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Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Enzymes of the 2-oxoacid:ferredoxin oxidoreductase (OFOR) superfamily catalyze the reversible oxidation of 2-oxoacids to acyl-coenzyme A esters and carbon dioxide (CO 2 )using ferredoxin or flavodoxin as the redox partner. Although members of the family share primary sequence identity, a variety of domain and subunit arrangements are known. Here, we characterize the structure of a four-subunit family member: the pyruvate:ferredoxin oxidoreductase (PFOR) from the methane producing archaeon Methanosarcina acetivorans (MaPFOR). The 1.92 Å resolution crystal structure of MaPFOR shows a protein fold like those of single- or two-subunit PFORs that function in 2-oxoacid oxidation, including the location of the requisite thiamine pyrophosphate (TPP), and three [4Fe-4S] clusters. Of note, MaPFOR typically functions in the CO 2 reductive direction, and structural comparisons to the pyruvate oxidizing PFORs show subtle differences in several regions of catalytical relevance. These studies provide a framework that may shed light on the biochemical mechanisms used to facilitate reductive pyruvate synthesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ribosome structure and activity are challenged at high temperatures, often demanding modifications to ribosomal RNAs (rRNAs) to retain translation fidelity. LC-MS/MS, bisulfite-sequencing, and high-resolution cryo-EM structures of the archaeal ribosome identified an RNA modification, N 4, N 4-dimethylcytidine (m 4 2 C), at the universally conserved C918 in the 16S rRNA helix 31 loop. Here, we characterize and structurally resolve a class of RNA methyltransferase that generates m 4 2 C whose function is critical for hyperthermophilic growth. m 4 2 C is synthesized by the activity of a unique family of RNA methyltransferase containing a Rossman-fold that targets only intact ribosomes. The phylogenetic distribution of the newly identified m 4 2 C synthase family implies that m 4 2 C is biologically relevant in each domain. Resistance of m 4 2 C to bisulfite-driven deamination suggests that efforts to capture m 5 C profiles via bisulfite sequencing are also capturing m 4 2 C., Competing Interests: Competing interests statement:K.A.F., P.S.H., V.T., L.E., J.S., H.P.F., R.C., and T.J.S. do not have any competing financial interests nor conflicts of interest to report. N.D., E.J.W., R.T.F., Y.-L.T., G.B.R., and I.R.C. are employed and funded by New England Biolabs, Inc., a manufacturer and vendor of molecular biology reagents, including nucleic acid modifying and synthesis enzymes. The authors state that this affiliation does not affect their impartiality, objectivity of data generation or interpretation, adherence to journal standards and policies, or availability of data.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity., (© 2024 The Protein Society.)

Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. In this study, the crystal structure of ApePCNA1, one of three PCNAs from the thermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols., (open access.)

The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea 1-3 . Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, functional homology between archaeal and eukaryotic factor-dependent termination, and fundamental mechanistic similarities in factor-dependent termination in bacteria, archaea, and eukaryotes., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The halophilic archaeon Haloarcula hispanica utilizes the sugar alcohols mannitol and sorbitol as carbon and energy sources. Genes, enzymes, and transcriptional regulators involved in uptake and degradation of these sugar alcohols were identified by growth experiments with deletion mutants and enzyme characterization. It is shown that both mannitol and sorbitol are taken up via a single ABC transporter of the CUT1 transporter family. Then, mannitol and sorbitol are oxidized to fructose by two distinct dehydrogenases. Fructose is further phosphorylated to fructose-1-phosphate by a haloarchaeal ketohexokinase, providing the first evidence for a physiological function of ketohexokinase in prokaryotes. Finally, fructose-1-phosphate is phosphorylated via fructose-1-phosphate kinase to fructose-1,6-bisphosphate, which is cleaved to triosephosphates by a Class I fructose-1,6-bisphosphate aldolase. Two distinct transcriptional regulators, acting as activators, have been identified: an IclR-like regulator involved in activating genes for sugar alcohol uptake and oxidation to fructose, and a GfcR-like regulator that likely activates genes involved in the degradation of fructose to pyruvate. This is the first comprehensive analysis of a sugar alcohol degradation pathway in Archaea., (© 2024. The Author(s).)

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota., (© 2024. The Author(s).)

