Uncultivated DPANN archaea are ubiquitous inhabitants of global oxygen-deficient zones with diverse metabolic potential.

Archaea belonging to the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota) superphylum have been found in an expanding number of environments and perform a variety of biogeochemical roles, including contributing to carbon, sulfur, and nitrogen cycling. Generally characterized by ultrasmall cell sizes and reduced genomes, DPANN archaea may form mutualistic, commensal, or parasitic interactions with various archaeal and bacterial hosts, influencing the ecology and functioning of microbial communities. While DPANN archaea reportedly comprise a sizeable fraction of the archaeal community within marine oxygen-deficient zone (ODZ) water columns, little is known about their metabolic capabilities in these ecosystems. We report 33 novel metagenome-assembled genomes (MAGs) belonging to the DPANN phyla Nanoarchaeota, Pacearchaeota, Woesearchaeota, Undinarchaeota, Iainarchaeota, and SpSt-1190 from pelagic ODZs in the Eastern Tropical North Pacific and the Arabian Sea. We find these archaea to be permanent, stable residents of all three major ODZs only within anoxic depths, comprising up to 1% of the total microbial community and up to 25%-50% of archaea as estimated from read mapping to MAGs. ODZ DPANN appear to be capable of diverse metabolic functions, including fermentation, organic carbon scavenging, and the cycling of sulfur, hydrogen, and methane. Within a majority of ODZ DPANN, we identify a gene homologous to nitrous oxide reductase. Modeling analyses indicate the feasibility of a nitrous oxide reduction metabolism for host-attached symbionts, and the small genome sizes and reduced metabolic capabilities of most DPANN MAGs suggest host-associated lifestyles within ODZs. IMPORTANCE: Archaea from the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota) superphylum have diverse metabolic capabilities and participate in multiple biogeochemical cycles. While metagenomics and enrichments have revealed that many DPANN are characterized by ultrasmall genomes, few biosynthetic genes, and episymbiotic lifestyles, much remains unknown about their biology. We report 33 new DPANN metagenome-assembled genomes originating from the three global marine oxygen-deficient zones (ODZs), the first from these regions. We survey DPANN abundance and distribution within the ODZ water column, investigate their biosynthetic capabilities, and report potential roles in the cycling of organic carbon, methane, and nitrogen. We test the hypothesis that nitrous oxide reductases found within several ODZ DPANN genomes may enable ultrasmall episymbionts to serve as nitrous oxide consumers when attached to a host nitrous oxide producer. Our results indicate DPANN archaea as ubiquitous residents within the anoxic core of ODZs with the potential to produce or consume key compounds.

Cell surface architecture of the cultivated DPANN archaeon Nanobdella aerobiophila.

The DPANN archaeal clade includes obligately ectosymbiotic species. Their cell surfaces potentially play an important role in the symbiotic interaction between the ectosymbionts and their hosts. However, little is known about the mechanism of ectosymbiosis. Here, we show cell surface structures of the cultivated DPANN archaeon Nanobdella aerobiophila strain MJ1T and its host Metallosphaera sedula strain MJ1HA, using a variety of electron microscopy techniques, i.e., negative-staining transmission electron microscopy, quick-freeze deep-etch TEM, and 3D electron tomography. The thickness, unit size, and lattice symmetry of the S-layer of strain MJ1T were different from those of the host archaeon strain MJ1HA. Genomic and transcriptomic analyses highlighted the most highly expressed MJ1T gene for a putative S-layer protein with multiple glycosylation sites and immunoglobulin-like folds, which has no sequence homology to known S-layer proteins. In addition, genes for putative pectin lyase- or lectin-like extracellular proteins, which are potentially involved in symbiotic interaction, were found in the MJ1T genome based on in silico 3D protein structure prediction. Live cell imaging at the optimum growth temperature of 65°C indicated that cell complexes of strains MJ1T and MJ1HA were motile, but sole MJ1T cells were not. Taken together, we propose a model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila.IMPORTANCEDPANN archaea are widely distributed in a variety of natural and artificial environments and may play a considerable role in the microbial ecosystem. All of the cultivated DPANN archaea so far need host organisms for their growth, i.e., obligately ectosymbiotic. However, the mechanism of the ectosymbiosis by DPANN archaea is largely unknown. To this end, we performed a comprehensive analysis of the cultivated DPANN archaeon, Nanobdella aerobiophila, using electron microscopy, live cell imaging, transcriptomics, and genomics, including 3D protein structure prediction. Based on the results, we propose a reasonable model of the symbiotic interaction and cell cycle of Nanobdella aerobiophila, which will enhance our understanding of the enigmatic physiology and ecological significance of DPANN archaea.

