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Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota., (© 2022. The Author(s).)

Thermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H 2 by fermenting carbohydrates. High concentration of H 2 inhibits the growth of T. maritima, and S 0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S 0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx , yeeE , and rnf genes when the strain grew in S 0 - or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments. IMPORTANCE The reduction of S 0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S 0 , polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S 0 - or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance., (© 2022. The Author(s).)

For more than 40 years, marine microorganisms have raised great interest because of their major ecological function and their numerous applications for biotechnology and pharmacology. Particularly, Archaea represent a resource of great potential for the identification of new metabolites because of their adaptation to extreme environmental conditions and their original metabolic pathways, allowing the synthesis of unique biomolecules. Studies on archaeal carotenoids are still relatively scarce and only a few works have focused on their industrial scale production and their biotechnological and pharmacological properties, while the societal demand for these bioactive pigments is growing. This article aims to provide a comprehensive review of the current knowledge on carotenoid metabolism in Archaea and the potential applications of these pigments in biotechnology and medicine. After reviewing the ecology and classification of these microorganisms, as well as their unique cellular and biochemical characteristics, this paper highlights the most recent data concerning carotenoid metabolism in Archaea, the biological properties of these pigments, and biotechnological considerations for their production at industrial scale.

Archaeal glycerophospholipids are the main constituents of the cytoplasmic membrane in the archaeal domain of life and fundamentally differ in chemical composition compared to bacterial phospholipids. They consist of isoprenyl chains ether-bonded to glycerol-1-phosphate. In contrast, bacterial glycerophospholipids are composed of fatty acyl chains ester-bonded to glycerol-3-phosphate. This largely domain-distinguishing feature has been termed the "lipid-divide". The chemical composition of archaeal membranes contributes to the ability of archaea to survive and thrive in extreme environments. However, ether-bonded glycerophospholipids are not only limited to extremophiles and found also in mesophilic archaea. Resolving the structural basis of glycerophospholipid biosynthesis is a key objective to provide insights in the early evolution of membrane formation and to deepen our understanding of the molecular basis of extremophilicity. Many of the glycerophospholipid enzymes are either integral membrane proteins or membrane-associated, and hence are intrinsically difficult to study structurally. However, in recent years, the crystal structures of several key enzymes have been solved, while unresolved enzymatic steps in the archaeal glycerophospholipid biosynthetic pathway have been clarified providing further insights in the lipid-divide and the evolution of early life., (© 2022. The Author(s).)

Radical S -adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5'-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions. These organisms are responsible for the yearly production of nearly 1 billion tons of methane, a potent greenhouse gas as well as a valuable energy source. Thus, understanding the details of methanogenic metabolism and elucidating the functions of essential enzymes in these organisms can provide insights into strategies to decrease greenhouse gas emissions as well as inform advances in bioenergy production processes. This minireview provides an overview of the current state of the field regarding the functions of radical SAM enzymes in methanogens and discusses gaps in knowledge that should be addressed.

Microbial denitrification is a crucial biological process for the treatment of nitrogen-polluted water. Traditional denitrification process consumes external organic carbon leading to an increase in treatment costs. We developed a novel sulfide-driven denitrification methane oxidation (SDMO) system that integrates autotrophic denitrification (AD) and denitrification anaerobic methane oxidation (DAMO) for cost-effective denitrification and biogas utilization in situ. Two SDMO systems were operated for 735 days, with nitrate and nitrite serving as electron acceptors, to explore the performance of sewage denitrification and characterize metabolic mechanisms. Results showed SDMO system could reach as high as 100% efficiency of nitrogen removal and biogas desulfurization without an external carbon source when HRT was 10 days and inflow nitrogen concentrations were 50-100 mgN·L -1 . Besides, nitrate was a preferable electron acceptor for SDMO system. Biogas not only enhanced nitrogen removal but also intensified the DAMO, nitrogen removed through DAMO contribution doubled as original period from 2.9 mgN·(L·d) -1 to 6.2 mgN·(L·d) -1 , and the ratio of nitrate removal through AD to DAMO was 1.2:1 with nitrate as electron acceptor. While nitrogen removed almost all through AD contribution and DAMO was weaken as before, the ratio of nitrate removal through AD to DAMO was 21.2:1 with nitrite as electron acceptor. Biogas introduced into SDMO system with nitrate inspired the growth of DAMO bacteria Candidatus Methylomirabilis from 0.3% to 19.6% and motivated its potentiality to remove nitrate without ANME archaea participation accompanying with gene mfnE upregulating ∼100 times. According to the reconstructed genome from binning analysis, the dramatically upregulated gene mfnE was derived from Candidatus Methylomirabilis, which may represent a novel metabolism pathway for DAMO bacteria to replace the role of archaea for nitrate reduction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microbial methane oxidation is the major biological methane (CH 4 ) sink in the carbon cycle. Methanotrophs can use various electron acceptors in addition to oxygen; understanding the role and contribution of methanotrophs is thus an important topic. However, anaerobic oxidation of methane (AOM) mediated by methanotrophs is poorly explored and understood. This article summarizes the role aerobic methanotrophic bacteria play in AOM. Though AOM was originally considered to be mediated by anaerobic methanotrophic archaea, intra-aerobic methane-oxidizing bacteria (Candidatus Methylomirabilis oxyfera) appear to be involved in nitrite-dependent AOM. In addition, aerobic methanotrophs of the Methylomonadaceae and Methylocystaceae, are more versatile than previously assumed and can also be involved in nitrate/nitrite- or mineral oxide-dependent AOM under oxygen-limitation. Furthermore, the simultaneous reduction of nitrous oxide and oxidation of CH 4 may be another new metabolic trait of aerobic methanotrophs. We discuss the potential metabolic pathways of CH 4 oxidation under hypoxic conditions. It is of great ecological importance not only for the quantification of CH 4 oxidation and emissions, but also for the definition of a new function of aerobic methanotrophs in anaerobic/hypoxic environments., 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 © 2022 Elsevier B.V. All rights reserved.)