The anaerobic oxidation of alkanes is a microbial process that mitigates the flux of hydrocarbon seeps into the oceans. In marine archaea, the process depends on sulphate-reducing bacterial partners to exhaust electrons, and it is generally assumed that the archaeal CO 2 -forming enzymes (CO dehydrogenase and formylmethanofuran dehydrogenase) are coupled to ferredoxin reduction. Here, we study the molecular basis of the CO 2 -generating steps of anaerobic ethane oxidation by characterising native enzymes of the thermophile Candidatus Ethanoperedens thermophilum obtained from microbial enrichment. We perform biochemical assays and solve crystal structures of the CO dehydrogenase and formylmethanofuran dehydrogenase complexes, showing that both enzymes deliver electrons to the F 420 cofactor. Both multi-metalloenzyme harbour electronic bridges connecting CO and formylmethanofuran oxidation centres to a bound flavin-dependent F 420 reductase. Accordingly, both systems exhibit robust coupled F 420 -reductase activities, which are not detected in the cell extract of related methanogens and anaerobic methane oxidisers. Based on the crystal structures, enzymatic activities, and metagenome mining, we propose a model in which the catabolic oxidising steps would wire electron delivery to F 420 in this organism. Via this specific adaptation, the indirect electron transfer from reduced F 420 to the sulphate-reducing partner would fuel energy conservation and represent the driving force of ethanotrophy., (© 2024. The Author(s).)

The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1 is crucial for climate and environmental research due to its ability to produce methane. Here, we establish a Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency and limitation). The translation of 93 previously annotated and 314 unannotated small ORFs, coding for proteins ≤ 70 amino acids, is predicted with high confidence based on Ribo-seq data. LC-MS analysis validates the translation for 62 annotated small ORFs and 26 unannotated small ORFs. Epitope tagging followed by immunoblotting analysis confirms the translation of 13 out of 16 selected unannotated small ORFs. A comprehensive differential transcription and translation analysis reveals that 29 of 314 unannotated small ORFs are differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. A high number of reported small RNAs are emerging as dual-function RNAs, including sRNA 154 , the central regulatory small RNA of nitrogen metabolism. Several unannotated small ORFs are conserved in Methanosarcina species and overproducing several (small ORF encoded) small proteins suggests key physiological functions. Overall, the comprehensive analysis opens an avenue to elucidate the function(s) of multitudinous small proteins and dual-function RNAs in M. mazei., (© 2024. The Author(s).)

Neurodegenerative diseases are characterized by the presence of misfolded and aggregated proteins which are thought to contribute to the development of the disease. In one form of inherited blinding disease, retinitis pigmentosa, a P23H mutation in the light-sensing receptor, rhodopsin causes rhodopsin misfolding resulting in complete vision loss. We investigated whether a xenogeneic protein-unfolding ATPase (unfoldase) from thermophilic Archaea, termed PANet, could counteract the proteotoxicity of P23H rhodopsin. We found that PANet increased the number of surviving photoreceptors in P23H rhodopsin mice and recognized rhodopsin as a substate in vitro. This data supports the feasibility and efficacy of using a xenogeneic unfoldase as a therapeutic approach in mouse models of human neurodegenerative diseases. We also showed that an archaeal proteasome, called the T20S can degrade rhodopsin in vitro and demonstrated that it is feasible and safe to express gateless T20S proteasomes in vivo in mouse rod photoreceptors. Expression of archaeal proteasomes may be an effective therapeutic approach to stimulate protein degradation in retinopathies and neurodegenerative diseases with protein-misfolding etiology., Competing Interests: No competing interests is declared., (Copyright: © 2024 Brooks et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp 154 -His 193 -Ser 348 . Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The K m , V max and K cat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg -1 and 1538.09 S -1 , respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Dimethyladenosine transferase 1 (DIMT1), an ortholog of bacterial KsgA is a conserved protein that assists in ribosome biogenesis by modifying two successive adenosine bases near the 3' end of small subunit (SSU) rRNA. Although KsgA/DIMT1 proteins have been characterized in bacteria and eukaryotes, they are yet unexplored in archaea. Also, their dynamics are not well understood. Here, we structurally and functionally characterized the apo and holo forms of archaeal DIMT1 from Pyrococcus horikoshii. Wild-type protein and mutants were analyzed to capture different transition states, including open, closed, and intermediate states. This study reports a unique inter-domain movement that is needed for substrate (RNA) positioning in the catalytic pocket, and is only observed in the presence of the cognate cofactors S-adenosyl-L-methionine (SAM) or S-adenosyl-L-homocysteine (SAH). The binding of the inhibitor sinefungine, an analog of SAM or SAH, to archaeal DIMT1 blocks the catalytic pocket and renders the enzyme inactive., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Elucidating the role of molecular chaperones in extremely thermophilic archaea, including the gamma prefoldin (γPFD) in the deep-sea methanogen Methanocaldococcus jannaschii, is integral to understanding microbial adaptation to hot environments. This study focuses on genetically engineered knock-out and overexpression strains to evaluate the importance of γPFD in the growth and thermal tolerance of M. jannaschii. An in-depth analysis of cell growth, morphology and transcriptional responses to heat stress revealed that although the gene encoding γPFD is substantially upregulated in response to heat shock, the γPFD is not indispensable for high-temperature survival. Instead, its absence in the knock-out strain is compensated for by the upregulation of several proteolytic proteins in the absence of heat shock, nearly matching the corresponding transcription profile of selected transcripts for proteins involved in protein synthesis and folding in the wild-type strain following heat shock, using quantitative reverse-transcription PCR (RT-qPCR). These findings bridge environmental adaptation with molecular biology, underscoring the versatility of extremophiles and providing a deeper mechanistic understanding of how they cope with stress., (© 2024 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

