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The world of microbes is diverse, but the majority of these fascinating organisms are not as famous (or perhaps infamous) as their pathogenic counterparts. Cell highlights six scientists whose work addresses some of the most fundamental questions in biology, even though their microbial models may seem a tad bit unconventional., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Lake Salda, a terrestrial analog for the paleolake in Jezero Crater on Mars, hosts active, subfossil, and fossil hydromagnesite microbialites, making it an ideal location to study microbialite formation and subsequent processes. Our understanding of this record is still limited by an incomplete knowledge of the macro- and mesoscale morphotypes of microbialites, along with their spatial distribution and correlation with microbial and geochemical processes that influence microbialite formation. In this study, we investigated the spatial distribution, morphotypes, mineralogy, geochemistry, and microbial diversity of the microbialites and identified six distinct zones (Zone I to Zone VI) with major microbialite build-ups in Lake Salda. Newly identified microbialites were classified based on the macro- and mesostructures. Our work shows that the lake contains stromatolites, thrombolites, stromatolitic thrombolites, dendrolites, and microbially induced sedimentary structures. At macroscale, Lake Salda microbialites exhibit hemispheres, stacked domes, and laterally linked columnar structures while minicolumns, knobs, mesoclots, laminae, and botryoidal structures are common at mesoscale. The macro- and mesoscale distribution of different microbialite types spatially correlates with microbial community composition and water depth. Deep-growing microbialites with a low abundance of Cyanobacteria (∼1%-4%) and high abundance of Firmicutes (28%-93%) exhibit steeply convex lamination, producing finger-like minicolumnar mesostructures. In contrast, shallow-growing microbialites with a low abundance of Firmicutes (0%-5%) and high abundance of Cyanobacteria (11%-37%) have well-preserved gently convex millimeter-scale lamination, resulting in cauliflower mesostructures. Palygorskite ((Mg, Al) 2 Si 4 O 10 (OH)) is identified in the diatom-rich microbial layer of the deep-growing microbialites. Regardless of the microbialite types, hydromagnesite and aragonite are present in the extracellular polymeric substance (EPS)-rich zone of the shallow and deep-growing microbialites. Overall, environmental changes (e.g., water depth and, accommodation space) play a major role in the formation and spatial distribution of different microbialite morphologies at the macro- and mesoscale. Differences in the relative abundance of dominant microorganisms between mesostructured types suggest that mesomorphology may be influenced by changes in microbial diversity. Spatial variations in the microbialite morphotypes, along with the abundant presence of entombed biomass (e.g., mineralized filaments), may indicate areas that have a high potential for the preservation of biosignatures., (© 2024 John Wiley & Sons Ltd.)

The origin of the eukaryotic cell, with its compartmentalized nature and generally large size compared with bacterial and archaeal cells, represents a cornerstone event in the evolution of complex life on Earth. In a process referred to as eukaryogenesis, the eukaryotic cell is believed to have evolved between approximately 1.8 and 2.7 billion years ago from its archaeal ancestors, with a symbiosis with a bacterial (proto-mitochondrial) partner being a key event. In the tree of life, the branch separating the first from the last common ancestor of all eukaryotes is long and lacks evolutionary intermediates. As a result, the timing and driving forces of the emergence of complex eukaryotic features remain poorly understood. During the past decade, environmental and comparative genomic studies have revealed vital details about the identity and nature of the host cell and the proto-mitochondrial endosymbiont, enabling a critical reappraisal of hypotheses underlying the symbiotic origin of the eukaryotic cell. Here we outline our current understanding of the key players and events underlying the emergence of cellular complexity during the prokaryote-to-eukaryote transition and discuss potential avenues of future research that might provide new insights into the enigmatic origin of the eukaryotic cell., (© 2024. Springer Nature Limited.)

Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades 1-4 , thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with 'Candidatus Verstraetearchaeota') in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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_2 TS , proposed to serve as the nomenclatural type for the species Asgardarchaeum abyssi TS according to the rules of the SeqCode. Based on protein content and compositional features, we infer that A. abyssi AB3033_2 TS 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon 'Candidatus Harpocratesius repetitus' shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs., (© 2024. The Author(s).)

