Minimal and hybrid hydrogenases are active from archaea.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

Scientists' call to action: Microbes, planetary health, and the Sustainable Development Goals.

Microorganisms, including bacteria, archaea, viruses, fungi, and protists, are essential to life on Earth and the functioning of the biosphere. Here, we discuss the key roles of microorganisms in achieving the United Nations Sustainable Development Goals (SDGs), highlighting recent and emerging advances in microbial research and technology that can facilitate our transition toward a sustainable future. Given the central role of microorganisms in the biochemical processing of elements, synthesizing new materials, supporting human health, and facilitating life in managed and natural landscapes, microbial research and technologies are directly or indirectly relevant for achieving each of the SDGs. More importantly, the ubiquitous and global role of microbes means that they present new opportunities for synergistically accelerating progress toward multiple sustainability goals. By effectively managing microbial health, we can achieve solutions that address multiple sustainability targets ranging from climate and human health to food and energy production. Emerging international policy frameworks should reflect the vital importance of microorganisms in achieving a sustainable future.

The Archaeal Cell Cycle.

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.

Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

Organelle Formation in Bacteria and Archaea.

Uncovering the mechanisms that underlie the biogenesis and maintenance of eukaryotic organelles is a vibrant and essential area of biological research. In comparison, little attention has been paid to the process of compartmentalization in bacteria and archaea. This lack of attention is in part due to the common misconception that organelles are a unique evolutionary invention of the "complex" eukaryotic cell and are absent from the "primitive" bacterial and archaeal cells. Comparisons across the tree of life are further complicated by the nebulous criteria used to designate subcellular structures as organelles. Here, with the aid of a unified definition of a membrane-bounded organelle, we present some of the recent findings in the study of lipid-bounded organelles in bacteria and archaea.

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization.

Hi-C has become a routine method for probing the 3D organization of genomes. However, when applied to prokaryotes and archaea, the current protocols are expensive and limited in their resolution. We develop a cost-effective Hi-C protocol to explore chromosome conformations of these two kingdoms at the gene or operon level. We first validate it on E. coli and V. cholera, generating sub-kilobase-resolution contact maps, and then apply it to the euryarchaeota H. volcanii, Hbt. salinarum, and T. kodakaraensis. With a resolution of up to 1 kb, we explore the diversity of chromosome folding in this phylum. In contrast to crenarchaeota, these euryarchaeota lack (active/inactive) compartment-like structures. Instead, their genomes are composed of self-interacting domains and chromatin loops. In H. volcanii, these structures are regulated by transcription and the archaeal structural maintenance of chromosomes (SMC) protein, further supporting the ubiquitous role of these processes in shaping the higher-order organization of genomes.

Diversity and Evolution of Methane-Related Pathways in Archaea.

Methane is one of the most important greenhouse gases on Earth and holds an important place in the global carbon cycle. Archaea are the only organisms that use methanogenesis to produce energy and rely on the methyl-coenzyme M reductase complex (Mcr). Over the last decade, new results have significantly reshaped our view of the diversity of methane-related pathways in the Archaea. Many new lineages that synthesize or use methane have been identified across the whole archaeal tree, leading to a greatly expanded diversity of substrates and mechanisms. In this review, we present the state of the art of these advances and how they challenge established scenarios of the origin and evolution of methanogenesis, and we discuss the potential trajectories that may have led to this strikingly wide range of metabolisms.

Horizontal Gene Transfer in Archaea-From Mechanisms to Genome Evolution.

Archaea remains the least-studied and least-characterized domain of life despite its significance not just to the ecology of our planet but also to the evolution of eukaryotes. It is therefore unsurprising that research into horizontal gene transfer (HGT) in archaea has lagged behind that of bacteria. Indeed, several archaeal lineages may owe their very existence to large-scale HGT events, and thus understanding both the molecular mechanisms and the evolutionary impact of HGT in archaea is highly important. Furthermore, some mechanisms of gene exchange, such as plasmids that transmit themselves via membrane vesicles and the formation of cytoplasmic bridges that allows transfer of both chromosomal and plasmid DNA, may be archaea-specific. This review summarizes what we know about HGT in archaea, and the barriers that restrict it, highlighting exciting recent discoveries and pointing out opportunities for future research.

Anaerobic Degradation of Alkanes by Marine Archaea.

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Euryarchaeota, using the model organism Haloferax volcanii. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.

Halofilins as emerging bactofilin families of archaeal cell shape plasticity orchestrators.

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 ΔhalA 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 ΔhalA and ΔhalB 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.

Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

A novel N4,N4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily.

