Chemotaxis shapes the microscale organization of the ocean's microbiome.

The capacity of planktonic marine microorganisms to actively seek out and exploit microscale chemical hotspots has been widely theorized to affect ocean-basin scale biogeochemistry1-3, but has never been examined comprehensively in situ among natural microbial communities. Here, using a field-based microfluidic platform to quantify the behavioural responses of marine bacteria and archaea, we observed significant levels of chemotaxis towards microscale hotspots of phytoplankton-derived dissolved organic matter (DOM) at a coastal field site across multiple deployments, spanning several months. Microscale metagenomics revealed that a wide diversity of marine prokaryotes, spanning 27 bacterial and 2 archaeal phyla, displayed chemotaxis towards microscale patches of DOM derived from ten globally distributed phytoplankton species. The distinct DOM composition of each phytoplankton species attracted phylogenetically and functionally discrete populations of bacteria and archaea, with 54% of chemotactic prokaryotes displaying highly specific responses to the DOM derived from only one or two phytoplankton species. Prokaryotes exhibiting chemotaxis towards phytoplankton-derived compounds were significantly enriched in the capacity to transport and metabolize specific phytoplankton-derived chemicals, and displayed enrichment in functions conducive to symbiotic relationships, including genes involved in the production of siderophores, B vitamins and growth-promoting hormones. Our findings demonstrate that the swimming behaviour of natural prokaryotic assemblages is governed by specific chemical cues, which dictate important biogeochemical transformation processes and the establishment of ecological interactions that structure the base of the marine food web.

GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy.

The Genome Taxonomy Database (GTDB; https://gtdb.ecogenomic.org) provides a phylogenetically consistent and rank normalized genome-based taxonomy for prokaryotic genomes sourced from the NCBI Assembly database. GTDB R06-RS202 spans 254 090 bacterial and 4316 archaeal genomes, a 270% increase since the introduction of the GTDB in November, 2017. These genomes are organized into 45 555 bacterial and 2339 archaeal species clusters which is a 200% increase since the integration of species clusters into the GTDB in June, 2019. Here, we explore prokaryotic diversity from the perspective of the GTDB and highlight the importance of metagenome-assembled genomes in expanding available genomic representation. We also discuss improvements to the GTDB website which allow tracking of taxonomic changes, easy assessment of genome assembly quality, and identification of genomes assembled from type material or used as species representatives. Methodological updates and policy changes made since the inception of the GTDB are then described along with the procedure used to update species clusters in the GTDB. We conclude with a discussion on the use of average nucleotide identities as a pragmatic approach for delineating prokaryotic species.

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

Insights into the phylogeny and coding potential of microbial dark matter.

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called 'microbial dark matter'. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

acdc - Automated Contamination Detection and Confidence estimation for single-cell genome data.

BACKGROUND: A major obstacle in single-cell sequencing is sample contamination with foreign DNA. To guarantee clean genome assemblies and to prevent the introduction of contamination into public databases, considerable quality control efforts are put into post-sequencing analysis. Contamination screening generally relies on reference-based methods such as database alignment or marker gene search, which limits the set of detectable contaminants to organisms with closely related reference species. As genomic coverage in the tree of life is highly fragmented, there is an urgent need for a reference-free methodology for contaminant identification in sequence data. RESULTS: We present acdc, a tool specifically developed to aid the quality control process of genomic sequence data. By combining supervised and unsupervised methods, it reliably detects both known and de novo contaminants. First, 16S rRNA gene prediction and the inclusion of ultrafast exact alignment techniques allow sequence classification using existing knowledge from databases. Second, reference-free inspection is enabled by the use of state-of-the-art machine learning techniques that include fast, non-linear dimensionality reduction of oligonucleotide signatures and subsequent clustering algorithms that automatically estimate the number of clusters. The latter also enables the removal of any contaminant, yielding a clean sample. Furthermore, given the data complexity and the ill-posedness of clustering, acdc employs bootstrapping techniques to provide statistically profound confidence values. Tested on a large number of samples from diverse sequencing projects, our software is able to quickly and accurately identify contamination. Results are displayed in an interactive user interface. Acdc can be run from the web as well as a dedicated command line application, which allows easy integration into large sequencing project analysis workflows. CONCLUSIONS: Acdc can reliably detect contamination in single-cell genome data. In addition to database-driven detection, it complements existing tools by its unsupervised techniques, which allow for the detection of de novo contaminants. Our contribution has the potential to drastically reduce the amount of resources put into these processes, particularly in the context of limited availability of reference species. As single-cell genome data continues to grow rapidly, acdc adds to the toolkit of crucial quality assurance tools.