Microbial communities are not only shaped by the diversity of microorganisms and their individual metabolic potential, but also by the vast amount of intra- and interspecies interactions that can occur pairwise interactions among microorganisms, we suggest that more attention should be drawn towards the effects on the entire microbiome that emerge from individual interactions between community members. The production of certain metabolites that can be tied to a specific microbe-microbe interaction might subsequently influence the physicochemical parameters of the habitat, stimulate a change in the trophic network of the community or create new micro-habitats through the formation of biofilms, similar to the production of antimicrobial substances which might negatively affect only one microorganism but cause a ripple effect on the abundance of other community members. Here, we argue that combining established as well as innovative laboratory and computational methods is needed to predict novel interactions and assess their secondary effects. Such efforts will enable future microbiome studies to expand our knowledge on the dynamics of complex microbial communities., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.

Allopatric speciation has been difficult to examine among microorganisms, with prior reports of endemism restricted to sub-genus level taxa. Previous microbial community analysis via 16S rRNA gene sequencing of 925 geothermal springs from the Taupō Volcanic Zone (TVZ), Aotearoa-New Zealand, revealed widespread distribution and abundance of a single bacterial genus across 686 of these ecosystems (pH 1.2-9.6 and 17.4-99.8 °C). Here, we present evidence to suggest that this genus, Venenivibrio (phylum Aquificota), is endemic to Aotearoa-New Zealand. A specific environmental niche that increases habitat isolation was identified, with maximal read abundance of Venenivibrio occurring at pH 4-6, 50-70 °C, and low oxidation-reduction potentials. This was further highlighted by genomic and culture-based analyses of the only characterised species for the genus, Venenivibrio stagnispumantis CP.B2 T , which confirmed a chemolithoautotrophic metabolism dependent on hydrogen oxidation. While similarity between Venenivibrio populations illustrated that dispersal is not limited across the TVZ, extensive amplicon, metagenomic, and phylogenomic analyses of global microbial communities from DNA sequence databases indicates Venenivibrio is geographically restricted to the Aotearoa-New Zealand archipelago. We conclude that geographic isolation, complemented by physicochemical constraints, has resulted in the establishment of an endemic bacterial genus., (© 2024. The Author(s).)

Members of the candidate phylum Dadabacteria, recently reassigned to the phylum Candidatus Desulfobacterota, are cosmopolitan in the marine environment found both free-living and associated with hosts that are mainly marine sponges. Yet, these microorganisms are poorly characterized, with no cultured representatives and an ambiguous phylogenetic position in the tree of life. Here, we performed genome-centric metagenomics to elucidate their phylogenomic placement and predict the metabolism of the sponge-associated members of this lineage. Rank-based phylogenomics revealed several new species and a novel family (Candidatus Spongomicrobiaceae) within a sponge-specific order, named here Candidatus Nemesobacterales. Metabolic reconstruction suggests that Ca. Nemesobacterales are aerobic heterotrophs, capable of synthesizing most amino acids, vitamins and cofactors and degrading complex carbohydrates. We also report functional divergence between sponge- and seawater-associated metagenome-assembled genomes. Niche-specific adaptations to the sponge holobiont were evident from significantly enriched genes involved in defense mechanisms against foreign DNA and environmental stressors, host-symbiont interactions and secondary metabolite production. Fluorescence in situ hybridization gave a first glimpse of the morphology and lifestyle of a member of Ca. Desulfobacterota. Candidatus Nemesobacterales spp. were found both inside sponge cells centred around sponge nuclei and in the mesohyl of the sponge Geodia barretti. This study sheds light on the enigmatic group Ca. Nemesobacterales and their functional characteristics that reflect a symbiotic lifestyle., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Motyčková et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning order-level divergence ( Pelagibacterales ). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales . We used a new isolate genome, recently released single-amplified genomes and metagenome-assembled genomes, and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with the recruitment of metagenomes spanning the open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade and support the contention that this group represents a taxonomic family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in the transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa. IMPORTANCE One goal of marine microbiologists is to uncover the roles various microorganisms are playing in biogeochemical cycles. Success in this endeavor relies on differentiating groups of microbes and circumscribing their relationships. An early-diverging group (subclade V) of the most abundant bacterioplankton, SAR11, has recently been proposed as a separate lineage that does not share a most recent common ancestor. But beyond phylogenetics, little has been done to evaluate how these organisms compare with SAR11. Our work leverages dozens of new genomes to demonstrate the similarities and differences between subclade V and SAR11. In our analysis, we also establish that subclade V is synonymous with a group of bacteria established from 16S rRNA gene sequences, AEGEAN-169. Subclade V/AEGEAN-169 has clear metabolic distinctions from SAR11 and their shared traits point to remarkable convergent evolution if they do not share a most recent common ancestor., Competing Interests: The authors declare no conflict of interest.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes 1 . However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved 2-4 . Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells., (© 2023. The Author(s).)

