Membrane regulators such as sterols and hopanoids play a major role in the physiological and physicochemical adaptation of the different plasmic membranes in Eukarya and Bacteria. They are key to the functionalization and the spatialization of the membrane, and therefore indispensable for the cell cycle. No archaeon has been found to be able to synthesize sterols or hopanoids to date. They also lack homologs of the genes responsible for the synthesis of these membrane regulators. Due to their divergent membrane lipid composition, the question whether archaea require membrane regulators, and if so, what is their nature, remains open. In this review, we review evidence for the existence of membrane regulators in Archaea, and propose tentative location and biological functions. It is likely that no membrane regulator is shared by all archaea, but that they may use different polyterpenes, such as carotenoids, polyprenols, quinones and apolar polyisoprenoids, in response to specific stressors or physiological needs.

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.

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 from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium 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 approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled 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 on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. 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. These data demonstrate that archaea tend to 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 origins and evolution.

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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.

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.

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.