Impact of single-cell genomics and metagenomics on the emerging view of extremophile "microbial dark matter".

Despite >130 years of microbial cultivation studies, many microorganisms remain resistant to traditional cultivation approaches, including numerous candidate phyla of bacteria and archaea. Unraveling the mysteries of these candidate phyla is a grand challenge in microbiology and is especially important in habitats where they are abundant, including some extreme environments and low-energy ecosystems. Over the past decade, parallel advances in DNA amplification, DNA sequencing and computing have enabled rapid progress on this problem, particularly through metagenomics and single-cell genomics. Although each approach suffers limitations, metagenomics and single-cell genomics are particularly powerful when combined synergistically. Studies focused on extreme environments have revealed the first substantial genomic information for several candidate phyla, encompassing putative acidophiles (Parvarchaeota), halophiles (Nanohaloarchaeota), thermophiles (Acetothermia, Aigarchaeota, Atribacteria, Calescamantes, Korarchaeota, and Fervidibacteria), and piezophiles (Gracilibacteria). These data have enabled insights into the biology of these organisms, including catabolic and anabolic potential, molecular adaptations to life in extreme environments, unique genomic features such as stop codon reassignments, and predictions about cell ultrastructure. In addition, the rapid expansion of genomic coverage enabled by these studies continues to yield insights into the early diversification of microbial lineages and the relationships within and between the phyla of Bacteria and Archaea. In the next 5 years, the genomic foliage within the tree of life will continue to grow and the study of yet-uncultivated candidate phyla will firmly transition into the post-genomic era.

Putative genome contamination has minimal impact on the GTDB taxonomy.

The Genome Taxonomy Database (GTDB) provides a species to domain classification of publicly available genomes based on average nucleotide identity (ANI) (for species) and a concatenated gene phylogeny normalized by evolutionary rates (for genus to phylum), which has been widely adopted by the scientific community. Here, we use the Genome UNClutterer (GUNC) software to identify putatively contaminated genomes in GTDB release 07-RS207. We found that GUNC reported 35,723 genomes as putatively contaminated, comprising 11.25 % of the 317,542 genomes in GTDB release 07-RS207. To assess the impact of this high level of inferred contamination on the delineation of taxa, we created 'clean' versions of the 34,846 putatively contaminated bacterial genomes by removing the most contaminated half. For each clean half, we re-calculated the ANI and concatenated gene phylogeny and found that only 77 (0.22 %) of the genomes were not consistent with their original classification. We conclude that the delineation of taxa in GTDB is robust to the putative contamination detected by GUNC.

Description of Asgardarchaeum abyssi gen. nov. spec. nov., a novel species within the class Asgardarchaeia and phylum Asgardarchaeota in accordance with the SeqCode.

Asgardarchaeota, commonly referred to as Asgard archaea, is a candidatus phylum-rank archaeal clade that includes the closest archaeal relatives of eukaryotes. Despite their prevalence in the scientific literature, the name Asgardarchaeota lacks nomenclatural validation. Here, we describe a novel high-quality metagenome-assembled genome (MAG), AB3033_2TS, proposed to serve as the nomenclatural type for the species Asgardarchaeum abyssiTS according to the rules of the SeqCode. Based on protein content and compositional features, we infer that A. abyssi AB3033_2TS is an acetogenic chemoheterotroph, possibly a facultative lithoautotroph, and is adapted to a thermophilic lifestyle. Utilizing genomes from Asgard archaea, TACK, and Euryarchaea, we perform phylogenomic reconstructions using the GTDB archaeal marker genes, the current reference set for taxonomic classification. Calibrating relative evolutionary divergence (RED) values for Asgardarchaeota using established Thermoproteota lineages in the GTDB r207 reference tree, we establish a robust classification and propose Asgardarchaeum as the type genus for the family Asgardarchaeaceae (fam. nov)., the order Asgardarchaeales (ord. nov.), the class Asgardarchaeia (class. nov.), and the phylum Asgardarchaeota (phyl. nov.). This effort aims to preserve taxonomic congruence in the scientific literature.