5-Methyluridine (m 5 U) rRNA modifications frequently occur at U747 and U1939 ( Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S -adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m 5 U (Arm 5 U) MTases. Among these, a tRNA-specific Arm 5 U MTase ( Pab TrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm 5 U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii ( Ph RlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron-sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of Ph RlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with Ph RlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm 5 U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.

Bactofilins are rigid, nonpolar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii , halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in Δ halA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while Δ halA and Δ halB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. The deletion of halA in the haloarchaeon Halobacterium salinarum , whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of the S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results suggest that halofilins might play a significant mechanical scaffolding role in addition to possibly directing envelope synthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Microbial rhodopsins are photoreceptive membrane proteins found in microorganisms with an all-trans-retinal chromophore. The function of many microbial rhodopsins is determined by three residues in the third transmembrane helix called motif residues. Here, we report a group of microbial rhodopsins with a novel Thr-Thr-Gly (TTG) motif. The ion-transport assay revealed that they function as light-driven inward anion pumps similar to halorhodopsins previously found in archaea and bacteria. Based on the characteristic glycine residue in their motif and light-driven anion-pumping function, these new rhodopsins are called glycylhalorhodopsins (GHRs). X-ray crystallographic analysis found large cavities on the cytoplasmic side, which are produced by the small side-chain volume of the glycine residue in the motif. The opened structure of GHR on the cytoplasmic side is related to the anion releasing process to the cytoplasm during the photoreaction compared to canonical halorhodopsin from Natronomonas pharaonis (NpHR). GHR also transports SO 4 2- and the extracellular glutamate residue plays an essential role in extracellular SO 4 2- uptake. In summary, we have identified TTG motif-containing microbial rhodopsins that display an anion-releasing mechanism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

HerA is an ATP-dependent translocase that is widely distributed in archaea and some bacteria. It belongs to the HerA/FtsK translocase bacterial family, which is a subdivision of the RecA family. Currently, it is identified that HerA participates in the repair of DNA double-strand breaks (DSBs) or confers anti-phage defense by assembling other proteins into large complexes. In recent years, there has been a growing understanding of the bioinformatics, biochemistry, structure, and function of HerA subfamily members in both archaea and bacteria. This comprehensive review compares the structural disparities among diverse HerAs and elucidates their respective roles in specific life processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca 2+ -activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Since first identified as a separate domain of life in the 1970s, it has become clear that archaea differ profoundly from both eukaryotes and bacteria. In this review, we look across the archaeal domain and discuss the diverse mechanisms by which archaea control cell cycle progression, DNA replication, and cell division. While the molecular and cellular processes archaea use to govern these critical cell biological processes often differ markedly from those described in bacteria and eukaryotes, there are also striking similarities that highlight both unique and common principles of cell cycle control across the different domains of life. Since much of the eukaryotic cell cycle machinery has its origins in archaea, exploration of the mechanisms of archaeal cell division also promises to illuminate the evolution of the eukaryotic cell cycle.