CPR bacteria and DPANN archaea play pivotal roles in response of microbial community to antibiotic stress in groundwater.

The accumulation of antibiotics in the natural environment can disrupt microbial population dynamics. However, our understanding of how microbial communities adapt to the antibiotic stress in groundwater ecosystems remains limited. By recovering 2675 metagenome-assembled genomes (MAGs) from 66 groundwater samples, we explored the effect of antibiotics on bacterial, archaeal, and fungal communities, and revealed the pivotal microbes and their mechanisms in coping with antibiotic stress. The results indicated that antibiotics had the most significant influence on bacterial and archaeal communities, while the impact on the fungal community was minimal. Analysis of co-occurrence networks between antibiotics and microbes revealed the critical roles of Candidate Phyla Radiation (CPR) bacteria and DPANN archaea, two representative microbial groups in groundwater ecosystem, in coping with antibiotic resistance and enhancing network connectivity and complexity. Further genomic analysis demonstrated that CPR bacteria carried approximately 6 % of the identified antibiotic resistance genes (ARGs), indicating their potential to withstand antibiotics on their own. Meanwhile, the genomes of CPR bacteria and DPANN archaea were found to encode diverse biosynthetic gene clusters (BGCs) responsible for producing antimicrobial metabolites, which could not only assist CPR and DPANN organisms but also benefit the surrounding microbes in combating antibiotic stress. These findings underscore the significant impact of antibiotics on prokaryotic microbial communities in groundwater, and highlight the importance of CPR bacteria and DPANN archaea in enhancing the overall resilience and functionality of the microbial community in the face of antibiotic stress.

The Archaeome's Role in Colorectal Cancer: Unveiling the DPANN Group and Investigating Archaeal Functional Signatures.

BACKGROUND AND AIMS: Gut microbial imbalances are linked to colorectal cancer (CRC), but archaea's role remains underexplored. Here, using previously published metagenomic data from different populations including Austria, Germany, Italy, Japan, China, and India, we performed bioinformatic and statistical analysis to identify archaeal taxonomic and functional signatures related to CRC. METHODS: We analyzed published fecal metagenomic data from 390 subjects, comparing the archaeomes of CRC and healthy individuals. We conducted a biostatistical analysis to investigate the relationship between Candidatus Mancarchaeum acidiphilum (DPANN superphylum) and other archaeal species associated with CRC. Using the Prokka tool, we annotated the data focusing on archaeal genes, subsequently linking them to CRC and mapping them against UniprotKB and GO databases for specific archaeal gene functions. RESULTS: Our analysis identified enrichment of methanogenic archaea in healthy subjects, with an exception for Methanobrevibacter smithii, which correlated with CRC. Notably, CRC showed a strong association with archaeal species, particularly Natrinema sp. J7-2, Ferroglobus placidus, and Candidatus Mancarchaeum acidiphilum. Furthermore, the DPANN archaeon exhibited a significant correlation with other CRC-associated archaea (p < 0.001). Functionally, we found a marked association between MvhB-type polyferredoxin and colorectal cancer. We also highlighted the association of archaeal proteins involved in the biosynthesis of leucine and the galactose metabolism process with the healthy phenotype. CONCLUSIONS: The archaeomes of CRC patients show identifiable alterations, including a decline in methanogens and an increase in Halobacteria species. MvhB-type polyferredoxin, linked with CRC and species like Candidatus Mancarchaeum acidiphilum, Natrinema sp. J7-2, and Ferroglobus placidus emerge as potential archaeal biomarkers. Archaeal proteins may also offer gut protection, underscoring archaea's role in CRC dynamics.

Metagenomic Discovery of "Candidatus Parvarchaeales"-Related Lineages Sheds Light on Adaptation and Diversification from Neutral-Thermal to Acidic-Mesothermal Environments.