Gene inversion led to the emergence of brackish archaeal heterotrophs in the aftermath of the Cryogenian Snowball Earth.

Land-ocean interactions greatly impact the evolution of coastal life on earth. However, the ancient geological forces and genetic mechanisms that shaped evolutionary adaptations and allowed microorganisms to inhabit coastal brackish waters remain largely unexplored. In this study, we infer the evolutionary trajectory of the ubiquitous heterotrophic archaea Poseidoniales (Marine Group II archaea) presently occurring across global aquatic habitats. Our results show that their brackish subgroups had a single origination, dated to over 600 million years ago, through the inversion of the magnesium transport gene corA that conferred osmotic-stress tolerance. The subsequent loss and gain of corA were followed by genome-wide adjustment, characterized by a general two-step mode of selection in microbial speciation. The coastal family of Poseidoniales showed a rapid increase in the evolutionary rate during and in the aftermath of the Cryogenian Snowball Earth (∼700 million years ago), possibly in response to the enhanced phosphorus supply and the rise of algae. Our study highlights the close interplay between genetic changes and ecosystem evolution that boosted microbial diversification in the Neoproterozoic continental margins, where the Cambrian explosion of animals soon followed.

Proposal of names for 329 higher rank taxa defined in the Genome Taxonomy Database under two prokaryotic codes.

The Genome Taxonomy Database (GTDB) is a taxonomic framework that defines prokaryotic taxa as monophyletic groups in concatenated protein reference trees according to systematic criteria. This has resulted in a substantial number of changes to existing classifications (https://gtdb.ecogenomic.org). In the case of union of taxa, GTDB names were applied based on the priority of publication. The division of taxa or change in rank led to the formation of new Latin names above the rank of genus that were only made publicly available via the GTDB website without associated published taxonomic descriptions. This has sometimes led to confusion in the literature and databases. A number of the provisional GTDB names were later published in other studies, while many still lack authorships. To reduce further confusion, here we propose names and descriptions for 329 GTDB-defined prokaryotic taxa, 223 of which are suitable for validation under the International Code of Nomenclature of Prokaryotes (ICNP) and 49 under the Code of Nomenclature of Prokaryotes described from Sequence Data (SeqCode). For the latter, we designated 23 genomes as type material. An additional 57 taxa that do not currently satisfy the validation criteria of either code are proposed as Candidatus.

Protecting the invisible: Establishing guideline values for copper toxicity to marine microbiomes.

Understanding the rapid responses of marine microbiomes to environmental disturbances is paramount for supporting early assessments of harm to high-value ecosystems, such as coral reefs. Yet, management guidelines aimed at protecting aquatic life from environmental pollution remain exclusively defined for organisms at higher trophic levels. In this study, 16S rRNA gene amplicon sequencing was applied in conjunction with propidium monoazide for cell-viability assessment as a sensitive tool to determine taxon- and community-level changes in a seawater microbial community under copper (Cu) exposure. Bayesian model averaging was used to establish concentration-response relationships to evaluate the effects of copper on microbial composition, diversity, and richness for the purpose of estimating microbiome Hazard Concentration (mHCx) values. Predicted mHC5 values at which a 5 % change in microbial composition, diversity, and richness occurred were 1.05, 0.72, and 0.38 µg Cu L-1, respectively. Threshold indicator taxa analysis was applied across the copper concentrations to identify taxon-specific change points for decreasing taxa. These change points were then used to generate a Prokaryotic Sensitivity Distribution (PSD), from which mHCxdec values were derived for copper, suitable for the protection of 99, 95, 90, and 80 % of the marine microbiome. The mHC5dec guideline value of 0.61 µg Cu L-1, protective of 95 % of the marine microbial community, was lower than the equivalent Australian water quality guideline value based on eukaryotic organisms at higher trophic levels. This suggests that marine microbial communities might be more vulnerable, highlighting potential insufficiencies in their protection against copper pollution. The mHCx values proposed here provide approaches to quantitatively assess the effects of contaminants on microbial communities towards the inclusion of prokaryotes in future water quality guidelines.