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60β homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality., (© 2024 Federation of European Biochemical Societies.)

Few described archaeal, and fewer bacterial, lineages thrive under salt-saturating conditions, such as solar saltern crystallizers (salinity above 30% w/v). They accumulate molar K + cytoplasmic concentrations to maintain osmotic balance ('salt-in' strategy) and have proteins adaptively enriched in negatively charged acidic amino acids. Here we analysed metagenomes and metagenome-assembled genomes from geothermally influenced hypersaline ecosystems with increasing chaotropicity in the Danakil Depression. Normalized abundances of universal single-copy genes confirmed that haloarchaea and Nanohaloarchaeota encompass 99% of microbial communities in the near-life-limiting conditions of the Western-Canyon Lakes. Danakil metagenome- and metagenome-assembled-genome-inferred proteomes, compared with those of freshwater, seawater and solar saltern ponds up to saturation (6-14-32% salinity), showed that Western-Canyon Lake archaea encode the most acidic proteomes ever observed (median protein isoelectric points ≤4.4). We identified previously undescribed haloarchaeal families as well as an Aenigmatarchaeota family and a bacterial phylum independently adapted to extreme halophily. Despite phylum-level diversity decreasing with increasing salinity-chaotropicity, and unlike in solar salterns, adapted archaea exceedingly diversified in Danakil ecosystems, challenging the notion of decreasing diversity under extreme conditions. Metabolic flexibility to utilize multiple energy and carbon resources generated by local hydrothermalism along feast-and-famine strategies seemingly shapes microbial diversity in these ecosystems near life limits., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

L-asparaginase (ASNase, E.C. 3.5.1.1) catalyzes the deamination of L-asparagine to L-aspartic acid and ammonia and is widely used in medicine to treat acute lymphocytic leukemia. It also has significant applications in the food industry by inhibiting acrylamide formation. In this study, we characterized a thermostable ASNase from the hyper thermophilic strain, Pyrococcus yayanosii CH1. The recombinant enzyme (PyASNase) exhibited maximal activity at pH 8.0 and 85 °C. Moreover, PyASNase demonstrated promising thermostability across temperatures ranging from 70 to 95 °C. The kinetic parameters of PyASNase for L-asparagine were a K m of 6.3 mM, a k cat of 1989s -1 , and a k cat /K m of 315.7 mM -1  s -1 . Treating potato samples with 10 U/mL of PyASNase at 85 °C for merely 10 min reduced the acrylamide content in the final product by 82.5%, demonstrating a high efficiency and significant advantage of PyASNase in acrylamide inhibition., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK.)

Inteins (intervening proteins), mobile genetic elements removed through protein splicing, often interrupt proteins required for DNA replication, recombination, and repair. An abundance of in vitro evidence implies that inteins may act as regulatory elements, whereby reduced splicing inhibits production of the mature protein lacking the intein, but in vivo evidence of regulatory intein excision in the native host is absent. The model archaeon Thermococcus kodakarensis encodes 15 inteins, and we establish the impacts of intein splicing inhibition on host physiology and replication in vivo. We report that a decrease in intein splicing efficiency of the recombinase RadA, a Rad51/RecA homolog, has widespread physiological consequences, including a general growth defect, increased sensitivity to DNA damage, and a switch in the mode of DNA replication from recombination-dependent replication toward origin-dependent replication.

