We investigated the influence of archaeal lipids (C25,25) isolated from thermophilic archaeon Aeropyrum pernix K1 on physicochemical properties of liposomes comprised of egg sphingomyelin (SM) and cholesterol (CH) using fluorescence emission anisotropy, calcein release studies, dynamic light scattering, transmission electron microscopy and phase analysis light scattering. The 2 mol% addition of archaeal lipids enabled formation of small unilamellar vesicles by sonication while also having significant effect on reducing mean size, polydispersity index and zeta potential of C25,25/SM/CH vesicles. Increasing the ratio of C25,25 lipids in mixture of C25,25/SM/CH decreased lipid ordering parameter in dose dependent manner at different temperatures. We also demonstrated that adding 15 mol% C25,25 to SM/CH mixture will cause it to notably interact with fetal bovine serum which could make them a viable alternative adjuvant to synthetic ether-linked lipids in development of advanced liposomal vaccine delivery systems. The prospect of combining the proven strengths of SM/CH mixtures with the unique properties of C25,25 opens exciting possibilities for advancing drug delivery technologies, promising to yield formulations that are both highly effective and adaptable to a range of therapeutic applications.

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

Deep-sea hydrothermal vents host archaeal and bacterial thermophilic communities, including taxonomically and functionally diverse Thermoproteota. Despite their prevalence in high-temperature submarine communities, Thermoproteota are chronically under-represented in genomic databases and issues have emerged regarding their nomenclature, particularly within the Aeropyrum-Thermodiscus-Caldisphaera. To resolve some of these problems, we identified 47 metagenome-assembled genomes (MAGs) within this clade, from 20 previously published deep-sea hydrothermal vent and submarine volcano metagenomes, and 24 MAGs from public databases. Using phylogenomic analysis, Genome Taxonomy Database Toolkit (GTDB-Tk) taxonomic assessment, 16S rRNA gene phylogeny, average amino acid identity (AAI) and functional gene patterns, we re-evaluated of the taxonomy of the Aeropyrum-Thermodiscus-Caldisphaera. At least nine genus-level clades were identified with two or more MAGs. In accordance with SeqCode requirements and recommendations, we propose names for three novel genera, viz. Tiamatella incendiivivens, Hestiella acidicharens and Calypsonella navitae. A fourth genus was also identified related to Thermodiscus maritimus, for which no available sequenced genome exists. We propose the novel species Thermodiscus eudorianus to describe our high-quality Thermodiscus MAG, which represents the type genome for the genus. All three novel genera and T. eudorianus are likely anaerobic heterotrophs, capable of fermenting protein-rich carbon sources, while some Tiamatella, Calypsonella and T. eudorianus may also reduce polysulfides, thiosulfate, sulfur and/or selenite, and the likely acidophile, Hestiella, may reduce nitrate and/or perchlorate. Based on phylogenomic evidence, we also propose the family Acidilobaceae be amended to include Caldisphaera, Aeropyrum, Thermodiscus and Stetteria and the novel genera described here.

Most life forms on earth live at temperatures below 50 °C. Within these organisms are proteins that form the three-dimensional structures essential to their biological activity and function. However, some thermophilic life forms can resist higher temperatures and have corresponding adaptations to preserve protein function at these high temperatures. Among the structural factors responsible for this resistance of thermophilic proteins to high temperatures is the presence of additional hydrogen bonds in the thermophilic proteins, which means that the structure of the protein is more resistant to unfolding. Similarly, thermostable proteins are rich in structure-stabilizing salt bridges and/or disulfide bridges. In this context, we perform multiple replica molecular dynamics simulations at different temperatures on the Aeropyrum pernix (L7Ae) protein (from the crenarchaeal species A. pernix), known for its high melting temperature, and this in the aim to elucidate the structural factors responsible for its high thermostability. The results reveal that between the most sensitive regions of the protein to the increase of temperature are the loops L1, and L5, which surround the hydrophobic core region of the protein, besides the loop L9, and the C-terminal α5 region. This latter is the longer alpha helix of the protein secondary structure motifs and it is the first to be denaturated at 450 K, while the rest of the protein secondary structure motifs at this temperature were intact. The mechanism of unfolding that follows this protein at 550 K is similar to other thermophile proteins found in literature, with the opening of the loops that surround the hydrophobic core of the protein. So, the latter is completely exposed to the solvent, and partially denatured. The total denaturation process of the protein takes an average time of 40 ns to be achieved. Our investigation also shows that all the calculated salt bridges, with distances less than or equal to 6 A°, are on the periphery part of the protein, exposed to the solvent. However, the hydrophobic core of the protein is not involved in the formation of salt bridges, but rather with formation of some important hydrogen bondings that still persist even at 450 K. So, optimizing hydrogen bonding, near or within the core region, at high temperatures is a strategy that follows this thermostable protein to protect its hydrophobic core from denaturation, and ensure the thermal stability of the protein.