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo., (© 2022. The Author(s).)

DNA gyrase is a type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. In bacteria, DNA gyrase has an essential role in the homeostatic regulation of supercoiling. While ubiquitous in bacteria, DNA gyrase was previously reported to have a patchy distribution in Archaea but its emergent function and evolutionary history in this domain of life remains elusive. In this study, we used phylogenomic approaches and an up-to date sequence dataset to establish global and archaea-specific phylogenies of DNA gyrases. The most parsimonious evolutionary scenario infers that DNA gyrase was introduced into the lineage leading to Euryarchaeal group II via a single horizontal gene transfer from a bacterial donor which we identified as an ancestor of Gracilicutes and/or Terrabacteria. The archaea-focused trees indicate that DNA gyrase spread from Euryarchaeal group II to some DPANN and Asgard lineages via rare horizontal gene transfers. The analysis of successful recent transfers suggests a requirement for syntropic or symbiotic/parasitic relationship between donor and recipient organisms. We further show that the ubiquitous archaeal Topoisomerase VI may have co-evolved with DNA gyrase to allow the division of labor in the management of topological constraints. Collectively, our study reveals the evolutionary history of DNA gyrase in Archaea and provides testable hypotheses to understand the prerequisites for successful establishment of DNA gyrase in a naive archaeon and the associated adaptations in the management of topological constraints., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

In high-load anaerobic digestion such as in kitchen waste, side-stream micro-aeration (SMA) shows excellent operational performance to direct micro-aeration (DMA). It immediately restores the acidification to stability. Methanogenic performance remained stable when organic load ratios (OLR) was further increased to 5.5 g VS/L. Enhanced enzyme activity, microbial aggregation, and proliferation of bacteria and archaea were observed in SMA. The results indicates that SMA enriched Methanosaeta (relative abundance exceeded 93%) and induced the change of the main methanogenic pathway to acetoclastic methanogenesis. Mechanisms was further explored by using metagenomic analysis, and the results show SMA avoids mass formation of ROS (reactive oxygen species) by cycling the aerated slurry, and retains benefits of trace O 2 on material and energic metabolism, which poses great application potentials and deserves further investigation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This review discusses the history of discovery and study of the operation of the two rotary ion-translocating ATPase nano-motors: (i) F-ATPase/synthase (holocomplex F 1 F O ) of mitochondria/bacteria and (ii) eukaryotic V-ATPase (holocomplex V 1 V O ). Vacuolar adenosine triphosphatase (V-ATPase) is a transmembrane multisubunit complex found in all eukaryotes from yeast to humans. It is structurally and functionally similar to the F-ATPase/synthase of mitochondria/bacteria and the A-ATPase/synthase of archaebacteria, which indicates a common evolutionary origin of the rotary ion-translocating nano-motors built into cell membranes and invented by Nature billions of years ago. Previously we have published several reviews on this topic with appropriate citations of our original research. This review is focused on the historical analysis of the discovery and study of transmembrane rotary ion-translocating ATPase nano-motors functioning in bacteria, eukaryotic cells and mitochondria of animals.

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH 4 ) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by 'Candidatus Methanoperedens nitroreducens' in a CH 4 -fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH 4 directly. In the absence of CH 4 , current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH 4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of 'Ca. M. nitroreducens' to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems., (Copyright © 2022. Published by Elsevier Ltd.)

Studies on the structure-function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a 'β-barrel' fold, formed by the double-stranded antiparallel β-sheet 1 tightly packing with the triple-stranded antiparallel β-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the β-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the β-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 °C), as compared with the wild-type Cren7 (Tm > 80 °C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins., (Copyright © 2022. Published by Elsevier B.V.)

Live-cell fluorescence imaging of methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization analysis, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F 420 -reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate-containing medium, consistent with previous studies suggesting the increased abundance of these proteins in the absence of H 2 . Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed a membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea. IMPORTANCE Methanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. The application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.

The Terrestrial Miscellaneous Euryarchaeota Group has been identified in various environments, and the single genome investigated thus far suggests that these archaea are anaerobic sulfite reducers. We assemble 35 new genomes from this group that, based on genome analysis, appear to possess aerobic and facultative anaerobic lifestyles and may oxidise rather than reduce sulfite. We propose naming this order (representing 16 genera) "Lutacidiplasmatales" due to their occurrence in various acidic environments and placement within the phylum Thermoplasmatota. Phylum-level analysis reveals that Thermoplasmatota evolution had been punctuated by several periods of high levels of novel gene family acquisition. Several essential metabolisms, such as aerobic respiration and acid tolerance, were likely acquired independently by divergent lineages through convergent evolution rather than inherited from a common ancestor. Ultimately, this study describes the terrestrially prevalent Lutacidiciplasmatales and highlights convergent evolution as an important driving force in the evolution of archaeal lineages., (© 2022. The Author(s).)