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, N4,N4-dimethylcytidine (m42C), 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 m42C whose function is critical for hyperthermophilic growth. m42C 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 m42C synthase family implies that m42C is biologically relevant in each domain. Resistance of m42C to bisulfite-driven deamination suggests that efforts to capture m5C profiles via bisulfite sequencing are also capturing m42C.

Expanding Archaeal Diversity and Phylogeny: Past, Present, and Future.

The discovery of the Archaea is a major scientific hallmark of the twentieth century. Since then, important features of their cell biology, physiology, ecology, and diversity have been revealed. Over the course of some 40 years, the diversity of known archaea has expanded from 2 to about 30 phyla comprising over 20,000 species. Most of this archaeal diversity has been revealed by environmental 16S rRNA gene amplicon sequencing surveys using a broad range of universal and targeted primers. Of the few primers that target a large fraction of known archaeal diversity, all display a bias against recently discovered lineages, which limits studies aiming to survey overall archaeal diversity. Induced by genomic exploration of archaeal diversity, and improved phylogenomics approaches, archaeal taxonomic classification has been frequently revised. Due to computational limitations and continued discovery of new lineages, a stable archaeal phylogeny is not yet within reach. Obtaining phylogenetic and taxonomic consensus of archaea should be a high priority for the archaeal research community.

McrD binds asymmetrically to methyl-coenzyme M reductase improving active-site accessibility during assembly.

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane, and its activity accounts for nearly all biologically produced methane released into the atmosphere. The assembly of MCR is an intricate process involving the installation of a complex set of posttranslational modifications and the unique Ni-containing tetrapyrrole called coenzyme F430. Despite decades of research, details of MCR assembly remain largely unresolved. Here, we report the structural characterization of MCR in two intermediate states of assembly. These intermediate states lack one or both F430 cofactors and form complexes with the previously uncharacterized McrD protein. McrD is found to bind asymmetrically to MCR, displacing large regions of the alpha subunit and increasing active-site accessibility for the installation of F430-shedding light on the assembly of MCR and the role of McrD therein. This work offers crucial information for the expression of MCR in a heterologous host and provides targets for the design of MCR inhibitors.

Respiration-driven methanotrophic growth of diverse marine methanogens.

Anaerobic marine environments are the third largest producer of the greenhouse gas methane. The release to the atmosphere is prevented by anaerobic 'methanotrophic archaea (ANME) dependent on a symbiotic association with sulfate-reducing bacteria or direct reduction of metal oxides. Metagenomic analyses of ANME are consistent with a reverse methanogenesis pathway, although no wild-type isolates have been available for validation and biochemical investigation. Herein is reported the characterization of methanotrophic growth for the diverse marine methanogens Methanosarcina acetivorans C2A and Methanococcoides orientis sp. nov. Growth was dependent on reduction of either ferrihydrite or humic acids revealing a respiratory mode of energy conservation. Acetate and/or formate were end products. Reversal of the well-characterized methanogenic pathways is remarkably like the consensus pathways for uncultured ANME based on extensive metagenomic analyses.

Pathways of N2O production by marine ammonia-oxidizing archaea determined from dual-isotope labeling.

The ocean is a net source of the greenhouse gas and ozone-depleting substance, nitrous oxide (N2O), to the atmosphere. Most of that N2O is produced as a trace side product during ammonia oxidation, primarily by ammonia-oxidizing archaea (AOA), which numerically dominate the ammonia-oxidizing community in most marine environments. The pathways to N2O production and their kinetics, however, are not completely understood. Here, we use 15N and 18O isotopes to determine the kinetics of N2O production and trace the source of nitrogen (N) and oxygen (O) atoms in N2O produced by a model marine AOA species, Nitrosopumilus maritimus. We find that during ammonia oxidation, the apparent half saturation constants of nitrite and N2O production are comparable, suggesting that both processes are enzymatically controlled and tightly coupled at low ammonia concentrations. The constituent atoms in N2O are derived from ammonia, nitrite, O2, and H2O via multiple pathways. Ammonia is the primary source of N atoms in N2O, but its contribution varies with ammonia to nitrite ratio. The ratio of 45N2O to 46N2O (i.e., single or double labeled N) varies with substrate ratio, leading to widely varying isotopic signatures in the N2O pool. O2 is the primary source for O atoms. In addition to the previously demonstrated hybrid formation pathway, we found a substantial contribution by hydroxylamine oxidation, while nitrite reduction is an insignificant source of N2O. Our study highlights the power of dual 15N-18O isotope labeling to disentangle N2O production pathways in microbes, with implications for interpretation of pathways and regulation of marine N2O sources.