"Candidatus Parvarchaeales" microbes, representing a DPANN archaeal group with limited metabolic potential and reliance on hosts for their growth, were initially found in acid mine drainage (AMD). Due to the lack of representatives, however, their ecological roles and adaptation to extreme habitats such as AMD as well as how they diverge across the lineage remain largely unexplored. By applying genome-resolved metagenomics, 28 Parvarchaeales-associated metagenome-assembled genomes (MAGs) representing two orders and five genera were recovered. Among them, we identified three new genera and proposed the names "Candidatus Jingweiarchaeum," "Candidatus Haiyanarchaeum," and "Candidatus Rehaiarchaeum," with the former two belonging to a new order, "Candidatus Jingweiarchaeales." Further analyses of the metabolic potentials revealed substantial niche differentiation between Jingweiarchaeales and Parvarchaeales. Jingweiarchaeales may rely on fermentation, salvage pathways, partial glycolysis, and the pentose phosphate pathway (PPP) for energy conservation reservation, while the metabolic potentials of Parvarchaeales might be more versatile. Comparative genomic analyses suggested that Jingweiarchaeales favor habitats with higher temperatures and that Parvarchaeales are better adapted to acidic environments. We further revealed that the thermal adaptation of these lineages, especially Haiyanarchaeum, might rely on genomic features such as the usage of specific amino acids, genome streamlining, and hyperthermophile featured genes such as rgy. Notably, the adaptation of Parvarchaeales to acidic environments was possibly driven by horizontal gene transfer (HGT). The reconstruction of ancestral states demonstrated that both may have originated from thermal and neutral environments and later spread to mesothermal and acidic environments. These evolutionary processes may also be accompanied by adaptation to oxygen-rich environments via HGT. IMPORTANCE "Candidatus Parvarchaeales" microbes may represent a lineage uniquely distributed in extreme environments such as AMD and hot springs. However, little is known about the strategies and processes of how they adapted to these extreme environments. By the discovery of potential new order-level lineages, "Ca. Jingweiarchaeales," and in-depth comparative genomic analysis, we unveiled the functional differentiation of these lineages. Furthermore, we show that the adaptation of these lineages to high-temperature and acidic environments was driven by different strategies, with the former relying more on genomic characteristics such as genome streamlining and amino acid compositions and the latter relying more on the acquisition of genes associated with acid tolerance. Finally, by the reconstruction of the ancestral states of the optimal growth temperature (OGT) and isoelectric point (pI), we showed the potential evolutionary process of Parvarchaeales-related lineages with regard to the shift from the high-temperature environment of their common ancestors to low-temperature (potentially acidic) environments.

Comparative Genomic Insights into the Evolution of Halobacteria-Associated "Candidatus Nanohaloarchaeota".

Members of the phylum "Candidatus Nanohaloarchaeota," a representative lineage within the DPANN superphylum, are characterized by their nanosized cells and symbiotic lifestyle with Halobacteria. However, the development of the symbiosis remains unclear. Here, we propose two novel families, "Candidatus Nanoanaerosalinaceae" and "Candidatus Nanohalalkaliarchaeaceae" in "Ca. Nanohaloarchaeota," represented by five dereplicated metagenome-assembled genomes obtained from hypersaline sediments or related enrichment cultures of soda-saline lakes. Phylogenetic analyses reveal that the two novel families are placed at the root of the family "Candidatus Nanosalinaceae," including the cultivated taxa. The two novel families prefer hypersaline sediments, and the acid shift of predicted proteomes indicates a "salt-in" strategy for hypersaline adaptation. They contain a lower proportion of putative horizontal gene transfers from Halobacteria than "Ca. Nanosalinaceae," suggesting a weaker association with Halobacteria. Functional prediction and historical events reconstruction disclose that they exhibit divergent potentials in carbohydrate and organic acid metabolism and environmental responses. Globally, comparative genomic analyses based on the new families enrich the taxonomic and functional diversity of "Ca. Nanohaloarchaeota" and provide insights into the evolutionary process of "Ca. Nanohaloarchaeota" and their symbiotic relationship with Halobacteria. IMPORTANCE The DPANN superphylum is a group of archaea widely distributed in various habitats. They generally have small cells and have a symbiotic lifestyle with other archaea. The archaeal symbiotic interaction is vital to understanding microbial communities. However, the formation and evolution of the symbiosis between the DPANN lineages and other diverse archaea remain unclear. Based on phylogeny, habitat distribution, hypersaline adaptation, host prediction, functional potentials, and historical events of "Ca. Nanohaloarchaeota," a representative phylum within the DPANN superphylum, we report two novel families representing intermediate stages, and we infer the evolutionary process of "Ca. Nanohaloarchaeota" and their Halobacteria-associated symbiosis. Altogether, this research helps in understanding the evolution of symbiosis in "Ca. Nanohaloarchaeota" and provides a model for the evolution of other DPANN lineages.