In the Anthropocene, nitrogen pollution is becoming an increasing challenge for both mankind and the Earth system. Microbial nitrogen cycling begins with aerobic nitrification, which is also the key rate-limiting step. For over a century, it has been accepted that nitrification occurs sequentially involving ammonia oxidation, which produces nitrite followed by nitrite oxidation, generating nitrate. This perception was changed by the discovery of comammox Nitrospira bacteria and their metabolic pathway. In addition, this also provided us with new knowledge concerning the complex nitrogen cycle network. In the comammox process, ammonia can be completely oxidized to nitrate in one cell via the subsequent activity of the enzyme complexes, ammonia monooxygenase, hydroxylamine dehydrogenase, and nitrite oxidodreductase. Over the past five years, research on comammox made great progress. However, there still exist a lot of questions, including how much does comammox contribute to nitrification? How large is the diversity and are there new strains to be discovered? Do comammox bacteria produce the greenhouse gas N 2 O, and how or to which extent may they contribute to global climate change? The above four aspects are of great significance on the farmland nitrogen management, aquatic environment restoration, and mitigation of global climate change. As large number of comammox bacteria and pathways have been detected in various terrestrial and aquatic ecosystems, indicating that the comammox process may exert an important role in the global nitrogen cycle., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Small regulatory RNAs (sRNAs) are present in almost all investigated microbes, regarded as modulators and regulators of gene expression and also known to play their regulatory role in the environmentally significant process. It has been estimated that less than 1% of the microbes in nature are culturable in the laboratory, hindering our understanding of their physiology, and living strategies. However, recent big advancing of DNA sequencing and omics-related data analysis makes the understanding of the genetics, metabolic potentials, even ecological roles of uncultivated microbes possible. In this study, we used a metagenome and metatranscriptome-based integrated approach to identify small RNAs in the microbiome of Guaymas Basin sediments. Hundreds of environmental sRNAs comprising 228 groups were identified based on their homology, 82% of which displayed high similarity with previously known small RNAs in Rfam database, whereas, "18%" are putative novel sRNA motifs. A putative cis-acting sRNA potentially binding to methyl coenzyme M reductase, a key enzyme in methanogenesis or anaerobic oxidation of methane (AOM), was discovered in the genome of ANaerobic MEthane oxidizing archaea group 1 (ANME-1), which were the dominate microbe in the sample. These sRNAs were actively expressed in local Guaymas Basin hydrothermal environment, suggesting important roles of sRNAs in regulating microbial activity in natural environments., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Compared with the extensive knowledge on cell division in model eukaryotes and bacteria, little is known about how archaea divide. Interestingly, both endosomal sorting complex required for transport (ESCRT)-based and FtsZ-based cell division systems are found in members of the Archaea. In the past couple of years, several studies have started to shed light on FtsZ-based cell division processes in members of the Euryarchaeota. In this review we highlight recent findings in this emerging field of research. We present current knowledge of the cell division machinery of halophiles which relies on two FtsZ proteins, and we compare it with that of methanobacteria, which relies on only one FtsZ. Finally, we discuss how these differences relate to the distinct cell envelopes of these two archaeal model systems., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This article describes a study of frozen volcanic deposits collected from volcanoes Tolbachik and Bezymianny on the Kamchatka Peninsula, Russia, and Deception Island volcano, Antarctica. In addition, we studied suprasnow ash layers deposited after the 2007 eruptions of volcanoes Shiveluch and Bezymianny on Kamchatka. The main objectives were to characterize the presence and survivability of thermophilic microorganisms in perennially frozen volcanic deposits. As opposed to permafrost from the polar regions, viable thermophiles were detected in volcanic permafrost by cultivation, microscopy, and sequencing. In the permafrost of Tolbachik volcano, we observed methane formation by both psychrophilic and thermophilic methanogenic archaea, while at 37°C, methane production was noticeably lower. Thermophilic bacteria isolated from volcanic permafrost from the Deception Island were 99.93% related to Geobacillus stearothermophilus . Our data showed biological sulfur reduction to sulfide at 85°C and even at 130°C, where hyperthermophilic archaea of the genus Thermoproteus were registered. Sequences of hyperthermophilic bacteria of the genus Caldicellulosiruptor were discovered in clone libraries from fresh volcanic ash deposited on snow. Microorganisms found in volcanic terrestrial permafrost may serve as a model for the alien inhabitants of Mars, a cryogenic planet with numerous volcanoes. Thermophiles and hyperthermophiles and their metabolic processes represent a guideline for the future exploration missions on Mars.