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been \'domesticated\', that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

Mismatch glycosylase (MIG) can excise thymine and uracil from mutagenic T:G and U:G mispairs, which arise from cytosine and 5-methylcytosine deamination, respectively. Here, we present evidence that a thermostable MIG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-MIG) can remove thymine and uracil from T:G and U:G mispairs at high temperature, albeit at a low efficiency for U:G mispair. The enzyme displays maximum efficiency at 70 oC - 75 °C and pH 7.0-8.0. Tb-MIG is extremely thermostable, retaining 50% activity after heating at 100 oC for 2 hrs. In addition, Tb-MIG is a bifunctional glycosylase with an AP lyase activity, then resembles the MIG from the hyperthermopilic crenarchaeon Pyrobaculum aerophilium, but contrasts with the MIG from the hyperthermopilic crenarchaeon Aeropyrum pernix. Importantly, we show that residues Y133 and D151 in Tb-MIG are essential for thymine removal, and that residues A58, N153 and R156 are involved in thymine removal. Compared with the wild-type protein, the A58D and Y133K mutants display the increased AP lyase activity, confirming the essential roles played by the correspondingly conserved Asp and Lys in endonuclease III for AP site cleavage. Overall, our work is the first biochemical characterization of a hypthermophilic euryarchaeal MIG, augmenting our understanding on archaeal MIG function.

Pyridoxal-5\'-phosphate-dependent cysteine synthases synthesize l-cysteine from their primary substrates, O-acetyl-l-serine (OAS) and O-phospho-l-serine (OPS), and their secondary substrate, sulfide. The mechanism by which cysteine synthases recognize OPS remains unclear; hence, we investigated the OPS recognition mechanism of the OPS sulfhydrylase obtained from Aeropyrum pernix K1 (ApOPSS) and the OAS sulfhydrylase-B obtained from Escherichia coli (EcOASS-B), using protein engineering methods. From the amino acid sequence alignment data, we found that some OPS sulfhydrylases (OPSSs) had a Tyr corresponding to the Phe225 and Phe141 residues in ApOPSS and EcOASS-B, respectively, and that the Tyr residue could facilitate OPS recognition. The enzymatic activity of the ApOPSS F225Y mutant toward OPS decreased compared with that of the wild-type; the kcat value decreased 2.3-fold during cysteine synthesis. X-ray crystallography results of the complex of ApOPSS F225Y and F225Y/R297A mutants bound to OPS and l-cysteine showed that kcat might have decreased because of the stronger interactions of the reaction product phosphate with Tyr225, Thr203, and Arg297, and that of the l-cysteine with Tyr225. The specific activity of the EcOASS-B F141Y mutant toward OPS increased by 50-fold compared with that of the wild-type. Thus, a Tyr within a cysteine synthase corresponding to the Phe225 in ApOPSS and Phe141 in EcOASS-B could act as a key residue for classifying an unknown cysteine synthase as an OPSS. The elucidation of the substrate recognition system of cysteine synthases would enable us to effectively classify cysteine synthases and develop pathogen-specific drug targets, as OPSS is absent in mammalian hosts.

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 °C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. KEY POINTS: • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability.

Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.