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Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Microbes that generate copious amounts of hydrogen (H 2 ) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis , produce generous quantities of H 2 and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H 2 output but additional improvements are desired. An unexamined and potentially valuable route towards increased H 2 production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H 2 production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H 2 producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H 2 output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H 2 per cell than the host strain. Eliminating H 2 consumption and alternative routes of electron sinks in concert with protein tethering further increased H 2 output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis . Key points Protein tethering between redox proteins can reroute electron flux in vivo. Enforced protein proximity results in ~ 40% increases in H 2 production per cell. Protein-tethering provides a generalizable framework to redirect redox metabolism.

Glutamate transporters catalyse the thermodynamically unfavourable transport of anionic amino acids across the cell membrane by coupling it to the downhill transport of cations. This coupling mechanism is still poorly understood, in part because the available crystal structures of these transporters are of relatively low resolution. Here we solve crystal structures of the archaeal transporter Glt in the presence and absence of aspartate and use molecular dynamics simulations and binding assays to show how strict coupling between the binding of three sodium ions and aspartate takes place.

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue Glt Tk , a Na + - L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na + ions. These structures explain how substrate-leakage is prevented – a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport. Glutamate transporters are membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft via a so-called elevator mechanism. Here the authors present five cryo-EM structures of the transporter homologue Glt Tk , which explain how substrate leakage is prevented.

Archaea thrive in extreme environments, exhibiting unique traits with significant biotechnological potential. In this study, we investigated whether Thermococcus onnurineus NA1 could stably integrate a large glycoside hydrolase (GH) gene cluster from T. pacificus P-4, enhancing β-linked polysaccharides degradation for hydrogen production. Among 35 Thermococcus genomes examined via OrthoFinder2 and OrthoVenn3, and selecting Tpa-GH gene clusters as the target, we cloned and integrated Tpa-GH into T. onnurineus NA1 using a fosmid-based system, creating the GH03 mutant. Cultivation in a modified MM1 medium supplemented with laminarin revealed significantly higher growth and hydrogen production in T. onnurineus GH03 than in the wild-type strain. Our findings demonstrate the feasibility of stable, large-fragment DNA integration in hyperthermophilic archaea and underscore the promise of T. onnurineus GH03 as a strain for high-temperature biomass conversion.

Branched-chain polyamines (BCPAs), exemplified by N ⁴-bis(aminopropyl)spermidine, are distinctive polycations that occur predominantly in thermophilic bacteria and euryarchaeal archaea. Their dedicated aminopropyltransferase, BpsA (EC 2.5.1.128), extends spermidine into branched architectures via sequential decarboxylated S -adenosylmethionine (dcSAM)-dependent reactions. Accumulated evidence demonstrates that BCPAs engage nucleic acids with substantially higher affinity than linear polyamines such as spermidine, and they uniquely induce strong DNA compaction accompanied by B→A→C structural transitions. These interactions greatly enhance the resistance of DNA to thermal, chemical, and physical damage. Genetic and physiological analyses in Thermococcus kodakarensis further show that loss of BCPA biosynthesis compromises growth at very high temperatures, disrupts temperature- and membrane-associated stress responses, and alters transcriptional and translational regulation; intriguingly, the linear tetraamine thermospermine can partially substitute for BCPA in several of these functions. Beyond cellular physiology, immobilized BCPAs enable sensitive nucleic-acid capture and direct PCR and isothermal DNA amplification from highly dilute solutions, demonstrating their potential utility in molecular diagnostics and environmental DNA workflows. This review synthesizes current knowledge of BCPA distribution, biosynthesis, structure–function relationships, cellular roles, and emerging biotechnological applications, and highlights key open questions in the field.

To understand the specific function of the HerA-NurA complex, which is believed to function in the end resection process to create a 3′-overhanging DNA for the following strand invasion in homologous recombination, we performed biochemical and structural analyses of this complex from a hyperthermophilic archaeon, Thermococcus kodakarensis , inhabiting a harsh environment where DNA is easily damaged. We found that the HerA-NurA complex cleaves both strands of double-stranded DNA in an exonucleolytic manner, regardless of the structure of the DNA end. Our structural analysis revealed the detailed characteristics of the nuclease activity exhibited by the HerA-NurA complex. Based on the presented information, it is unlikely that the HerA-NurA complex directly functions in end resection, but rather is involved in other functions, possibly in defense against viral infections.

In eukaryotes, structural maintenance of chromosomes (SMC) complexes form topologically associating domains (TADs) by extruding DNA loops and being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using high-resolution chromosome conformation capture sequencing, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We find that TrmBL2, a nucleoid-associated protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif . TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with structural properties of underlying DNA sequences. Our study illuminates a eukaryotic-like mechanism of domain formation in archaea and a role of intrinsic DNA structure in large-scale genome organization. Eukaryotic chromosomes are organized into arrays of compact domain structures called TADs. Here the authors show that a member of the Archaea, the prokaryotic domain closest to the Eukarya, uses a eukaryotic-like mechanism of chromosomal domain formation.

DNA polymerase substrate specificity is fundamental to genome integrity and to polymerase applications in biotechnology. In the current paradigm, active site geometry is the main site of specificity control. Here, we describe the discovery of a distinct specificity checkpoint located over 25 Å from the active site in the polymerase thumb subdomain. In Tgo, the replicative DNA polymerase from Thermococcus gorgonarius, we identify a single mutation (E664K) within this region that enables translesion synthesis across a template abasic site or a cyclobutane thymidine dimer. In conjunction with a classic \"steric-gate\" mutation (Y409G) in the active site, E664K transforms Tgo DNA polymerase into an RNA polymerase capable of synthesizing RNAs up to 1.7 kb long as well as fully pseudouridine-, 5-methyl-C–, 2'-fluoro–, or 2'-azido–modified RNAs primed from a wide range of primer chemistries comprising DNA, RNA, locked nucleic acid (LNA), or 2'O-methyl–DNA. We find that E664K enables RNA synthesis by selectively increasing polymerase affinity for the noncognate RNA/DNA duplex as well as lowering the Km for ribonucleotide triphosphate incorporation. This gatekeeper mutation therefore identifies a key missing step in the adaptive path from DNA to RNA polymerases and defines a previously unknown postsynthetic determinant of polymerase substrate specificity with implications for the synthesis and replication of noncognate nucleic acid polymers.

Sulfur 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.