Ascetosporea are endoparasites of marine invertebrates that include economically important pathogens of aquaculture species. Owing to their often-minuscule cell sizes, strict intracellular lifestyle, lack of cultured representatives and minimal availability of molecular data, these unicellular parasites remain poorly studied. Here, we sequenced and assembled the genome and transcriptome of Paramikrocytos canceri, an endoparasite isolated from the European edible crab Cancer pagurus. Using bioinformatic predictions, we show that P. canceri likely possesses a mitochondrion-related organelle (MRO) with highly reduced metabolism, resembling the mitosomes of other parasites but with key differences. Like other mitosomes, this MRO is predicted to have reduced metabolic capacity and lack an organellar genome and function in iron-sulfur cluster (ISC) pathway-mediated Fe-S cluster biosynthesis. However, the MRO in P. canceri is uniquely predicted to produce ATP via a partial glycolytic pathway and synthesize phospholipids de novo through the CDP-DAG pathway. Heterologous gene expression confirmed that proteins from the ISC and CDP-DAG pathways retain mitochondrial targeting sequences that are recognized by yeast mitochondria. This represents a unique combination of metabolic pathways in an MRO, including the first reported case of a mitosome-like organelle able to synthesize phospholipids de novo. Some of these phospholipids, such as phosphatidylserine, are vital in other protist endoparasites that invade their host through apoptotic mimicry., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Chlamydiae is a bacterial phylum composed of obligate animal and protist endosymbionts. However, other members of the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum are primarily free living. How Chlamydiae transitioned to an endosymbiotic lifestyle is still largely unresolved. Here we reconstructed Planctomycetes-Verrucomicrobia-Chlamydiae species relationships and modelled superphylum genome evolution. Gene content reconstruction from 11,996 gene families suggests a motile and facultatively anaerobic last common Chlamydiae ancestor that had already gained characteristic endosymbiont genes. Counter to expectations for genome streamlining in strict endosymbionts, we detected substantial gene gain within Chlamydiae. We found that divergence in energy metabolism and aerobiosis observed in extant lineages emerged later during chlamydial evolution. In particular, metabolic and aerobic genes characteristic of the more metabolically versatile protist-infecting chlamydiae were gained, such as respiratory chain complexes. Our results show that metabolic complexity can increase during endosymbiont evolution, adding an additional perspective for understanding symbiont evolutionary trajectories across the tree of life., (© 2023. The Author(s).)

Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems., (© 2022. The Author(s).)