Nitrosopumilus maritimus is a marine ammonia-oxidizing archaeon with a high affinity for ammonia. It fixes carbon via a modified hydroxypropionate/hydroxybutyrate cycle and shows weak utilization of cyanate as a supplementary energy and nitrogen source. When oxygen is depleted, N. maritimus produces its own oxygen, which may explain its regular occurrence in anoxic waters. Several enzymes of the ammonia oxidation and oxygen production pathways remain to be identified.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Asgardarchaeota harbour many eukaryotic signature proteins and are widely considered to represent the closest archaeal relatives of eukaryotes. Whether similarities between Asgard archaea and eukaryotes extend to their viromes remains unknown. Here we present 20 metagenome-assembled genomes of Asgardarchaeota from deep-sea sediments of the basin off the Shimokita Peninsula, Japan. By combining a CRISPR spacer search of metagenomic sequences with phylogenomic analysis, we identify three family-level groups of viruses associated with Asgard archaea. The first group, verdandiviruses, includes tailed viruses of the class Caudoviricetes (realm Duplodnaviria); the second, skuldviruses, consists of viruses with predicted icosahedral capsids of the realm Varidnaviria; and the third group, wyrdviruses, is related to spindle-shaped viruses previously identified in other archaea. More than 90% of the proteins encoded by these viruses of Asgard archaea show no sequence similarity to proteins encoded by other known viruses. Nevertheless, all three proposed families consist of viruses typical of prokaryotes, providing no indication of specific evolutionary relationships between viruses infecting Asgard archaea and eukaryotes. Verdandiviruses and skuldviruses are likely to be lytic, whereas wyrdviruses potentially establish chronic infection and are released without host cell lysis. All three groups of viruses are predicted to play important roles in controlling Asgard archaea populations in deep-sea ecosystems., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Asgard archaea are globally distributed prokaryotic microorganisms related to eukaryotes; however, viruses that infect these organisms have not been described. Here, using metagenome sequences recovered from deep-sea hydrothermal sediments, we characterize six relatively large (up to 117 kb) double-stranded DNA (dsDNA) viral genomes that infected two Asgard archaeal phyla, Lokiarchaeota and Helarchaeota. These viruses encode Caudovirales-like structural proteins, as well as proteins distinct from those described in known archaeal viruses. Their genomes contain around 1-5% of genes associated with eukaryotic nucleocytoplasmic large DNA viruses (NCLDVs) and appear to be capable of semi-autonomous genome replication, repair, epigenetic modifications and transcriptional regulation. Moreover, Helarchaeota viruses may hijack host ubiquitin systems similar to eukaryotic viruses. Genomic analysis of these Asgard viruses reveals that they contain features of both prokaryotic and eukaryotic viruses, and provides insights into their potential infection and host interaction mechanisms., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Stored topsoil acts as a microbial inoculant for ecological restoration of land after disturbance, but the altered circumstances frequently create unfavourable conditions for microbial survival. Nitrogen cycling is a critical indicator for ecological success and this study aimed to investigate the cornerstone taxa driving the process. Previous in silico studies investigating stored topsoil discovered persistent archaeal taxa with the potential for re-establishing ecological activity. Ammonia oxidization is the limiting step in nitrification and as such, ammonia-oxidizing archaea (AOA) can be considered one of the gatekeepers for the re-establishment of the nitrogen cycle in disturbed soils. Semi-arid soil samples were enriched with ammonium sulfate to promote the selective enrichment of ammonia oxidizers for targeted genomic recovery, and to investigate the microbial response of the microcosm to nitrogen input. Ammonia addition produced an increase in AOA population, particularly within the genus Candidatus Nitrosotalea, from which metagenome-assembled genomes (MAGs) were successfully recovered. The Ca. Nitrosotalea archaeon candidates' ability to survive in extreme conditions and rapidly respond to ammonia input makes it a potential bioprospecting target for application in ecological restoration of semi-arid soils and the recovered MAGs provide a metabolic blueprint for developing potential strategies towards isolation of these acclimated candidates., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Studies have shown that dissolved organic matters (DOMs) may affect soil nutrient availability to plants due to their effect on microbial communities; however, the relationships of soil DOM-bacterial community-N function in response to root exudates remains poorly understand. Here, we evaluated the DOM composition, bacterial taxonomic variation and nitrogen transformation rates in both acidic and alkaline soils, with or without the typical nitrate preference plant (wheat, Triticum aestivum L.). After 30 days' cultivation, DOM compositions such as sugars, amines, amino acids, organic acid, and ketone were significantly increased in soil with wheat vs. bare soil, and these compounds were mainly involved in nitrogen metabolism pathways. Soil core bacterial abundance was changed while bacterial community diversity decreased in response to wheat planting. Function prediction analysis based on FAPROTAX software showed that the bacterial community were significantly (p < 0.05) affiliated with nitrification and organic compound degradation. Additionally, db-RDA and VPA analysis suggested that the contribution of soil DOM to the variance of bacterial community was stronger than that of soil available nutrients. Furthermore, the N-transformation related bacteria like Burkholderiales and ammonia-oxidizing bacteria (AOB) were positively correlated with soil gross nitrification rate, confirming that the soil N transformation was enhanced in both acidic and alkaline soils. Our results provide insight into how soil DOM affects the community structure and function of bacteria to regulate the process of nitrogen transformation in plant-soil system., Competing Interests: Declaration of competing interest The author(s) 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 © 2022 Elsevier B.V. All rights reserved.)

Lost Hammer Spring, located in the High Arctic of Nunavut, Canada, is one of the coldest and saltiest terrestrial springs discovered to date. It perennially discharges anoxic (<1 ppm dissolved oxygen), sub-zero (~-5 °C), and hypersaline (~24% salinity) brines from the subsurface through up to 600 m of permafrost. The sediment is sulfate-rich (1 M) and continually emits gases composed primarily of methane (~50%), making Lost Hammer the coldest known terrestrial methane seep and an analog to extraterrestrial habits on Mars, Europa, and Enceladus. A multi-omics approach utilizing metagenome, metatranscriptome, and single-amplified genome sequencing revealed a rare surface terrestrial habitat supporting a predominantly lithoautotrophic active microbial community driven in part by sulfide-oxidizing Gammaproteobacteria scavenging trace oxygen. Genomes from active anaerobic methane-oxidizing archaea (ANME-1) showed evidence of putative metabolic flexibility and hypersaline and cold adaptations. Evidence of anaerobic heterotrophic and fermentative lifestyles were found in candidate phyla DPANN archaea and CG03 bacteria genomes. Our results demonstrate Mars-relevant metabolisms including sulfide oxidation, sulfate reduction, anaerobic oxidation of methane, and oxidation of trace gases (H 2 , CO 2 ) detected under anoxic, hypersaline, and sub-zero ambient conditions, providing evidence that similar extant microbial life could potentially survive in similar habitats on Mars., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Trace metals have been an important ingredient for life throughout Earth's history. Here, we describe the genome-guided cultivation of a member of the elusive archaeal lineage Caldarchaeales (syn. Aigarchaeota), Wolframiiraptor gerlachensis, and its growth dependence on tungsten. A metagenome-assembled genome (MAG) of W. gerlachensis encodes putative tungsten membrane transport systems, as well as pathways for anaerobic oxidation of sugars probably mediated by tungsten-dependent ferredoxin oxidoreductases that are expressed during growth. Catalyzed reporter deposition-fluorescence in-situ hybridization (CARD-FISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) show that W. gerlachensis preferentially assimilates xylose. Phylogenetic analyses of 78 high-quality Wolframiiraptoraceae MAGs from terrestrial and marine hydrothermal systems suggest that tungsten-associated enzymes were present in the last common ancestor of extant Wolframiiraptoraceae. Our observations imply a crucial role for tungsten-dependent metabolism in the origin and evolution of this lineage, and hint at a relic metabolic dependence on this trace metal in early anaerobic thermophiles., (© 2022. The Author(s).)