Progress and Challenges in Studying the Ecophysiology of Archaea.

It has been less than two decades since the study of archaeal ecophysiology has become unshackled from the limitations of cultivation and amplicon sequencing through the advent of metagenomics. As a primer to the guide on producing archaeal genomes from metagenomes, we briefly summarize here how different meta'omics, imaging, and wet lab methods have contributed to progress in understanding the ecophysiology of Archaea. We then peer into the history of how our knowledge on two particularly important lineages was assembled: the anaerobic methane and alkane oxidizers, encountered primarily among Euryarchaeota, and the nanosized, mainly parasitic, members of the DPANN superphylum.

Nanobdella aerobiophila gen. nov., sp. nov., a thermoacidophilic, obligate ectosymbiotic archaeon, and proposal of Nanobdellaceae fam. nov., Nanobdellales ord. nov. and Nanobdellia class. nov.

A co-culture of a novel thermoacidophilic, obligate symbiotic archaeon, designated as strain MJ1T, with its specific host archaeon Metallosphaera sedula strain MJ1HA was obtained from a terrestrial hot spring in Japan. Strain MJ1T grew in the co-culture under aerobic conditions. Coccoid cells of strain MJ1T were 200-500 nm in diameter, and attached to the MJ1HA cells in the co-culture. The ranges and optima of the growth temperature and pH of strain MJ1T in the co-culture were 60-75 °C (optimum, 65-70 °C) and pH 1.0-4.0 (optimum, pH 2.5), respectively. Core lipids of dialkyl glycerol tetraethers (GDGT)-3 and GDGT-4 were highly abundant in MJ1T cells concentrated from the co-culture. Strain MJ1T has a small genome (0.67 Mbp) lacking genes for biosynthesis of essential biomolecules, such as nucleotides, lipids and ATP. The genomic DNA G+C content was 24.9 mol%. The 16S rRNA gene sequence of strain MJ1T was most closely related to that of the cultivated species, 'Nanopusillus acidilobi' strain N7A (85.8 % similarity). Based on phylogenetic and physiological characteristics, we propose the name Nanobdella aerobiophila gen. nov., sp. nov. to accommodate the strain MJ1T (=JCM 33616T=DSM 111728T). In addition, we propose the names Nanobdellaceae fam. nov., Nanobdellales ord. nov., and Nanobdellia class. nov. to accommodate the novel genus.

Looking through the Lens of the Ribosome Biogenesis Evolutionary History: Possible Implications for Archaeal Phylogeny and Eukaryogenesis.

Our understanding of microbial diversity and its evolutionary relationships has increased substantially over the last decade. Such an understanding has been greatly fueled by culture-independent metagenomics analyses. However, the outcome of some of these studies and their biological and evolutionary implications, such as the origin of the eukaryotic lineage from the recently discovered archaeal Asgard superphylum, is debated. The sequences of the ribosomal constituents are amongst the most used phylogenetic markers. However, the functional consequences underlying the analysed sequence diversity and their putative evolutionary implications are essentially not taken into consideration. Here, we propose to exploit additional functional hallmarks of ribosome biogenesis to help disentangle competing evolutionary hypotheses. Using selected examples, such as the multiple origins of halophily in archaea or the evolutionary relationship between the Asgard archaea and Eukaryotes, we illustrate and discuss how function-aware phylogenetic framework can contribute to refining our understanding of archaeal phylogeny and the origin of eukaryotic cells.

A Micrarchaeon Isolate Is Covered by a Proteinaceous S-Layer.

