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Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This 'microbial dark matter' has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order (‘ Candidatus Altiarchaeales’). The representative, double-membraned species ‘ Candidatus Altiarchaeum hamiconexum’ has an autotrophic metabolism that uses a not-yet-reported Factor 420 -free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nano-grappling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink. Research on microbes that inhabit the Earth's subsurface is mostly based on metagenomic information only. Here, Probst et al . combine metagenomics with ultrastructural and functional analyses to study the biology of a group of uncultivated subsurface archaea, the SM1 Euryarchaeon lineage.

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins. The electron cryo-microscopy structure of Sulfolobus islandicus pili enabled the identification of SiL_2606 as the main pilin in these filaments and revealed that the pili are glycosylated, which probably explains how these structures remain soluble and stable even when cells grow at pH 3 and 80 °C.

The great majority of microbial species remains uncultured, severely limiting their taxonomic characterization and thus communication among scientists. Although Candidatus was devised as a provisional category to classify uncultured taxa, it has not been widely accepted owing to technical limitations and lack of priority of Candidatus names in the official nomenclature. High-throughput sequencing provides the potential for data-rich taxonomic descriptions of uncultivated microbes, comparable in quality to those of cultured organisms. In order to fully realize this potential, standards and guidelines on how to perform these descriptions are needed. Here we aimed to outline these standards and draw the roadmap for a new genome-based taxonomy that, at least initially, would be parallel but highly convergent to the one in existence for isolates. In particular, we recommend the use of DNA genome sequences, recovered by population binning or single-cell techniques, as the basis for (i) identification and phylogenetic placement, (ii) bioinformatics-based functional and thus phenotypic predictions, as well as (iii) type material. We also recommend the implementation of an independent nomenclatural system for uncultivated taxa, following the same nomenclature rules as those for cultured Bacteria and Archaea but with its own list of validly published names. If widely adopted, this system will not only facilitate a comprehensive characterization of the ‘uncultivated majority’, but also provide a unified catalogue of validly published names, thereby avoiding synonyms and confusion. We also suggest that a committee of experts, supported by an international microbiological society, should be formed to govern the new classification system.

N 4 -acetylcytidine (ac 4 C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA 1 – 3 . However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac 4 C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac 4 C at single-nucleotide resolution. In human and yeast mRNAs, ac 4 C sites are not detected but can be induced—at a conserved sequence motif—via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac 4 C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac 4 C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac 4 C and its potential thermoadaptive role. Our studies quantitatively define the ac 4 C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease 4 – 6 . A method termed ac 4 C-seq is introduced for the transcriptome-wide mapping of the RNA modification N 4 -acetylcytidine, revealing widespread temperature-dependent acetylation that facilitates thermoadaptation in hyperthermophilic archaea.

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell 1 – 3 . A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated 4 – 6 , but never visualized. Here we describe a highly enriched culture of ‘ Candidatus Lokiarchaeum ossiferum’, a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7–14 days, reaches cell densities of up to 5 × 10 7 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain 7 . ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying ‘ Ca . L. ossiferum’ cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin—one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures. Culture and analysis of ‘ Candidatus  Lokiarchaeum ossiferum’—a member of the Asgard phylum—reveals an elaborate cell architecture with extensive membranous protrusions.

Despite the surge of microbial genome data, experimental testing is important to confirm inferences about the cell biology, ecological roles and evolution of microorganisms. As the majority of archaeal and bacterial diversity remains uncultured and poorly characterized, culturing is a priority. The growing interest in and need for efficient cultivation strategies has led to many rapid methodological and technological advances. In this Review, we discuss common barriers that can hamper the isolation and culturing of novel microorganisms and review emerging, innovative methods for targeted or high-throughput cultivation. We also highlight recent examples of successful cultivation of novel archaea and bacteria, and suggest key microorganisms for future cultivation attempts.Culturing microorganisms is a priority to complement the flood of genomic data illuminating archaeal and bacterial diversity. In this Review, Ettema and colleagues highlight recent successes in culturing elusive lineages, innovative methods and key targets for future cultivation.

Compared to bacteria, our knowledge of archaeal biology is limited. Historically, microbiologists have mostly relied on culturing and single-gene diversity surveys to understand Archaea in nature. However, only six of the 27 currently proposed archaeal phyla have cultured representatives. Advances in genomic sequencing and computational approaches are revolutionizing our understanding of Archaea. The recovery of genomes belonging to uncultured groups from the environment has resulted in the description of several new phyla, many of which are globally distributed and are among the predominant organisms on the planet. In this Review, we discuss how these genomes, together with long-term enrichment studies and elegant in situ measurements, are providing insights into the metabolic capabilities of the Archaea. We also debate how such studies reveal how important Archaea are in mediating an array of ecological processes, including global carbon and nutrient cycles, and how this increase in archaeal diversity has expanded our view of the tree of life and early archaeal evolution, and has provided new insights into the origin of eukaryotes. Recent advances in genome sequencing have started to reveal an increasing diversity and distribution of archaeal genomes across multiple ecosystems. In this Review, Baker and colleagues discuss how these genomes elucidate the metabolic capabilities of the Archaea and their ecological roles, while also expanding our view of the tree of life and of eukaryogenesis.

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H 2 ) is a niche process. To gain a broader insight into the importance of microbial H 2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H 2 . The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H 2 -based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H 2 -sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content ( p O 2 ) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H 2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H 2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H 2 metabolism.

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH 4 + -N per gram of soil) and high (200 μg NH 4 + -N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.

Nitrification is a key process of the nitrogen (N) cycle in soil with major environmental implications. The recent discovery of ammonia-oxidizing archaea (AOA) questions the traditional assumption of the dominant role of ammonia-oxidizing bacteria (AOB) in nitrification. We investigated AOB and AOA growth and nitrification rate in two different layers of three grassland soils treated with animal urine substrate and a nitrification inhibitor [dicyandiamide (DCD)]. We show that AOB were more abundant in the topsoils than in the subsoils, whereas AOA were more abundant in one of the subsoils. AOB grew substantially when supplied with a high dose of urine substrate, whereas AOA only grew in the Controls without the urine-N substrate. AOB growth and the amoA gene transcription activity were significantly inhibited by DCD. Nitrification rates were much higher in the topsoils than in the subsoils and were significantly related to AOB abundance, but not to AOA abundance. These results suggest that AOB and AOA prefer different soil N conditions to grow: AOB under high ammonia (NH₃) substrate and AOA under low NH₃ substrate conditions.