Selenium (Se) is an important trace element that mainly occurs in the form of selenocysteine in selected proteins. In prokaryotes, Se is also required for the synthesis of selenouridine and Se-containing cofactor. A large number of selenoprotein families have been identified in diverse prokaryotic organisms, most of which are thought to be involved in various redox reactions. In the last decade or two, computational prediction of selenoprotein genes and comparative genomics of Se metabolic pathways and selenoproteomes have arisen, providing new insights into the metabolism and function of Se and their evolutionary trends in bacteria and archaea. This review aims to offer an overview of recent advances in bioinformatics analysis of Se utilization in prokaryotes. We describe current computational strategies for the identification of selenoprotein genes and generate the most comprehensive list of prokaryotic selenoproteins reported to date. Furthermore, we highlight the latest research progress in comparative genomics and metagenomics of Se utilization in prokaryotes, which demonstrates the divergent and dynamic evolutionary patterns of different Se metabolic pathways, selenoprotein families, and selenoproteomes in sequenced organisms and environmental samples. Overall, bioinformatics analyses of Se utilization, function, and evolution may contribute to a systematic understanding of how this micronutrient is used in nature.

This study describes the phylogenomic analysis and metabolic insights of metagenome-assembled genomes (MAGs) retrieved from hot spring sediment samples. The metagenome-assembled sequences recovered three near-complete genomes belonging to the archaeal phylum. Analysis of genome-wide core genes and 16S rRNA-based phylogeny placed the ILS200 and ILS300 genomes within the uncultivated and largely understudied bathyarchaeal phylum, whereas ILS100 represented the phylum Thaumarchaeota . The average nucleotide identity (ANI) of the bin ILS100 was 76% with Nitrososphaeria&#95;archaeon&#95;isolate&#95;SpSt-1069. However, the bins ILS200 and ILS300 showed ANI values of 75% and 70% with Candidatus&#95;Bathyarchaeota&#95;archaeon&#95;isolate&#95;DRTY-6&#95;2&#95;bin&#95;115 and Candidatus&#95;Bathyarchaeota&#95;archaeon&#95;BA1&#95;ba1&#95;01, respectively. The genomic potential of Bathyarchaeota bins ILS200 and ILS300 showed genes necessary for the Wood-Ljungdahl pathway, and the gene encoding the methyl coenzyme M reductase ( mcr ) complex essential for methanogenesis was absent. The metabolic potential of the assembled genomes included genes involved in nitrogen assimilation, including nitrogenase and the genes necessary for the urea cycle. The presence of these genes suggested the metabolic potential of Bathyarchaeota to fix nitrogen under extreme environments. In addition, the ILS200 and ILS300 genomes carried genes involved in the tricarboxylic acid (TCA) cycle, glycolysis, and degradation of organic carbons. Finally, we conclude that the reconstructed Bathyarchaeota bins are autotrophic acetogens and organo-heterotrophs. IMPORTANCE We describe the Bathyarchaeota bins that are likely to be acetogens with a wide range of metabolic potential. These bins did not exhibit methanogenic machinery, suggesting methane production may not occur by all subgroup lineages of Bathyarchaeota . Phylogenetic analysis support that both ILS200 and ILS300 belonged to the Bathyarchaeota . The discovery of new bathyarchaeotal MAGs provides additional knowledge for understanding global carbon and nitrogen metabolism under extreme conditions.

Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth's largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) " Candidatus Nitrotoga" and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change. IMPORTANCE Microorganisms play critical roles in Earth's nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earth's largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low-nutrient waters and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy by using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals the unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers.

Subcellular compartmentalization is a defining feature of all cells. In prokaryotes, compartmentalization is generally achieved via protein-based strategies. The two main classes of microbial protein compartments are bacterial microcompartments and encapsulin nanocompartments. Encapsulins self-assemble into proteinaceous shells with diameters between 24 and 42 nm and are defined by the viral HK97-fold of their shell protein. Encapsulins have the ability to encapsulate dedicated cargo proteins, including ferritin-like proteins, peroxidases, and desulfurases. Encapsulation is mediated by targeting sequences present in all cargo proteins. Encapsulins are found in many bacterial and archaeal phyla and have been suggested to play roles in iron storage, stress resistance, sulfur metabolism, and natural product biosynthesis. Phylogenetic analyses indicate that they share a common ancestor with viral capsid proteins. Many pathogens encode encapsulins, and recent evidence suggests that they may contribute toward pathogenicity. The existing information on encapsulin structure, biochemistry, biological function, and biomedical relevance is reviewed here.