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in "Ca. Micrarchaeum harzensis A_DKE," and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated, and the micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described coculture, containing "Ca. Micrarchaeum harzensis A_DKE" and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of "Ca. Micrarchaeum harzensis A_DKE." Both organisms are clearly distinguishable by cell size, shape, and surface structure. IMPORTANCE Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member "Candidatus Micrarchaeum harzensis A_DKE," an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of "Ca. Micrarchaeum harzensis A_DKE" is an important step toward understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

A divide-and-conquer phylogenomic approach based on character supermatrices resolves early steps in the evolution of the Archaea.

BACKGROUND: The recent rise in cultivation-independent genome sequencing has provided key material to explore uncharted branches of the Tree of Life. This has been particularly spectacular concerning the Archaea, projecting them at the center stage as prominently relevant to understand early stages in evolution and the emergence of fundamental metabolisms as well as the origin of eukaryotes. Yet, resolving deep divergences remains a challenging task due to well-known tree-reconstruction artefacts and biases in extracting robust ancient phylogenetic signal, notably when analyzing data sets including the three Domains of Life. Among the various strategies aimed at mitigating these problems, divide-and-conquer approaches remain poorly explored, and have been primarily based on reconciliation among single gene trees which however notoriously lack ancient phylogenetic signal. RESULTS: We analyzed sub-sets of full supermatrices covering the whole Tree of Life with specific taxonomic sampling to robustly resolve different parts of the archaeal phylogeny in light of their current diversity. Our results strongly support the existence and early emergence of two main clades, Cluster I and Cluster II, which we name Ouranosarchaea and Gaiarchaea, and we clarify the placement of important novel archaeal lineages within these two clades. However, the monophyly and branching of the fast evolving nanosized DPANN members remains unclear and worth of further study. CONCLUSIONS: We inferred a well resolved rooted phylogeny of the Archaea that includes all recently described phyla of high taxonomic rank. This phylogeny represents a valuable reference to study the evolutionary events associated to the early steps of the diversification of the archaeal domain. Beyond the specifics of archaeal phylogeny, our results demonstrate the power of divide-and-conquer approaches to resolve deep phylogenetic relationships, which should be applied to progressively resolve the entire Tree of Life.

Insight into the symbiotic lifestyle of DPANN archaea revealed by cultivation and genome analyses.

Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum "Candidatus Micrarchaeota" (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera-Metallosphaera, Acidianus, and Saccharolobus-originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

Genomic Insights Into the Archaea Inhabiting an Australian Radioactive Legacy Site.

During the 1960s, small quantities of radioactive materials were co-disposed with chemical waste at the Little Forest Legacy Site (LFLS, Sydney, Australia). The microbial function and population dynamics in a waste trench during a rainfall event have been previously investigated revealing a broad abundance of candidate and potentially undescribed taxa in this iron-rich, radionuclide-contaminated environment. Applying genome-based metagenomic methods, we recovered 37 refined archaeal MAGs, mainly from undescribed DPANN Archaea lineages without standing in nomenclature and 'Candidatus Methanoperedenaceae' (ANME-2D). Within the undescribed DPANN, the newly proposed orders 'Ca. Gugararchaeales', 'Ca. Burarchaeales' and 'Ca. Anstonellales', constitute distinct lineages with a more comprehensive central metabolism and anabolic capabilities within the 'Ca. Micrarchaeota' phylum compared to most other DPANN. The analysis of new and extant 'Ca. Methanoperedens spp.' MAGs suggests metal ions as the ancestral electron acceptors during the anaerobic oxidation of methane while the respiration of nitrate/nitrite via molybdopterin oxidoreductases would have been a secondary acquisition. The presence of genes for the biosynthesis of polyhydroxyalkanoates in most 'Ca. Methanoperedens' also appears to be a widespread characteristic of the genus for carbon accumulation. This work expands our knowledge about the roles of the Archaea at the LFLS, especially, DPANN Archaea and 'Ca. Methanoperedens', while exploring their diversity, uniqueness, potential role in elemental cycling, and evolutionary history.

Soil Candidate Phyla Radiation Bacteria Encode Components of Aerobic Metabolism and Co-occur with Nanoarchaea in the Rare Biosphere of Rhizosphere Grassland Communities.