We previously identified M.ApeKI from Aeropyum pernix K1 as a highly thermostable DNA (cytosine-5)-methyltransferase. M.ApeKI uses the type II restriction-modification system (R-M system), among the best-studied R-M systems. Although endonucleases generally utilize Mg (II) as a cofactor, several reports have shown that MTases exhibit different reactions in the presence of metal ions. This study aim was to evaluate the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions. We evaluated the influence of metal ions on the catalytic activity and DNA binding of M.ApeKI. The catalytic activity was inhibited by Cu (II), Mg (II), Mn (II), and Zn (II), each at 5 m m. DNA binding was more strongly inhibited by 5 m m Cu (II) and 10 m m Zn (II). To our knowledge, this is the first report showing that DNA binding of type II MTase is inhibited by metal ions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Members of the kingdom Nanobdellati , previously known as DPANN archaea, are characterized by ultrasmall cell sizes and reduced genomes. They primarily thrive through ectosymbiotic interactions with specific hosts in diverse environments. Recent successful cultivations have emphasized the importance of adhesion to host cells for understanding the ecophysiology of Nanobdellati . Cell adhesion is often mediated by cell surface carbohydrates, and in archaea, this may be facilitated by the glycosylated S-layer protein that typically coats their cell surface. In this study, we conducted glycoproteomic analyses on two co-cultures of Nanobdellati with their host archaea, as well as on pure cultures of both host and non-host archaea. Nanobdellati exhibited various glycoproteins, including archaellins and hypothetical proteins, with glycans that were structurally distinct from those of their hosts. This indicated that Nanobdellati autonomously synthesize their glycans for protein modifications probably using host-derived substrates, despite the high energy cost. Glycan modifications on Nanobdellati proteins consistently occurred on asparagine residues within the N-X-S/T sequon, consistent with patterns observed across archaea, bacteria, and eukaryotes. In both host and non-host archaea, S-layer proteins were commonly modified with hexose, N -acetylhexosamine, and sulfonated deoxyhexose. However, the N -glycan structures of host archaea, characterized by distinct sugars such as deoxyhexose, nonulosonate sugar, and pentose at the nonreducing ends, were implicated in enabling Nanobdellati to differentiate between host and non-host cells. Interestingly, the specific sugar, xylose, was eliminated from the N -glycan in a host archaeon when co-cultured with Nanobdella . These findings enhance our understanding of the role of protein glycosylation in archaeal interactions.IMPORTANCE Nanobdellati archaea, formerly known as DPANN, are phylogenetically diverse, widely distributed, and obligately ectosymbiotic. The molecular mechanisms by which Nanobdellati recognize and adhere to their specific hosts remain largely unexplored. Protein glycosylation, a fundamental biological mechanism observed across all domains of life, is often crucial for various cell-cell interactions. This study provides the first insights into the glycoproteome of Nanobdellati and their host and non-host archaea. We discovered that Nanobdellati autonomously synthesize glycans for protein modifications, probably utilizing substrates derived from their hosts. Additionally, we identified distinctive glycosylation patterns that suggest mechanisms through which Nanobdellati differentiate between host and non-host cells. This research significantly advances our understanding of the molecular basis of microbial interactions in extreme environments., Competing Interests: The authors declare no conflict of interest.

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a superfamily of histones, termed α3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones., (© 2024. The Author(s).)