S-TGA-1 and PGP-Me are native archaeal lipids associated with the bacteriorhodopsin (bR) trimer and contribute to protein stabilization and native dynamics for proton transfer. However, little is known about the underlying molecular mechanism of how these lipids regulate bR trimerization and efficient photocycling. Here, we explored the specific binding of S-TGA-1 and PGP-Me with the bR trimer and elucidated how specific interactions modulate the bR trimeric structure and proton release and uptake using long-term atomistic molecular dynamic simulations. Our results showed that S-TGA-1 and PGP-Me are essential for stabilizing the bR trimer and maintaining the coherent conformational dynamics necessary for proton transfer. The specific binding of S-TGA-1 with W80 and K129 regulates proton release on the extracellular surface by forming a "Glu-shared" model. The interaction of PGP-Me with K40 ensures proton uptake by accommodating the conformation of the helices to recruit enough water molecules on the cytoplasmic side. The present study results could fill in the theoretical gaps of studies on the functional role of archaeal lipids and could provide a reference for other membrane proteins containing similar archaeal lipids.

Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (" Candidatus Methanocomedenaceae") and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides , Anaerolineales , and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. IMPORTANCE Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

Marine cold seeps are natural sites of methane emission and harbor distinct microbial communities capable of oxidizing methane. The majority of known cold seeps are on tectonically active continental margins, but recent discoveries have revealed abundant seeps on passive margins as well, including on the U.S. Atlantic Margin (USAM). We sampled in and around four USAM seeps and combined pore water geochemistry measurements with amplicon sequencing of 16S rRNA and mcrA (DNA and RNA) to investigate the microbial communities present, their assembly processes, and how they compare to communities at previously studied sites. We found that the USAM seeps contained communities consistent with the canonical seep microbiome at the class and order levels but differed markedly at the sequence variant level, especially within the anaerobic methanotrophic (ANME) archaea. The ANME populations were highly uneven, with just a few dominant mcrA sequence variants at each seep. Interestingly, the USAM seeps did not form a distinct phylogenetic cluster when compared with other previously described seeps around the world. Consistent with this, we found only a very weak (though statistically significant) distance-decay trend in seep community similarity across a global data set. Ecological assembly indices suggest that the USAM seep communities were assembled primarily deterministically, in contrast to the surrounding nonseep sediments, where stochastic processes dominated. Together, our results suggest that the primary driver of seep microbial community composition is local geochemistry-specifically methane, sulfide, nitrate, acetate, and ammonium concentrations-rather than the geologic context, the composition of nearby seeps, or random events of dispersal. IMPORTANCE Cold seeps are now known to be widespread features of passive continental margins, including the northern U.S. Atlantic Margin (USAM). Methane seepage is expected to intensify at these relatively shallow seeps as bottom waters warm and underlying methane hydrates dissociate. While methanotrophic microbial communities might reduce or prevent methane release, microbial communities on passive margins have rarely been characterized. In this study, we investigated the Bacteria and Archaea at four cold seeps on the northern USAM and found that despite being colocated on the same continental slope, the communities significantly differ by site at the sequence variant level, particularly methane-cycling community members. Differentiation by site was not observed in similarly spaced background sediments, raising interesting questions about the dispersal pathways of cold seep microorganisms. Understanding the genetic makeup of these discrete seafloor ecosystems and how their microbial communities develop will be increasingly important as the climate changes.

The ESCRT machinery, comprising of multiple proteins and subcomplexes, is crucial for membrane remodelling in eukaryotic cells, in processes that include ubiquitin-mediated multivesicular body formation, membrane repair, cytokinetic abscission, and virus exit from host cells. This ESCRT system appears to have simpler, ancient origins, since many archaeal species possess homologues of ESCRT-III and Vps4, the components that execute the final membrane scission reaction, where they have been shown to play roles in cytokinesis, extracellular vesicle formation and viral egress. Remarkably, metagenome assemblies of Asgard archaea, the closest known living relatives of eukaryotes, were recently shown to encode homologues of the entire cascade involved in ubiquitin-mediated membrane remodelling, including ubiquitin itself, components of the ESCRT-I and ESCRT-II subcomplexes, and ESCRT-III and Vps4. Here, we explore the phylogeny, structure, and biochemistry of Asgard homologues of the ESCRT machinery and the associated ubiquitylation system. We provide evidence for the ESCRT-I and ESCRT-II subcomplexes being involved in ubiquitin-directed recruitment of ESCRT-III, as it is in eukaryotes. Taken together, our analyses suggest a pre-eukaryotic origin for the ubiquitin-coupled ESCRT system and a likely path of ESCRT evolution via a series of gene duplication and diversification events., (© 2022. The Author(s).)

The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Deep oligotrophic lakes sustain large populations of the class Nitrososphaeria (Thaumarchaeota) in their hypolimnion. They are thought to be the key ammonia oxidizers in this habitat, but their impact on N-cycling in lakes has rarely been quantified. We followed this archaeal population in one of Europe's largest lakes, Lake Constance, for two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. An abundant (8-39% of picoplankton) and transcriptionally active archaeal ecotype dominated the nitrifying community. It represented a freshwater-specific species present in major inland water bodies, for which we propose the name "Candidatus Nitrosopumilus limneticus". Its biomass corresponded to 12% of carbon stored in phytoplankton over the year´s cycle. Ca. N. limneticus populations incorporated significantly more ammonium than most other microorganisms in the hypolimnion and were driving potential ammonia oxidation rates of 6.0 ± 0.9 nmol l ‒1 d ‒1 , corresponding to potential cell-specific rates of 0.21 ± 0.11 fmol cell -1 d -1 . At the ecosystem level, this translates to a maximum capacity of archaea-driven nitrification of 1.76 × 10 9 g N-ammonia per year or 11% of N-biomass produced annually by phytoplankton. We show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems., (© 2022. The Author(s).)