Candidate Phyla Radiation (CPR) bacteria and nanoarchaea populate most ecosystems but are rarely detected in soil. We concentrated particles of less than 0.2 µm in size from grassland soil, enabling targeted metagenomic analysis of these organisms, which are almost totally unexplored in largely oxic environments such as soil. We recovered a diversity of CPR bacterial and some archaeal sequences but no sequences from other cellular organisms. The sampled sequences include Doudnabacteria (SM2F11) and Pacearchaeota, organisms rarely reported in soil, as well as Saccharibacteria, Parcubacteria, and Microgenomates. CPR and archaea of the phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) were enriched 100- to 1,000-fold compared to that in bulk soil, in which we estimate each of these organisms comprises approximately 1 to 100 cells per gram of soil. Like most CPR and DPANN sequenced to date, we predict these microorganisms live symbiotic anaerobic lifestyles. However, Saccharibacteria, Parcubacteria, and Doudnabacteria genomes sampled here also harbor ubiquinol oxidase operons that may have been acquired from other bacteria, likely during adaptation to aerobic soil environments. We conclude that CPR bacteria and DPANN archaea are part of the rare soil biosphere and harbor unique metabolic platforms that potentially evolved to live symbiotically under relatively oxic conditions. IMPORTANCE Here, we investigated overlooked microbes in soil, Candidate Phyla Radiation (CPR) bacteria and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) archaea, by size fractionating small particles from soil, an approach typically used for the recovery of viral metagenomes. Concentration of these small cells (<0.2 µm) allowed us to identify these organisms as part of the rare soil biosphere and to sample genomes that were absent from non-size-fractionated metagenomes. We found that some of these predicted symbionts, which have been largely studied in anaerobic systems, have acquired aerobic capacity via lateral transfer that may enable adaptation to oxic soil environments. We estimate that there are approximately 1 to 100 cells of each of these lineages per gram of soil, highlighting that the approach provides a window into the rare soil biosphere and its associated genetic potential.

Comparative Genomics Provides Insights into the Genetic Diversity and Evolution of the DPANN Superphylum.

DPANN is known as highly diverse, globally widespread, and mostly ectosymbiotic archaeal superphylum. However, this group of archaea was overlooked for a long time, and there were limited in-depth studies reported. In this investigation, 41 metagenome-assembled genomes (MAGs) belonging to the DPANN superphylum were recovered (18 MAGs had average nucleotide identity [ANI] values of <95% and a percentage of conserved proteins [POCP] of >50%, while 14 MAGs showed a POCP of <50%), which were analyzed comparatively with 515 other published DPANN genomes. Mismatches to known 16S rRNA gene primers were identified among 16S rRNA genes of DPANN archaea. Numbers of gene families lost (mostly related to energy and amino acid metabolism) were over three times greater than those gained in the evolution of DPANN archaea. Lateral gene transfer (LGT; ∼45.5% was cross-domain) had facilitated niche adaption of the DPANN archaea, ensuring a delicate equilibrium of streamlined genomes with efficient niche-adaptive strategies. For instance, LGT-derived cytochrome bd ubiquinol oxidase and arginine deiminase in the genomes of "Candidatus Micrarchaeota" could help them better adapt to aerobic acidic mine drainage habitats. In addition, most DPANN archaea acquired enzymes for biosynthesis of extracellular polymeric substances (EPS) and transketolase/transaldolase for the pentose phosphate pathway from Bacteria. IMPORTANCE The domain Archaea is a key research model for gaining insights into the origin and evolution of life, as well as the relevant biogeochemical processes. The discovery of nanosized DPANN archaea has overthrown many aspects of microbiology. However, the DPANN superphylum still contains a vast genetic novelty and diversity that need to be explored. Comprehensively comparative genomic analysis on the DPANN superphylum was performed in this study, with an attempt to illuminate its metabolic potential, ecological distribution and evolutionary history. Many interphylum differences within the DPANN superphylum were found. For example, Altiarchaeota had the biggest genome among DPANN phyla, possessing many pathways missing in other phyla, such as formaldehyde assimilation and the Wood-Ljungdahl pathway. In addition, LGT acted as an important force to provide DPANN archaeal genetic flexibility that permitted the occupation of diverse niches. This study has advanced our understanding of the diversity and genome evolution of archaea.

Deciphering Symbiotic Interactions of "Candidatus Aenigmarchaeota" with Inferred Horizontal Gene Transfers and Co-occurrence Networks.