The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales are an alphaproteobacterial order of obligate endosymbionts and parasites that infect a wide variety of eukaryotic hosts, including humans, livestock, insects and protists. Induced by their host-associated lifestyle, Rickettsiales genomes have undergone reductive evolution, leading to small, AT-rich genomes with limited metabolic capacities. Here we uncover eleven deep-branching alphaproteobacterial metagenome assembled genomes from aquatic environments, including data from the Tara Oceans initiative and other publicly available datasets, distributed over three previously undescribed Rickettsiales-related clades. Phylogenomic analyses reveal that two of these clades, Mitibacteraceae and Athabascaceae, branch sister to all previously sampled Rickettsiales. The third clade, Gamibacteraceae, branch sister to the recently identified ectosymbiotic 'Candidatus Deianiraea vastatrix'. Comparative analyses indicate that the gene complement of Mitibacteraceae and Athabascaceae is reminiscent of that of free-living and biofilm-associated bacteria. Ancestral genome content reconstruction across the Rickettsiales species tree further suggests that the evolution of host association in Rickettsiales was a gradual process that may have involved the repurposing of a type IV secretion system., (© 2022. The Author(s).)

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology, and particularly their virome, remain enigmatic. We reassembled and closed the chromosome of Candidatus Odinarchaeum yellowstonii LCB_4, through long-range PCR, revealing CRISPR spacers targeting viral contigs. We found related viruses in the genomes of diverse prokaryotes from geothermal environments, including other Asgard archaea. These viruses open research avenues into the ecology and evolution of Asgard archaea., (© 2022. The Author(s).)

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

The origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1-2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50-280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells., (© 2021. The Author(s).)

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids., (© 2021. The Author(s).)

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.

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.

Halobacteria (henceforth: Haloarchaea) are predominantly aerobic halophiles that are thought to have evolved from anaerobic methanogens. This remarkable transformation most likely involved an extensive influx of bacterial genes. Whether it entailed a single massive transfer event or a gradual stream of transfers remains a matter of debate. To address this, genomes that descend from methanogen-to-halophile intermediates are necessary. Here, we present five such near-complete genomes of Marine Group IV archaea (Hikarchaeia), the closest known relatives of Haloarchaea. Their inclusion in gene tree-aware ancestral reconstructions reveals an intermediate stage that had already lost a large number of genes, including nearly all of those involved in methanogenesis and the Wood-Ljungdahl pathway. In contrast, the last Haloarchaea common ancestor gained a large number of genes and expanded its aerobic respiration and salt/UV resistance gene repertoire. Our results suggest that complex and gradual patterns of gain and loss shaped the methanogen-to-halophile transition.

Microbe-mediated precipitation of Mn-oxides enriched in rare earth elements (REE) and other trace elements was discovered in tunnels leading to the main shaft of the Ytterby mine, Sweden. Defining the spatial distribution of microorganisms and elements in this ecosystem provide a better understanding of specific niches and parameters driving the emergence of these communities and associated mineral precipitates. Along with elemental analyses, high-throughput sequencing of the following four subsystems were conducted: (i) water seeping from a rock fracture into the tunnel, (ii) Mn-oxides and associated biofilm; referred to as the Ytterby Black Substance (YBS) biofilm (iii) biofilm forming bubbles on the Mn-oxides; referred to as the bubble biofilm and (iv) fracture water that has passed through the biofilms. Each subsystem hosts a specific collection of microorganisms. Differentially abundant bacteria in the YBS biofilm were identified within the Rhizobiales (e.g. Pedomicrobium), PLTA13 Gammaproteobacteria, Pirellulaceae, Hyphomonadaceae, Blastocatellia and Nitrospira. These taxa, likely driving the Mn-oxide production, were not detected in the fracture water. This biofilm binds Mn, REE and other trace elements in an efficient, dynamic process, as indicated by substantial depletion of these metals from the fracture water as it passes through the Mn deposit zone. Microbe-mediated oxidation of Mn(II) and formation of Mn(III/IV)-oxides can thus have considerable local environmental impact by removing metals from aquatic environments., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