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludi s can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis . RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function., Competing Interests: The authors declare no conflict of interest.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate the inheritance of eukaryotic Sec61/oligosaccharyltransferase/translocon-associated protein translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of Candidatus Prometheoarchaeum syntrophicum strain MK-D1 oligosaccharyltransferase 1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet-to-be-translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Salt-tolerant proteases with remarkable stability are highly desirable biocatalysts in the salt-fermented food industry. In this study, the undigested autocleavage product of HlyA (halolysin A), a low-salt adapted halolysin from halophilic archaeon Halococcus salifodinae, was investigated. HlyA underwent autocleavage of its C-terminal extension (CTE) at temperatures over 40 °C or NaCl concentrations below 2 M to yield HlyAΔCTE. HlyAΔCTE demonstrated robust stability over a wide range of -20-60 °C, 0.5-4 M NaCl, and pH 6.0-10.0 for at least 72 h. Notably, HlyAΔCTE is the first reported halolysin with such exceptional stability. Compared with HlyA, HlyAΔCTE preferred high temperatures (50-75 °C), low salinities (0.5-2.5 M NaCl), and near-neutral (pH 6.5-8.0) conditions to achieve high activity, consistently with its production conditions. HlyAΔCTE displayed a higher V max value against azocasein than HlyA. During fish sauce fermentation, HlyAΔCTE significantly enhanced fish protein hydrolysis, indicating its potential as a robust biocatalyst in the salt-fermented food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, k cat and k cat /K m values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Multi-antimicrobial extrusion (MATE) transporter membrane proteins provide drug and toxin resistivity by expelling compounds from cells. MATE proteins can be pictured as V-shaped. To regulate its functioning, the protein structure can switch between outward-facing (OF) and inward-facing (IF). Pyrococcus furiosus MATE (PfMATE) is the only member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily that has available both the IF and OF crystal structures. With the availability of both the IF and OF structures, we are able to perform computational investigations to determine how protonation of specific amino acids causes a cascade of changes in the protein conformation that allow PfMATE to change its state from OF to IF in order to regulate its antiporter function. Using a variety of computational and theoretical techniques, we investigated four different systems of IF and OF PfMATE along with the native archaeal lipid bilayer, without or with protonation at the experimentally determined locations within the protein. We performed molecular dynamics (MD) simulations to investigate the flexibility of the four different PfMATE structures and also performed targeted molecular dynamics (TMD) simulations, during which we observed occluded conformations. Our analysis of hydrogen bond changes, potential of mean force, dynamic network analysis, and transfer entropy analysis provides information on how protonation can induce cascading structural changes responsible for the transition between the IF and OF configurations., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Adenosine triphosphate (ATP) is the energy currency of all living organisms and can be used as an indicator for cell proliferation and cytotoxicity. In the present work, we have developed a novel ATP detection system by combining the biotinylation reaction from archaeon Sulfolobus tokodaii with fluorescence resonance energy transfer (FRET). In biotinylation from S. tokodaii, an enzyme known as biotin protein ligase (BPL) forms a very stable complex with its product, biotinylated substrate protein (BCCP). Here, BPL and BCCP were fused to the fluorescent proteins Cerulean and Clover, respectively, and ATP detection was accomplished by monitoring the FRET signal between the two fluorescent proteins, since ATP is an essential component for biotinylation and the tight BPL-BCCP complex is formed only after biotinylation. Using this system, we have succeeded in detecting 5 nM of ATP by biotinylation reaction with 50 nM of each fusion protein. Our method has a characteristic that the signal does not decay for at least 2 h after the start of the reaction, unlike in the case of the luminescence-based assay with luciferase commonly used for the ATP detection. Thus, our system allows for ATP detection which is not significantly constrained by measurement timing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Methanogenic archaea are chemolithotrophic prokaryotes that can reduce carbon dioxide with hydrogen gas to form methane. These microorganisms make a significant contribution to the global carbon cycle, with methanogenic archaea from anoxic environments estimated to contribute > 500 million tons of global methane annually. Archaeal methanogenesis is dependent on the methanofurans; aminomethylfuran containing coenzymes that act as the primary C 1 acceptor molecule during carbon dioxide fixation. Although the biosynthetic pathway to the methanofurans has been elucidated, structural adaptations which confer thermotolerance to Mfn enzymes from extremophilic archaea are yet to be investigated. Here we focus on the methanofuran biosynthetic enzyme MfnB, which catalyses the condensation of two molecules of glyceralde-3-phosphate to form 4‑(hydroxymethyl)-2-furancarboxaldehyde-phosphate. In this study, MfnB enzymes from the hyperthermophile Methanocaldococcus jannaschii and the mesophile Methanococcus maripaludis have been recombinantly overexpressed and purified to homogeneity. Thermal unfolding studies, together with steady-state kinetic assays, demonstrate thermoadaptation in the M. jannaschii enzyme. Molecular dynamics simulations have been used to provide a structural explanation for the observed properties. These reveal a greater number of side chain interactions in the M. jannaschii enzyme, which may confer protection from heating effects by enforcing spatial residue constraints., (© 2024. The Author(s).)

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN) 5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN) 5 . These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN) 5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN) 4 and (GlcN) 5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNAs are often modified to invoke new activities. While many modifications are limited in frequency, restricted to non-coding RNAs, or present only in select organisms, 5-methylcytidine (m 5 C) is abundant across diverse RNAs and fitness-relevant across Domains of life, but the synthesis and impacts of m 5 C have yet to be fully investigated. Here, we map m 5 C in the model hyperthermophile, Thermococcus kodakarensis. We demonstrate that m 5 C is ~25x more abundant in T. kodakarensis than human cells, and the m 5 C epitranscriptome includes ~10% of unique transcripts. T. kodakarensis rRNAs harbor tenfold more m 5 C compared to Eukarya or Bacteria. We identify at least five RNA m 5 C methyltransferases (R5CMTs), and strains deleted for individual R5CMTs lack site-specific m 5 C modifications that limit hyperthermophilic growth. We show that m 5 C is likely generated through partial redundancy in target sites among R5CMTs. The complexity of the m 5 C epitranscriptome in T. kodakarensis argues that m 5 C supports life in the extremes., (© 2024. The Author(s).)