Insights into plastic biodegradation: community composition and functional capabilities of the superworm (Zophobas morio) microbiome in styrofoam feeding trials.

Plastics are inexpensive and widely used organic polymers, but their high durability hinders biodegradation. Polystyrene, including extruded polystyrene (also known as styrofoam), is among the most commonly produced plastics worldwide and is recalcitrant to microbial degradation. In this study, we assessed changes in the gut microbiome of superworms (Zophobas morio) reared on bran, polystyrene or under starvation conditions over a 3 weeks period. Superworms on all diets were able to complete their life cycle to pupae and imago, although superworms reared on polystyrene had minimal weight gains, resulting in lower pupation rates compared to bran reared worms. The change in microbial gut communities from baseline differed considerably between diet groups, with polystyrene and starvation groups characterized by a loss of microbial diversity and the presence of opportunistic pathogens. Inferred microbial functions enriched in the polystyrene group included transposon movements, membrane restructuring and adaptations to oxidative stress. We detected several encoded enzymes with reported polystyrene and styrene degradation abilities, supporting previous reports of polystyrene-degrading bacteria in the superworm gut. By recovering metagenome-assembled genomes (MAGs) we linked phylogeny and functions and identified genera including Pseudomonas, Rhodococcus and Corynebacterium that possess genes associated with polystyrene degradation. In conclusion, our results provide the first metagenomic insights into the metabolic pathways used by the gut microbiome of superworms to degrade polystyrene. Our results also confirm that superworms can survive on polystyrene feed, but this diet has considerable negative impacts on host gut microbiome diversity and health.

Three families of Asgard archaeal viruses identified in metagenome-assembled genomes.

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.

Microvolume DNA extraction methods for microscale amplicon and metagenomic studies.

Investigating the composition and metabolic capacity of aquatic microbial assemblages usually requires the filtration of multi-litre samples, which are up to 1 million-fold larger than the microenvironments within which microbes are predicted to be spatially organised. To determine if community profiles can be reliably generated from microlitre volumes, we sampled seawater at a coastal and an oceanic site, filtered and homogenised them, and extracted DNA from bulk samples (2 L) and microvolumes (100, 10 and 1 µL) using two new approaches. These microvolume DNA extraction methods involve either physical or chemical lysis (through pH/thermal shock and lytic enzymes/surfactants, respectively), directly followed by the capture of DNA on magnetic beads. Downstream analysis of extracted DNA using both amplicon sequencing and metagenomics, revealed strong correlation with standard large volume approaches, demonstrating the fidelity of taxonomic and functional profiles of microbial communities in as little as 1 µL of seawater. This volume is six orders of magnitude smaller than most standard operating procedures for marine metagenomics, which will allow precise sampling of the heterogenous landscape that microbes inhabit.

Recoding of stop codons expands the metabolic potential of two novel Asgardarchaeota lineages.

Asgardarchaeota have been proposed as the closest living relatives to eukaryotes, and a total of 72 metagenome-assembled genomes (MAGs) representing six primary lineages in this archaeal phylum have thus far been described. These organisms are predicted to be fermentative heterotrophs contributing to carbon cycling in sediment ecosystems. Here, we double the genomic catalogue of Asgardarchaeota by obtaining 71 MAGs from a range of habitats around the globe, including the deep subsurface, brackish shallow lakes, and geothermal spring sediments. Phylogenomic inferences followed by taxonomic rank normalisation confirmed previously established Asgardarchaeota classes and revealed four additional lineages, two of which were consistently recovered as monophyletic classes. We therefore propose the names Candidatus Sifarchaeia class nov. and Ca. Jordarchaeia class nov., derived from the gods Sif and Jord in Norse mythology. Metabolic inference suggests that both classes represent hetero-organotrophic acetogens, which also have the ability to utilise methyl groups such as methylated amines, with acetate as the probable end product in remnants of a methanogen-derived core metabolism. This inferred mode of energy conservation is predicted to be enhanced by genetic code expansions, i.e., stop codon recoding, allowing the incorporation of the rare 21st and 22nd amino acids selenocysteine (Sec) and pyrrolysine (Pyl). We found Sec recoding in Jordarchaeia and all other Asgardarchaeota classes, which likely benefit from increased catalytic activities of Sec-containing enzymes. Pyl recoding, on the other hand, is restricted to Sifarchaeia in the Asgardarchaeota, making it the first reported non-methanogenic archaeal lineage with an inferred complete Pyl machinery, likely providing members of this class with an efficient mechanism for methylamine utilisation. Furthermore, we identified enzymes for the biosynthesis of ester-type lipids, characteristic of bacteria and eukaryotes, in both newly described classes, supporting the hypothesis that mixed ether-ester lipids are a shared feature among Asgardarchaeota.