The origin of eukaryotes is a major open question in evolutionary biology. Multiple hypotheses posit that eukaryotes likely evolved from a syntrophic relationship between an archaeon and an alphaproteobacterium based on H 2 exchange. However, there are no strong indications that modern eukaryotic H 2 metabolism originated from archaea or alphaproteobacteria. Here, we present evidence for the origin of H 2 metabolism genes in eukaryotes from an ancestor of the Anoxychlamydiales-a group of anaerobic chlamydiae, newly described here, from marine sediments. Among Chlamydiae, these bacteria uniquely encode genes for H 2 metabolism and other anaerobiosis-associated pathways. Phylogenetic analyses of several components of H 2 metabolism reveal that Anoxychlamydiales homologs are the closest relatives to eukaryotic sequences. We propose that an ancestor of the Anoxychlamydiales contributed these key genes during the evolution of eukaryotes, supporting a mosaic evolutionary origin of eukaryotic metabolism., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

We identified an alphaproteobacterium in the digestive gland of the abalone species Haliotis discus hannai. This phylotype dominated our 16S rRNA clone libraries from the digestive gland of H. discus hannai. Diversity surveys revealed that this phylotype was associated with H. discus hannai and also in another host species, H. gigantea. Whole genome phylogenies placed this bacterium as a new member affiliated with the family Rhodospirillaceae in Alphaproteobacteria. Gene annotation revealed a nearly complete glycolysis pathway but no TCA cycle, but the presence of anaerobic ribonucleoside-triphosphate reductase and oxygen-insensitive NAD(P)H-dependent nitroreductase, which show the genomic potential for anaerobic metabolism. A large cluster of genes encoding ankyrin repeat proteins (ANK) of eukaryotic-like repeat domains and a large gene set for the flagellar system were also detected. Alginate-binding periplasmic proteins and key genes responsible for alginate assimilation were found in the genome, which could potentially contribute to the breakdown of the host's alginate-rich macroalgal diet. These results raise the possibility that this novel alphaproteobacterium is a widespread member of the abalone microbiome that may use polysaccharides derived from its host's macroalgal diet., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

Background: Most diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed., Results: In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5' biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency., Conclusions: The presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.

Microbial mats or biofilms are known to colonize a wide range of substrates in aquatic environments. These dense benthic communities efficiently recycle nutrients and often exhibit high tolerance to environmental stressors, characteristics that enable them to inhabit harsh ecological niches. In some special cases, floating biofilms form at the air-water interface residing on top of a hydrophobic microlayer. Here, we describe biofilms that reside at the air-air interface by forming gas bubbles (bubble biofilms) in the former Ytterby mine, Sweden. The bubbles are built by micrometer thick membrane-like biofilm that holds enough water to sustain microbial activity. Molecular identification shows that the biofilm communities are dominated by the neuston bacterium Nevskia . Gas bubbles contain mostly air with a slightly elevated concentration of carbon dioxide. Biofilm formation and development was monitored in situ using a time-lapse camera over one year, taking one image every second hour. The bubbles were stable over long periods of time (weeks, even months) and gas build-up occurred in pulses as if the bedrock suddenly exhaled. The result was however not a passive inflation of a dying biofilm becoming more fragile with time (as a result of overstretching of the organic material). To the contrary, microbial growth lead to a more robust, hydrophobic bubble biofilm that kept the bubbles inflated for extended periods (several weeks, and in some cases even months)., Competing Interests: The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors., (© 2020 The Author(s).)