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance (≤1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13 C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34 S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface., (© 2022. The Author(s).)

Lytic polysaccharide monooxygenases could enhance the enzymatic conversion of recalcitrant polysaccharides by glycoside hydrolases. This study reports the expression and identification of a novel AA10 LPMO from Natrialbaceae archaeon, named NaLPMO10A, as a C1 oxidizer of chitin. The optimal temperature and pH for NaLPMO10A activity were 40 °C and 9.0, respectively, and NaLPMO10A exhibited high thermostability and pH stability under alkaline conditions. NaLPMO10A was also highly tolerant and stable when treated with high concentration of metal ions (1 M). Moreover, metal ions (Na + , K + , Ca 2+ and Mg 2+ ) significantly promoted NaLPMO10A activity and improved the saccharification efficiency of chitin by 22.6%, 45.9%, 36.7% and 53.9%, respectively, compared to commercial chitinase alone. Together, the findings of this study fill a gap in archaeal LPMO research, and for the first time demonstrate that archaeal NaLPMO10A could be a promising enzyme for improving saccharification under extreme condition, with potential applications in biorefineries., (Copyright © 2022. Published by Elsevier Ltd.)

Methanogenic archaea carry homologs of dissimilatory sulfite reductase (Dsr), called Dsr Like proteins (DsrLP). Dsr reduces sulfite to sulfide, a key step in an Earth's ancient metabolic process called dissimilatory sulfate reduction. The DsrLPs do not function as Dsr, and a computational approach is needed to develop hypotheses for guiding wet bench investigations on DsrLP's function. To make the computational analysis process efficient, the DsrLP amino acid sequences were transformed using only eight alphabets functionally representing twenty amino acids. The resultant reduced amino acid sequences were analyzed to identify conserved signature patterns in DsrLPs. Many of these patterns mapped on critical structural elements of Dsr and some were associated tightly with particular DsrLP groups. A search into the UniProtKB database identified several proteins carrying DsrLP's signature patterns; cysteine desulfurase, nucleosidase, and uroporphyrinogen III methylase were such matches. These outcomes provided clues to the functions of DsrLPs and highlighted the utility of the computational approach used., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Utilization of hyperthermophilic electro-active microorganisms in microbial electrolysis cells (MECs) that are used for hydrogen production from organic wastes offers significant advantages, such as increased reaction rate and enhanced degradation of insoluble materials. However, only a limited number of hyperthermophilic bioelectrochemical systems have been investigated so far. This study is the first to illustrate hydrogen production in hyperthermophilic MECs with a maximum rate of 0.57 ± 0.06 m 3 H 2 /m 3 d, where an iron reducing archaeon, Geoglobus acetivorans, was used as inoculum. In fact, this is the first study to report that G. acetivorans, as the fourth hyperthermophilic electro-active archaeon. In single chamber MECs operated at 80 °C with a set potential of 0.7 V, a peak current density of 1.53 ± 0.24 A/m 2 has been attained and this is the highest record of current produced by pure culture hyperthermophilic microorganisms. Turnover cyclic voltammetry curve illustrated a sigmoidal shape (midpoint of -0.40 V vs. Ag/AgCl), and together with linear relation of scan rate and peak anodic current, proves the biofilm attachment to the anode and its capability of direct electron transfer. Along with simple substrate (acetate), G. acetivorans effectively utilized dark fermentation effluent for hydrogen production in MECs., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The magnetite@polyaniline (Fe 3 O 4 @PANI) composites with different Fe 3 O 4 loadings were prepared, and their effect on methane production in anaerobic systems was investigated. The Fe 3 O 4 @PANI composite with a 40% loading of Fe 3 O 4 showed a better performance on accelerating methane production rate than other composites. The methane production rate was increased by 26.98% at the Fe 3 O 4 @PANI dosage of 0.6 g L -1 . The results of the contact angle and CLSM revealed that Fe 3 O 4 @PANI had a good bio-affinity and contact directly with bacteria and archaea. Then the mechanisms related to the enhancement of methane production by the composites were explored by the species annotation and enzyme activity. It showed that Fe 3 O 4 @PANI promoted the enrichment of DIET-related functional bacteria and archaea and improved the enzyme activity related to the acetoclastic methanogenic pathway., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

