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Three GPN-loop GTPases, GPN1-GPN3, are central to the maturation and trafficking of eukaryotic RNA polymerase II. This GTPase family is widely represented in archaea but typically occurs as single paralogs. Structural analysis of the GTP- and GDP-bound states of theSulfolobus acidocaldariusGPN enzyme (SaGPN) showed that this central GPN-loop GTPase adopts two distinct quaternary structures. In the GTP-bound form the γ-phosphate induces a tensed dimeric arrangement by interacting with the GPN region that is relaxed upon hydrolysis to GDP. Consequently, a rocking-like motion of the two protomers causes a major allosteric structural change towards the roof-like helices. Using a lock-switch-rock (LSR) mechanism, homo- and heterodimeric GPN-like GTPases are locked in the GTP-bound state and undergo large conformational changes upon GTP hydrolysis. AΔsaGPNstrain ofS. acidocaldariuswas characterized by impaired motility and major changes in the proteome underscoring its functional relevance forS. acidocaldarius in vivo.Significance StatementGPN-loop GTPases have been found to be crucial for eukaryotic RNA polymerase II assembly and nuclear trafficking. Despite their ubiquitous occurrence in eukaryotes and archaea the mechanism by which these self-activating GTPases mediate their function is unknown. Our study on an archaeal representative fromSulfolobus acidocaldariusshowed that these dimeric GTPases undergo large-scale conformational changes upon GTP hydrolysis, which can be summarized as a lock-switch-rock mechanism. The observed requirement ofSaGPN for motility appears to be due to its large footprint on the archaeal proteome.

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 core lipid composition of the hyperthermophilic neutrophilic marine archaeon Pyrococcus furiosus. Every growth parameter tested affected the core lipid 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 tetrathers (GDGT) 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.Significance statementWe describe here the membrane adaptive strategies the hyperthermophilic, neutrophilic, and marine model archaeon Pyrococcus furiosus implements in response to one of the largest sets of environmental stressors tested to date, including temperature, pH, salinity, presence/absence of elemental sulfur, carbon source, and genetic background. In contrast to the other archaea investigated so far, which response mainly involves the modulation of their diether/tetraether ratio and/or of their average number of cyclopentane rings, P. furiosus regulates its monoalkyl (so called H-shaped) to dialkyl tetraether ratio. Our study thus provides for the first time evidence of adaptive functions of archaeal monoalkyl tetraethers towards low temperature and pH and high salinity.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted fromEscherichia colifor example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted fromHalobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here we develop a new approach for assessing the membrane permeability of ~10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of eighteen metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of known archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches present on the archaeal phospholipid tails and the ether bond between the tails and the head group. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. Archaea have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell evolution.

Summary Sterols and hopanoids, the typical membrane regulators of Eukaryotes and Bacteria, are absent from Archaea, which might instead use acyclic polyisoprenoid hydrocarbons (APH). Although a few studies evidenced that APH do modulate membrane physicochemical properties as other membrane regulators, little is known about these intriguing compounds in the third domain of life. In an effort to elucidate APH distribution, physiological and adaptive functions, and biosynthetic pathway in Archaea, we collected all the literature data available and evaluated the APH composition of 65 archaeal species homogeneously distributed within the phylogenetic tree and ecological niches of Archaea. Fully saturated to fully unsaturated 30, 35, and 40-carbon long APH were detected in most species investigated, regardless of their phylogenetic position, lipid composition, or preferential environmental conditions, although the chain length could be correlated with growth conditions. Despite such a wide occurrence of APH in Archaea, only rare homologues of the eukaryotic and bacterial APH biosynthetic pathways were found. How they synthesize these compounds thus remains elusive and lipid analysis still prevails as the only mean to assess the production and functions of these essential archaeal membrane components.

Summary Microbes preserve membrane functionality under fluctuating environmental conditions by modulating their membrane lipid composition, a strategy termed homeoviscous adaptation. Although several studies have documented this strategy in Archaea, the influence of the majority of biotic and abiotic factors on archaeal lipid compositions remains unexplored. To constrain the homeoviscous adaptation strategies in Archaea, we studied the influence of temperature, pH, salinity, elemental sulfur, carbon source, and genetic background on the remarkable core lipid composition of the hyperthermophilic and neutrophilic marine archaeon Pyrococcus furiosus. Most growth parameters tested here affected its core lipid composition, the carbon source and the genetic background having the greatest influences. Surprisingly, P. furiosus membrane adaptation appears to marginally rely on the two major homeoviscous responses implemented by Archaea, i.e., the regulation of the ratio between diethers and tetraethers and of the number of cyclopentane-rings in tetraethers. Instead, it increases the ratio of monoalkyl (GMGT) over dialkyl (GDGT) tetrathers in response to decreasing temperature and pH and increasing salinity. Besides P. furiosus, numerous other species synthesize significant proportions of GMGT, which indicates that this unprecedented homeoviscous strategy might be common in Archaea. This paves the way for developing novel, GMGT-based environmental proxies.