It is generally well accepted that eukaryotes evolved from the symbiosis of an archaeal host cell and an alphaproteobacterium, a union that ultimately gave rise to the complex, eukaryotic cells we see today. However, the catalyst of this merger, the exact nature of the cellular biology of either partner, or how this event spawned the vast majority of complex life on Earth remains enigmatic. In recent years, the discovery of the Asgard archaea, the closest known prokaryotic relatives of eukaryotes, has been monumental for addressing these unanswered questions. These prokaryotes seem to encode an unprecedented number of genes related to features typically descriptive of eukaryotes, including intracellular trafficking, vesicular transport and a dynamic actin-based cytoskeleton. Collectively, these features imply that the Asgard archaea have the potential for cellular complexity previously thought to be unique to eukaryotes. Here, we review the most recent advances in our understanding of the archaeal cytoskeleton and its implications for determining the origin of eukaryotic cellular complexity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The bacterial phylum Chlamydiae is so far composed of obligate symbionts of eukaryotic hosts. Well known for Chlamydiaceae, pathogens of humans and other animals, Chlamydiae also include so-called environmental lineages that primarily infect microbial eukaryotes. Environmental surveys indicate that Chlamydiae are found in a wider range of environments than anticipated previously. However, the vast majority of this chlamydial diversity has been underexplored, biasing our current understanding of their biology, ecological importance, and evolution. Here, we report that previously undetected and active chlamydial lineages dominate microbial communities in deep anoxic marine sediments taken from the Arctic Mid-Ocean Ridge. Reaching relative abundances of up to 43% of the bacterial community, and a maximum diversity of 163 different species-level taxonomic units, these Chlamydiae represent important community members. Using genome-resolved metagenomics, we reconstructed 24 draft chlamydial genomes, expanding by over a third the known genomic diversity in this phylum. Phylogenomic analyses revealed several novel clades across the phylum, including a previously unknown sister lineage of the Chlamydiaceae, providing new insights into the origin of pathogenicity in this family. We were unable to identify putative eukaryotic hosts for these marine sediment chlamydiae, despite identifying genomic features that may be indicative of host-association. The high abundance and genomic diversity of Chlamydiae in these anoxic marine sediments indicate that some members could play an important, and thus far overlooked, ecological role in such environments and may indicate alternate lifestyle strategies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Motivation: Metagenomic and metatranscriptomic sequencing have become increasingly popular tools for producing massive amounts of short-read data, often used for the reconstruction of draft genomes or the detection of (active) genes in microbial communities. Unfortunately, sequence assemblies of such datasets generally remain a computationally challenging task. Frequently, researchers are only interested in a specific group of organisms or genes; yet, the assembly of multiple datasets only to identify candidate sequences for a specific question is sometimes prohibitively slow, forcing researchers to select a subset of available datasets to address their question. Here, we present PhyloMagnet, a workflow to screen meta-omics datasets for taxa and genes of interest using gene-centric assembly and phylogenetic placement of sequences., Results: Using PhyloMagnet, we could identify up to 87% of the genera in an in vitro mock community with variable abundances, while the false positive predictions per single gene tree ranged from 0 to 23%. When applied to a group of metagenomes for which a set of metagenome assembled genomes (MAGs) have been published, we could detect the majority of the taxonomic labels that the MAGs had been annotated with. In a metatranscriptomic setting, the phylogenetic placement of assembled contigs corresponds to that of transcripts obtained from transcriptome assembly., Availability and Implementation: PhyloMagnet is built using Nextflow, available at github.com/maxemil/PhyloMagnet and is developed and tested on Linux. It is released under the open source GNU GPL licence and documentation is available at phylomagnet.readthedocs.io. Version 0.5 of PhyloMagnet was used for all benchmarking experiments., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)