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS 2 ). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS - ) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS 2 to sustain growth. As such, medium supplied with FeS 2 should lead to higher availability of transition metals when compared to medium supplied with HS - . Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS - as sole S source) versus non-sulfidic (cells provided with FeS 2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS 2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.IMPORTANCEProteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments., Competing Interests: The authors declare no conflict of interest.

d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni 2+ . Its k cat and k cat / K m values toward d-fructose were measured to be 248.5 min -1 and 2.117 mM -1 ·min -1 , respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t 1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships., (© 2024. Crown.)

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1, N 6 -ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ubiquitin-like proteins (Ubls) in eukaryotes and bacteria mediate sulfur transfer for the biosynthesis of sulfur-containing biomolecules and form conjugates with specific protein targets to regulate their functions. Here, we investigated the functions and physiological importance of Ubls in a hyperthermophilic archaeon by constructing a series of deletion mutants. We found that the Ubls (TK1065, TK1093, and TK2118) in Thermococcus kodakarensis are conjugated to their specific target proteins, and all three are involved in varying degrees in the biosynthesis of sulfur-containing biomolecules such as tungsten cofactor (Wco) and tRNA thiouridines. TK2118 (named UblB) is involved in the biosynthesis of Wco in a glyceraldehyde 3-phosphate:ferredoxin oxidoreductase, which is required for glycolytic growth, whereas TK1093 (named UblA) plays a key role in the efficient thiolation of tRNAs, which contributes to cellular thermotolerance. Intriguingly, in the presence of elemental sulfur (S 0 ) in the culture medium, defective synthesis of these sulfur-containing molecules in Ubl mutants was restored, indicating that T. kodakarensis can use S 0 as an alternative sulfur source without Ubls. Our analysis indicates that the Ubl-mediated sulfur-transfer system in T. kodakarensis is important for efficient sulfur assimilation, especially under low S 0 conditions, which may allow this organism to survive in a low sulfur environment.IMPORTANCESulfur is a crucial element in living organisms, occurring in various sulfur-containing biomolecules including iron-sulfur clusters, vitamins, and RNA thionucleosides, as well as the amino acids cysteine and methionine. In archaea, the biosynthesis routes and sulfur donors of sulfur-containing biomolecules are largely unknown. Here, we explored the functions of Ubls in the deep-blanched hyperthermophilic archaeon, Thermococcus kodakarensis . We demonstrated functional redundancy of these proteins in the biosynthesis of tungsten cofactor and tRNA thiouridines and the significance of these sulfur-carrier functions, especially in low sulfur environments. We propose that acquisition of a Ubl sulfur-transfer system, in addition to an ancient inorganic sulfur assimilation pathway, enabled the primordial archaeon to advance into lower-sulfur environments and expand their habitable zone., Competing Interests: The authors declare no conflict of interest.

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5-6.0 (CIB12) and 4.5-5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na + /H + influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H + antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H + -coupled substrate excretion by the transporter., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sorting signals are crucial for the anchoring of proteins to the cell surface in archaea and bacteria. These proteins often feature distinct motifs at their C-terminus, cleaved by sortase or sortase-like enzymes. Gram-positive bacteria exhibit the LPXTGX consensus motif, cleaved by sortases, while Gram-negative bacteria employ exosortases recognizing motifs like PEP. Archaea utilize exosortase homologs known as archaeosortases for signal anchoring. Traditionally identification of such C-terminal sorting signals was performed with profile Hidden Markov Models (pHMMs). The C ell- W all PRE Diction (CW-PRED) method introduced for the first time a custom-made class HMM for proteins in Gram-positive bacteria that contain a cell wall sorting signal which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues. Here we present a new and updated version of CW-PRED for predicting C-terminal sorting signals in Archaea, Gram-positive, and Gram-negative bacteria. We used a large training set and several model enhancements that improve motif identification in order to achieve better discrimination between C-terminal signals and other proteins. Cross-validation demonstrates CW-PRED's superiority in sensitivity and specificity compared to other methods. Application of the method in reference proteomes reveals a large number of potential surface proteins not previously identified. The method is available for academic use at http://195.251.108.230/apps.compgen.org/CW-PRED/ and as standalone software.

The Pyrococcus horikoshii amino acid transporter Glt Ph revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFS open-1 , to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on Glt Ph are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N 2 ) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· AlF 4 - is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N 2 -machinery from these ancient archaea., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)