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.

International audience; The diether core membrane lipid sesterterpanyl-phytanyl-glycerol (so-called extended archaeol and often abbreviated C 20-C 25) 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 C 20-C 25 is most commonly produced. The proportion of C 20-C 25 relative to that of archaeol (C 20-C 20) 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, C 20-C 25 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 C 20-C 25 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 C 20-C 25 within Haloarchaea.

International audience; Bacteria and Eukarya organize their plasma membrane spatially into domains of distinct functions. Due to the uniqueness of their lipids, membrane functionalization in Archaea remains a debated area. A novel membrane ultrastructure predicts that monolayer and bilayer domains would be laterally segregated in the hyperthermophilic archaeon Thermococcus barophilus. With very different physico-chemical parameters of the mono-and bilayer, each domain type would thus allow the docking of different membrane proteins and express different biological functions in the membrane. To estimate the ubiquity of this putative membrane ultrastructure in and out of the order Thermococcales, we re-analyzed the core lipid composition of all the Thermococcales type species and collected all the literature data available for isolated archaea. We show that all species of Thermococcales synthesize a mixture of diether bilayer forming and tetraether monolayer forming lipids, in various ratio from 10 to 80% diether in Pyrococcus horikoshii and Thermococcus gorgonarius, respectively. Since the domain formation prediction rests only on the coexistence of di-and tetraether lipids, we show that all Thermococcales have the ability for domain formation, i.e., differential functionalization of their membrane. Extrapolating this view to the whole Archaea domain, we show that almost all archaea also have the ability to synthesize di-and tetraether lipids, which supports the view that functionalized membrane domains may be shared between all Archaea. Hence domain formation and membrane compartmentalization may have predated the separation of the three domains of life and be essential for the cell cycle.

Elucidating the lipidome of Archaea is essential to understand their tolerance to extreme environmental conditions. Previous characterizations of the lipid composition of Pyrococcus species, a model genus of hyperthermophilic archaea belonging to the Thermococcales order, led to conflicting results, which hindered the comprehension of their membrane structure and the putative adaptive role of their lipids. In an effort to clarify the lipid composition data of the Pyrococcus genus, we thoroughly investigated the distribution of both the core lipids (CL) and intact polar lipids (IPL) of the model Pyrococcus furiosus and, for the first time, of Pyrococcus yayanosii, the sole obligate piezophilic hyperthermophilic archaeon known to date. We showed a low diversity of IPL in the lipid extract of P. furiosus, which nonetheless allowed the first report of phosphatidyl inositol-based glycerol mono- and trialkyl glycerol tetraethers. With up to 13 different CL structures identified, the acid methanolysis of Pyrococcus furiosus revealed an unprecedented CL diversity and showed strong discrepancies with the IPL compositions reported here and in previous studies. By contrast, P. yayanosii displayed fewer CL structures but a much wider variety of polar heads. Our results showed severe inconsistencies between IPL and CL relative abundances. Such differences highlight the diversity and complexity of the Pyrococcus plasma membrane composition and demonstrate that a large part of its lipids remains uncharacterized. Reassessing the lipid composition of model archaea should lead to a better understanding of the structural diversity of their lipidome and of their physiological and adaptive functions.

International audience; Archaea, the most extremophilic domain of life, contain ether and branched lipids which provide extraordinary bilayer properties. We determined the structural characteristics of diether archaeal-like phospholipids as functions of hydration and temperature by neutron diffraction. Hydration and temperature are both crucial parameters for the self-assembly and physicochemical properties of lipid bilayers. In this study, we detected non-lamellar phases of archaeal-like lipids at low hydration levels, and lamellar phases at levels of 90% relative humidity or more exclusively. Moreover, at 90% relative humidity, a phase transition between two lamellar phases was discernible. At full hydration, lamellar phases were present up to 70ᵒC and no phase transition was observed within the temperature range studied (from 25 °C to 70 °C). In addition, we determined the neutron scattering length density and the bilayer's structural parameters from different hydration and temperature conditions. At the highest levels of hydration, the system exhibited rearrangements on its corresponding hydrophobic region. Furthermore, the water uptake of the lipids examined was remarkably high. We discuss the effect of ether linkages and branched lipids on the exceptional characteristics of archaeal phospholipids.

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.