Hydrogenosomes are H2-producing mitochondrial homologs found in some anaerobic microbial eukaryotes that provide a rare intracellular niche for H2-utilizing endosymbiotic archaea. Among ciliates, anaerobic and aerobic lineages are interspersed, demonstrating that the switch to an anaerobic lifestyle with hydrogenosomes has occurred repeatedly and independently. To investigate the molecular details of this transition, we generated genomic and transcriptomic data sets from anaerobic ciliates representing three distinct lineages. Our data demonstrate that hydrogenosomes have evolved from ancestral mitochondria in each case and reveal different degrees of independent mitochondrial genome and proteome reductive evolution, including the first example of complete mitochondrial genome loss in ciliates. Intriguingly, the FeFe-hydrogenase used for generating H2 has a unique domain structure among eukaryotes and appears to have been present, potentially through a single lateral gene transfer from an unknown donor, in the common aerobic ancestor of all three lineages. The early acquisition and retention of FeFe-hydrogenase helps to explain the facility whereby mitochondrial function can be so radically modified within this diverse and ecologically important group of microbial eukaryotes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The origin of eukaryotes represents an unresolved puzzle in evolutionary biology. Current research suggests that eukaryotes evolved from a merger between a host of archaeal descent and an alphaproteobacterial endosymbiont. The discovery of the Asgard archaea, a proposed archaeal superphylum that includes Lokiarchaeota, Thorarchaeota, Odinarchaeota and Heimdallarchaeota suggested to comprise the closest archaeal relatives of eukaryotes, has helped to elucidate the identity of the putative archaeal host. Whereas Lokiarchaeota are assumed to employ a hydrogen-dependent metabolism, little is known about the metabolic potential of other members of the Asgard superphylum. We infer the central metabolic pathways of Asgard archaea using comparative genomics and phylogenetics to be able to refine current models for the origin of eukaryotes. Our analyses indicate that Thorarchaeota and Lokiarchaeota encode proteins necessary for carbon fixation via the Wood-Ljungdahl pathway and for obtaining reducing equivalents from organic substrates. By contrast, Heimdallarchaeum LC2 and LC3 genomes encode enzymes potentially enabling the oxidation of organic substrates using nitrate or oxygen as electron acceptors. The gene repertoire of Heimdallarchaeum AB125 and Odinarchaeum indicates that these organisms can ferment organic substrates and conserve energy by coupling ferredoxin reoxidation to respiratory proton reduction. Altogether, our genome analyses suggest that Asgard representatives are primarily organoheterotrophs with variable capacity for hydrogen consumption and production. On this basis, we propose the 'reverse flow model', an updated symbiogenetic model for the origin of eukaryotes that involves electron or hydrogen flow from an organoheterotrophic archaeal host to a bacterial symbiont.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Large reservoirs of natural gas in the oceanic subsurface sustain complex communities of anaerobic microbes, including archaeal lineages with potential to mediate oxidation of hydrocarbons such as methane and butane. Here we describe a previously unknown archaeal phylum, Helarchaeota, belonging to the Asgard superphylum and with the potential for hydrocarbon oxidation. We reconstruct Helarchaeota genomes from metagenomic data derived from hydrothermal deep-sea sediments in the hydrocarbon-rich Guaymas Basin. The genomes encode methyl-CoM reductase-like enzymes that are similar to those found in butane-oxidizing archaea, as well as several enzymes potentially involved in alkyl-CoA oxidation and the Wood-Ljungdahl pathway. We suggest that members of the Helarchaeota have the potential to activate and subsequently anaerobically oxidize hydrothermally generated short-chain hydrocarbons.

The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, "Megavirales") include the families Poxviridae , Asfarviridae , Iridoviridae , Ascoviridae , Phycodnaviridae , Marseilleviridae , and Mimiviridae , as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki's Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae , have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki's Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome. IMPORTANCE Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches., (Copyright © 2019 Bäckström et al.)

Background: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive., Results: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis., Conclusion: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Endosymbiosis is a widespread phenomenon in the microbial world and can be based on diverse interactions between endosymbiont and host cell. The vast majority of the known endosymbiotic interactions involve bacteria that have invaded eukaryotic host cells. However, methanogenic archaea have been found to thrive in anaerobic, hydrogenosome-containing protists and it was suggested that this symbiosis is based on the transfer of hydrogen. Here, we used culture-independent genomics approaches to sequence the genomes of two distantly related methanogenic endosymbionts that have been acquired in two independent events by closely related anaerobic ciliate hosts Nyctotherus ovalis and Metopus contortus, respectively. The sequences obtained were then validated as originating from the ciliate endosymbionts by in situ probing experiments. Comparative analyses of these genomes and their closest free-living counterparts reveal that the genomes of both endosymbionts are in an early stage of adaptation towards endosymbiosis as evidenced by the large number of genes undergoing pseudogenization. For instance, the observed loss of genes involved in amino acid biosynthesis in both endosymbiont genomes indicates that the endosymbionts rely on their hosts for obtaining several essential nutrients. Furthermore, the endosymbionts appear to have gained significant amounts of genes of potentially secreted proteins, providing targets for future studies aiming to elucidate possible mechanisms underpinning host-interactions. Altogether, our results provide the first genomic insights into prokaryotic endosymbioses from the archaeal domain of life.