"Candidatus Aenigmarchaeota" ("Ca. Aenigmarchaeota") represents one of the earliest proposed evolutionary branches within the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, their ecological roles and potential host-symbiont interactions are still poorly understood. Here, eight metagenome-assembled genomes (MAGs) were reconstructed from hot spring ecosystems, and further in-depth comparative and evolutionary genomic analyses were conducted on these MAGs and other genomes downloaded from public databases. Although with limited metabolic capacities, we reported that "Ca. Aenigmarchaeota" in thermal environments harbor more genes related to carbohydrate metabolism than "Ca. Aenigmarchaeota" in nonthermal environments. Evolutionary analyses suggested that members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota contribute substantially to the niche expansion of "Ca. Aenigmarchaeota" via horizontal gene transfer (HGT), especially genes related to virus defense and stress responses. Based on co-occurrence network results and recent genetic exchanges among community members, we conjectured that "Ca. Aenigmarchaeota" may be symbionts associated with one MAG affiliated with the genus Pyrobaculum, though host specificity might be wide and variable across different "Ca. Aenigmarchaeota" organisms. This study provides significant insight into possible DPANN-host interactions and ecological roles of "Ca. Aenigmarchaeota." IMPORTANCE Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community. By integrating horizontal gene transfer (HGT) detection and co-occurrence network analysis on "Candidatus Aenigmarchaeota" ("Ca. Aenigmarchaeota"), we made one of the first attempts to infer their putative interaction partners and further decipher the potential functional and genetic interactions between the symbionts. We revealed that HGTs contributed by members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota conferred "Ca. Aenigmarchaeota" with the ability to survive under different environmental stresses, such as virus infection, high temperature, and oxidative stress. This study demonstrates that the interaction partners might be inferable by applying informatics analyses on metagenomic sequencing data.

Protein Family Content Uncovers Lineage Relationships and Bacterial Pathway Maintenance Mechanisms in DPANN Archaea.

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 3,197 genomes including 569 newly reconstructed genomes. Protein family content clearly separates the final set of 390 DPANN genomes from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

Metagenomic Insights into the Metabolic and Ecological Functions of Abundant Deep-Sea Hydrothermal Vent DPANN Archaea.

Due to their unique metabolism and important ecological roles, deep-sea hydrothermal archaea have attracted great scientific interest. Among these archaea, DPANN superphylum archaea are widely distributed in hydrothermal vent environments. However, DPANN metabolism and ecology remain largely unknown. In this study, we assembled 20 DPANN genomes among 43 reconstructed genomes obtained from deep-sea hydrothermal vent sediments. Phylogenetic analysis suggests 6 phyla, comprised of Aenigmarchaeota, Diapherotrites, Nanoarchaeota, Pacearchaeota, Woesearchaeota, and a new candidate phylum we have designated Kexuearchaeota These are included in the 20 DPANN archaeal members, indicating their broad diversity in this special environment. Analyses of their metabolism reveal deficiencies due to their reduced genome size, including gluconeogenesis and de novo nucleotide and amino acid biosynthesis. However, DPANN archaea possess alternate strategies to address these deficiencies. DPANN archaea also have the potential to assimilate nitrogen and sulfur compounds, indicating an important ecological role in the hydrothermal vent system.IMPORTANCE DPANN archaea show high distribution in the hydrothermal system, although they display small genome size and some incomplete biological processes. Exploring their metabolism is helpful to understand how such small forms of life adapt to this unique environment and what ecological roles they play. In this study, we obtained 20 high-quality metagenome-assembled genomes (MAGs) corresponding to 6 phyla of the DPANN group (Aenigmarchaeota, Diapherotrites, Nanoarchaeota, Pacearchaeota, Woesearchaeota, and a new candidate phylum designated Kexuearchaeota). Further metagenomic analyses provided insights on the metabolism and ecological functions of DPANN archaea to adapt to deep-sea hydrothermal environments. Our study contributes to a deeper understanding of their special lifestyles and should provide clues to cultivate this important archaeal group in the future.

Rich Repertoire of Quorum Sensing Protein Coding Sequences in CPR and DPANN Associated with Interspecies and Interkingdom Communication.