As a hallmark of Archaea, their cell membranes are comprised of ether lipids. However, Archaea-type ether lipids have recently been identified in Bacteria as well, with a somewhat different composition: In Bacillales, sn-glycerol 1-phosphate is etherified with one C35 isoprenoid chain, which is longer than the typical C20 chain in Archaea, and instead of a second isoprenoid chain, the product heptaprenylglyceryl phosphate becomes dephosphorylated and afterward diacetylated by the O-acetyltransferase YvoF. Interestingly, database searches have revealed YvoF homologs in Halobacteria (Archaea), too. Here, we demonstrate that YvoF from Haloferax volcanii can acetylate geranylgeranylglycerol in vitro. Additionally, we present the first-time identification of acetylated diether lipids in H. volcanii and Halobacterium salinarum by mass spectrometry. A variety of different acetylated lipids, namely acetylated archaeol, and acetylated archaetidylglycerol, were found, suggesting that halobacterial YvoF has a broad substrate range. We suppose that the acetyl group might serve to modify the polarity of the lipid headgroup, with still unknown biological effects., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Theionarchaea is a recently described archaeal class within the Euryarchaeota. While it is widely distributed in sediment ecosystems, little is known about its metabolic potential and ecological features. Here, we used metagenomics and metatranscriptomics to characterize 12 theionarchaeal metagenome-assembled genomes, which were further divided into two subgroups, from coastal mangrove sediments of China and seawater columns of the Yap Trench. Genomic analysis revealed that apart from the canonical sulfhydrogenase, Theionarchaea harbor genes encoding heliorhodopsin, group 4 [NiFe]-hydrogenase, and flagellin, in which genes for heliorhodopsin and group 4 [NiFe]-hydrogenase were transcribed in mangrove sediment. Further, the theionarchaeal substrate spectrum may be broader than previously reported as revealed by metagenomics and metatranscriptomics, and the potential carbon substrates include detrital proteins, hemicellulose, ethanol, and CO 2 . The genes for organic substrate metabolism (mainly detrital protein and amino acid metabolism genes) have relatively higher transcripts in the top sediment layers in mangrove wetlands. In addition, co-occurrence analysis suggested that the degradation of these organic compounds by Theionarchaea might be processed in syntrophy with fermenters (e.g., Chloroflexi) and methanogens. Collectively, these observations expand the current knowledge of the metabolic potential of Theionarchaea, and shed light on the metabolic strategies and roles of these archaea in the marine ecosystems., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Metagenomic analysis has facilitated prediction of a variety of carbon utilization potentials by uncultivated archaea including degradation of protein, which is a wide-spread carbon polymer in marine sediments. However, the activity of detrital catabolic protein degradation is mostly unknown for the vast majority of archaea. Here, we show actively executed protein catabolism in three archaeal phyla (uncultivated Thermoplasmata, SG8-5; Bathyarchaeota subgroup 15; Lokiarchaeota subgroup 2c) by RNA- and lipid-stable isotope probing in incubations with different marine sediments. However, highly abundant potential protein degraders Thermoprofundales (MBG-D) and Lokiarchaeota subgroup 3 were not incorporating 13 C-label from protein during incubations. Nonetheless, we found that the pathway for protein utilization was present in metagenome associated genomes (MAGs) of active and inactive archaea. This finding was supported by screening extracellular peptidases in 180 archaeal MAGs, which appeared to be widespread but not correlated to organisms actively executing this process in our incubations. Thus, our results have important implications: (i) multiple low-abundant archaeal groups are actually catabolic protein degraders; (ii) the functional role of widespread extracellular peptidases is not an optimal tool to identify protein catabolism, and (iii) catabolic degradation of sedimentary protein is not a common feature of the abundant archaeal community in temperate and permanently cold marine sediments., (© 2022. The Author(s).)

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT., (Copyright © 2022 Elsevier B.V. All rights reserved.)

cis-Prenyltransferases (cPTs) form linear polyprenyl pyrophosphates, the precursors of polyprenyl or dolichyl phosphates that are essential for cell function in all living organisms. Polyprenyl phosphate serves as a sugar carrier for peptidoglycan cell wall synthesis in bacteria, a role that dolichyl phosphate performs analogously for protein glycosylation in eukaryotes and archaea. Bacterial cPTs are characterized by their homodimeric structure, while cPTs from eukaryotes usually require two distantly homologous subunits for enzymatic activity. This study identifies the subunits of heteromeric cPT, Af1219 and Af0707, from a thermophilic sulphur-reducing archaeon, Archaeoglobus fulgidus. Both subunits are indispensable for cPT activity, and their protein-protein interactions were demonstrated by a pulldown assay. Gel filtration chromatography and chemical cross-linking experiments suggest that Af1219 and Af0707 likely form a heterotetramer complex. Although this expected subunit composition agrees with a reported heterotetrameric structure of human hCIT/NgBR cPT complex, the similarity of the quaternary structures is likely a result of convergent evolution., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum., (© 2022. The Author(s).)

Anaerobic sludge from sewage treatment was employed to derive a microbial colony that is capable of anaerobic oxidation of methane coupled with sulfate reduction and denitrification. Investigations revealed that methane can be oxidized with sulfate reduction and denitrification. When sulfate and nitrite acted as electron acceptors together, the rates and amount of methane conversion were higher than that when sulfate or nitrite alone was employed as an electron acceptor. The oxidation rate and amount of methane conversion reached 1.9 mg/(d•gVSS) and 22.24 mg, respectively. Methanotrophic bacteria, such as M. oxyfera, and Methylocystis sp., sulfate-reducing bacteria (SRB), e.g. Desulfosporosinus sp., and Desulfuromonas sp.; and denitrification bacteria, such as Hyphomicrobium sp., and Diaphorobacter sp., presented in the bacterial community. Anaerobic methanotrophic archaea (ANME), including Methanosaeta sp. and Methanobacterium sp. were found in the archaeal community. These findings indicate the coexistence of ANME, SRB and denitrification bacteria in the system. Nitrite reduction coupled with methane oxidation was performed independently by M. oxyfera during which limited oxygen generated. The oxygen released may be utilized by methanotrophic bacteria to produce organics, which could be used by denitrifying bacteria to reduce nitrite. Methanotrophic archaea could also oxidize methane to carbon dioxide or organics by reverse methanogenesis whereas sulfate was reduced to sulfide by SRB. This study opens possibility for biotechnological process of sulfate reduction and denitrification with methane as electron donor and provides a method for the synergistic treatment of wastewater containing sulfate/nitrite and waste gas containing methane., (Copyright © 2022 Elsevier Inc. All rights reserved.)