the physicochemical properties and functions of lipids as membrane constituents.
Lipid-water interaction plays an important role in the properties of lipid bilayers, cryoprotectants, and membrane-associated peptides and proteins. The temperature at which water bound to lipid bilayers freezes is lower than that of free water. Here, we report a solid-state NMR investigation on the freezing point depression of water in phospholipid bilayers in the presence and absence of cholesterol. Deuterium NMR spectra at different temperatures ranging from -75 to +10°C were obtained from fully 2H2O-hydrated POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) multilamellar vesicles (MLVs), prepared with and without cholesterol, to determine the freezing temperature of water and the effect of cholesterol on the freezing temperature of water in POPC bilayers. Our 2H NMR experiments reveal the motional behavior of unfrozen water molecules in POPC bilayers even at temperatures significantly below 0°C and show that the presence of cholesterol further lowered the freezing temperature of water in POPC bilayers. These results suggest that in the presence of cholesterol the fluidity and dynamics of lipid bilayers can be retained even at very low temperatures as exist in the liquid crystalline phase of the lipid. Therefore, bilayer samples prepared with a cryoprotectant like cholesterol should enable the performance of multidimensional solid-state NMR experiments to investigate the structure, dynamics, and topology of membrane proteins at a very low temperature with enhanced sample stability and possibly a better sensitivity. Phosphorus-31 NMR data suggest that lipid bilayers can be aligned at low temperatures, while 15N NMR experiments demonstrate that such aligned samples can be used to enhance the signal-to-noise ratio of 15N chemical shift spectra of a 37-residue human antimicrobial peptide, LL-37.
Reverse gyrase, an enzyme of uncertain funtion, is present in all
hyperthermophilic archaea and bacteria. Previous phylogenetic studies
have suggested that the gene for reverse gyrase has an archaeal origin
and was transferred laterally (LGT) to the ancestors of the two
bacterial hyperthermophilic phyla, Thermotogales and Aquificales.
Here, we performed an in-depth analysis of the evolutionary history of
reverse gyrase in light of genomic progress. We found genes coding for
reverse gyrase in the genomes of several thermophilic bacteria that
belong to phyla other than Aquificales and Thermotogales. Several of
these bacteria are not, strictly speaking, hyperthermophiles because
their reported optimal growth temperatures are below 80 °C.
Furthermore, we detected a reverse gyrase gene in the sequence of the
large plasmid of Thermus thermophilus strain HB8,
suggesting a possible mechanism of transfer to the T.
thermophilus strain HB8 involving plasmids and transposases.
The archaeal part of the reverse gyrase tree is congruent with recent
phylogenies of the archaeal domain based on ribosomal proteins or RNA
polymerase subunits. Although poorly resolved, the complete reverse
gyrase phylogeny suggests an ancient acquisition of the gene by
bacteria via one or two LGT events, followed by its secondary
distribution by LGT within bacteria. Finally, several genes of
archaeal origin located in proximity to the reverse gyrase gene in
bacterial genomes have bacterial homologues mostly in thermophiles or
hyperthermophiles, raising the possibility that they were
co-transferred with the reverse gyrase gene. Our new analysis of the
reverse gyrase history strengthens the hypothesis that the acquisition
of reverse gyrase may have been a crucial evolutionary step in the
adaptation of bacteria to high-temperature environments. However, it
also questions the role of this enzyme in thermophilic bacteria and
the selective advantage its presence could provide.
adaptation; Archaea; evolution; genome context; HGT; hyperthermophily; plasmid; thermophily; Thermus thermophilus
Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75–88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and “Bison Pool,” using various 13C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest 13C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. 13C-glucose showed a similar, but a 10–30 times lower uptake across most fatty acids. 13C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at “Bison Pool” and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of 13C-formate occurred only at very low rates at “Bison Pool” and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. 13C-uptake into archaeal lipids occurred predominantly with 13C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth.
hot springs; streamer biofilm communities; stable isotope probing; Archaea; heterotrophy; Aquificae; Yellowstone National Park
Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.
Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. 31P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is ~50 K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid 13C’-31P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular 13C-31P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced 31P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.
trehalose; lipid bilayers; 13C-31P distances; membrane peptide structure; solid-state NMR
Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase (GGR) family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with FAD and a bacterial lipid. The DGGR structure can be assigned to the well-studied, para-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two narrow curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the structure and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of GGRs. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.
The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.
The cell membrane is composed of a wide variety of lipids and proteins. Until recently, these were thought to be mixed evenly, but we now have evidence of the existence of “lipid rafts” — small, slow-moving areas of membrane in which certain types of lipid and protein accumulate. Rafts have many important biological functions in healthy cells, but also play a role in the assembly of influenza virus. For example, after the viral protein hemagglutinin is made inside the host cell, it accumulates in rafts. Exiting virus particles then take these portions of cell membrane with them as they leave the host cell. However, the mechanism by which proteins associate with lipid rafts is unclear. Here, we have used computers to simulate lipid membranes containing hemagglutinin. The simulations allow us to look in detail at the motions and interactions of individual proteins and lipids. We found that clusters of proteins altered the properties of nearby lipids, leading to accumulation of raft-type lipids. It therefore appears that aggregation of hemagglutinin may be enough to drive its association with rafts. This helps us to better understand both the influenza assembly process and the properties of lipid rafts.
The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.
cholesterol; membrane domains; fluidity; hydrophobic barrier; oxygen permeation; spin labeling
Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.
Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (a.k.a., bolalipids) have been prepared to determine 1) the influence of molecular structure on the physical properties of bolalipid membranes and 2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. The three bolalipids synthesized were: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 °C; C32phytBAS Tm = 14 °C; C20BAS Tm = 17°C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 20 °C, were 1.5 × 10−8 and 1.8 × 10−9 cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS:E. coli polar lipid, C20BAS:POPC, C32phytBAS:E. coli polar lipid, and C32phytBAS:POPC. Ste14p activity was lost in vesicles composed of 75–100 mol% C20BAS and 0–100 mol% C32BAS, but retained in vesicles with 0–50 mol% C20BAS and 0–100 mol% C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol% C20BAS and 100 mol% C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 Å. Since Ste14p activity was not affected by either the gel to liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75–100 mol% C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.
Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from Gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.
Lipopolysaccharide (LPS) is the major cell surface molecule of Gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three components: the distal O-polysaccharide, a core oligosaccharide, and the lipid A hydrophobic anchor, where lipid A is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from Gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In additional to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of Gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response and have provided many new targets in the development of antibacterial compounds.
lipid A; Bligh-Dyer; thin layer chromatography (TLC); lipopolysaccharide; mass spectrometry; Collision Induced Dissociation (CID); Photodissociation (PD)
Given the complexity of cell membranes, there is a need for an analytical technique which can explore the physical properties of lipid membranes in a high-throughput and noninvasive manner. A simplified sum-frequency vibrational imaging (SFVI) setup has been developed and characterized using asymmetrically prepared 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):1,2-distearoyl(d70)-sn-glycero-3-phosphocholine (DSPC-d70) lipid bilayer arrays. Exploiting the vibrational selectivity and inherent symmetry constraints of sum-frequency generation, SFVI was successfully used to probe the transition temperature of a patterned DSPC:DSPC-d70 lipid bilayer array. SFVI was also used to study the phase behavior in a multi-component micropatterned lipid bilayer array (MLBA) prepared using three different binary lipid mixtures (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DSPC, DOPC:1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC:DSPC). This paper demonstrates that a simplified SFVI setup provides the necessary chemical imaging capabilities with the spatial resolution, sensitivity and field of view required for exploring lipid membrane properties in a high-throughput array based assay.