The bacterial candidate phyla radiation (CPR) and the archaeal DPANN superphylum are two novel lineages that have substantially expanded the tree of life due to their large phylogenetic diversity. Because of their ultrasmall size, reduced genome, and lack of core biosynthetic capabilities, most CPR and DPANN members are predicted to be sustained through their interactions with other species. How the few characterized CPR and DPANN symbionts achieve these critical interactions is, however, poorly understood. Here, we conducted an in silico analysis on 2,597 CPR/DPANN genomes to test whether these ultrasmall microorganisms might encode homologs of reference proteins involved in the synthesis and/or the detection of 26 different types of communication molecules (quorum sensing [QS] signals), since QS signals are well-known mediators of intra- and interorganismic relationships. We report the discovery of 5,693 variants of QS proteins distributed across 63 CPR and 6 DPANN phyla and associated with 14 distinct types of communication molecules, most of which were characterized as interspecies QS signals.IMPORTANCE The selection of predicted genes for interspecies communication within the CPR and DPANN genomes sheds some light onto the underlying mechanisms supporting their inferred symbiotic lifestyle. Also, considering the lack of core pathways such as the de novo synthesis of nucleotides or amino acids in the CPR and DPANN lineages, the persistence of these genes highlights how determinant social traits can be for the survival of some microorganisms. Finally, the considerable number of variants of QS proteins identified among the 69 CPR and DPANN phyla substantially expands our knowledge of prokaryotic communication across the tree of life and suggests that the multiplicity of "dialects" in the microbial world is probably larger than previously appreciated.

Metabolic Diversity and Evolutionary History of the Archaeal Phylum "Candidatus Micrarchaeota" Uncovered from a Freshwater Lake Metagenome.

Acidophilic archaea of the archaeal Richmond Mine acidophilic nanoorganisms (ARMAN) group from the uncultured candidate phylum "Candidatus Micrarchaeota" have small genomes and cell sizes and are known to be metabolically dependent and physically associated with their Thermoplasmatales hosts. However, phylogenetically diverse "Ca Micrarchaeota" are widely distributed in various nonacidic environments, and it remains uncertain because of the lack of complete genomes whether they are also devoted to a partner-dependent lifestyle. Here, we obtained nine metagenome-assembled genomes of "Ca Micrarchaeota" from the sediments of a meromictic freshwater lake, including a complete, closed 1.2 Mbp genome of "Ca Micrarchaeota" Sv326, an archaeon phylogenetically distant from the ARMAN lineage. Genome analysis revealed that, contrary to ARMAN "Ca Micrarchaeota," the Sv326 archaeon has complete glycolytic pathways and ATP generation mechanisms in substrate phosphorylation reactions, the capacities to utilize some sugars and amino acids as substrates, and pathways for de novo nucleotide biosynthesis but lacked an aerobic respiratory chain. We suppose that Sv326 is a free-living scavenger rather than an obligate parasite/symbiont. Comparative analysis of "Ca Micrarchaeota" genomes representing different order-level divisions indicated that evolution of the "Ca Micrarchaeota" from a free-living "Candidatus Diapherotrites"-like ancestor involved losses of important metabolic pathways in different lineages and gains of specific functions in the course of adaptation to a partner-dependent lifestyle and specific environmental conditions. The ARMAN group represents the most pronounced case of genome reduction and gene loss, while the Sv326 lineage appeared to be rather close to the ancestral state of the "Ca Micrarchaeota" in terms of metabolic potential.IMPORTANCE The recently described superphylum DPANN includes several phyla of uncultivated archaea with small cell sizes, reduced genomes, and limited metabolic capabilities. One of these phyla, "Ca Micrarchaeota," comprises an enigmatic group of archaea found in acid mine drainage environments, the archaeal Richmond Mine acidophilic nanoorganisms (ARMAN) group. Analysis of their reduced genomes revealed the absence of key metabolic pathways consistent with their partner-associated lifestyle, and physical associations of ARMAN cells with their hosts were documented. However, "Ca Micrarchaeota" include several lineages besides the ARMAN group found in nonacidic environments, and none of them have been characterized. Here, we report a complete genome of "Ca Micrarchaeota" from a non-ARMAN lineage. Analysis of this genome revealed the presence of metabolic capacities lost in ARMAN genomes that could enable a free-living lifestyle. These results expand our understanding of genetic diversity, lifestyle, and evolution of "Ca Micrarchaeota."