A standardized archaeal taxonomy for the Genome Taxonomy Database.

The accrual of genomic data from both cultured and uncultured microorganisms provides new opportunities to develop systematic taxonomies based on evolutionary relationships. Previously, we established a bacterial taxonomy through the Genome Taxonomy Database. Here, we propose a standardized archaeal taxonomy that is derived from a 122-concatenated-protein phylogeny that resolves polyphyletic groups and normalizes ranks based on relative evolutionary divergence. The resulting archaeal taxonomy, which forms part of the Genome Taxonomy Database, is stable for a range of phylogenetic variables including marker gene selection, inference methods, corrections for rate heterogeneity and compositional bias, tree rooting scenarios and expansion of the genome database. Rank normalization is shown to robustly correct for substitution rates varying up to 30-fold using simulated datasets. Taxonomic curation follows the rules of the International Code of Nomenclature of Prokaryotes while taking into account proposals to formally recognize the rank of phylum and to use genome sequences as type material. This taxonomy is based on 2,392 archaeal genomes, 93.3% of which required one or more changes to their existing taxonomy, mainly owing to incomplete classification. We identify 16 archaeal phyla and reclassify 3 major monophyletic units from the former Euryarchaeota and one phylum that unites the Thaumarchaeota-Aigarchaeota-Crenarchaeota-Korarchaeota (TACK) superphylum into a single phylum.

Undinarchaeota illuminate DPANN phylogeny and the impact of gene transfer on archaeal evolution.

The recently discovered DPANN archaea are a potentially deep-branching, monophyletic radiation of organisms with small cells and genomes. However, the monophyly and early emergence of the various DPANN clades and their role in life's evolution are debated. Here, we reconstructed and analysed genomes of an uncharacterized archaeal phylum (Candidatus Undinarchaeota), revealing that its members have small genomes and, while potentially being able to conserve energy through fermentation, likely depend on partner organisms for the acquisition of certain metabolites. Our phylogenomic analyses robustly place Undinarchaeota as an independent lineage between two highly supported 'DPANN' clans. Further, our analyses suggest that DPANN have exchanged core genes with their hosts, adding to the difficulty of placing DPANN in the tree of life. This pattern can be sufficiently dominant to allow identifying known symbiont-host clades based on routes of gene transfer. Together, our work provides insights into the origins and evolution of DPANN and their hosts.

A complete domain-to-species taxonomy for Bacteria and Archaea.

The Genome Taxonomy Database is a phylogenetically consistent, genome-based taxonomy that provides rank-normalized classifications for ~150,000 bacterial and archaeal genomes from domain to genus. However, almost 40% of the genomes in the Genome Taxonomy Database lack a species name. We address this limitation by using commonly accepted average nucleotide identity criteria to set bounds on species and propose species clusters that encompass all publicly available bacterial and archaeal genomes. Unlike previous average nucleotide identity studies, we chose a single representative genome to serve as the effective nomenclatural 'type' defining each species. Of the 24,706 proposed species clusters, 8,792 are based on published names. We assigned placeholder names to the remaining 15,914 species clusters to provide names to the growing number of genomes from uncultivated species. This resource provides a complete domain-to-species taxonomic framework for bacterial and archaeal genomes, which will facilitate research on uncultivated species and improve communication of scientific results.

Author Correction: A complete domain-to-species taxonomy for Bacteria and Archaea.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.