A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

Genomic remnants of ancestral methanogenesis and hydrogenotrophy in Archaea drive anaerobic carbon cycling.

Anaerobic methane metabolism is among the hallmarks of Archaea, originating very early in their evolution. Here, we show that the ancestor of methane metabolizers was an autotrophic CO2-reducing hydrogenotrophic methanogen that possessed the two main complexes, methyl-CoM reductase (Mcr) and tetrahydromethanopterin-CoM methyltransferase (Mtr), the anaplerotic hydrogenases Eha and Ehb, and a set of other genes collectively called "methanogenesis markers" but could not oxidize alkanes. Overturning recent inferences, we demonstrate that methyl-dependent hydrogenotrophic methanogenesis has emerged multiple times independently, either due to a loss of Mtr while Mcr is inherited vertically or from an ancient lateral acquisition of Mcr. Even if Mcr is lost, Mtr, Eha, Ehb, and the markers can persist, resulting in mixotrophic metabolisms centered around the Wood-Ljungdahl pathway. Through their methanogenesis remnants, Thorarchaeia and two newly reconstructed order-level lineages in Archaeoglobi and Bathyarchaeia act as metabolically versatile players in carbon cycling of anoxic environments across the globe.

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.

Reconstruction of Archaeal Genomes from Short-Read Metagenomes.

As the majority of biological diversity remains unexplored and uncultured, investigating it requires culture-independent approaches. Archaea in particular suffer from a multitude of issues that make their culturing problematic, from them being frequently members of the rare biosphere, to low growth rates, to them thriving under very specific and often extreme environmental and community conditions that are difficult to replicate. OMICs techniques are state of the art approaches that allow direct high-throughput investigations of environmental samples at all levels from nucleic acids to proteins, lipids, and secondary metabolites. Metagenomics, as the foundation for other OMICs techniques, facilitates the identification and functional characterization of the microbial community members and can be combined with other methods to provide insights into the microbial activities, both on the RNA and protein levels. In this chapter, we provide a step-by-step workflow for the recovery of archaeal genomes from metagenomes, starting from raw short-read sequences. This workflow can be applied to recover bacterial genomes as well.

Agl24 is an ancient archaeal homolog of the eukaryotic N-glycan chitobiose synthesis enzymes.

Protein N-glycosylation is a post-translational modification found in organisms of all domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in Eukaryotes, although the enzyme catalyzing this reaction remains unknown. Here, we report the identification of a thermostable archaeal ß-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed its function as an inverting ß-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed similarities of Agl24 to the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which are catalyzing the same or a similar reaction, respectively. Phylogenetic analysis of Alg14/13 homologs indicates that they are ancient in Eukaryotes, either as a lateral transfer or inherited through eukaryogenesis.

Genetic diversity in terrestrial subsurface ecosystems impacted by geological degassing.

Earth's mantle releases 38.7 ± 2.9 Tg/yr CO2 along with other reduced and oxidized gases to the atmosphere shaping microbial metabolism at volcanic sites across the globe, yet little is known about its impact on microbial life under non-thermal conditions. Here, we perform comparative metagenomics coupled to geochemical measurements of deep subsurface fluids from a cold-water geyser driven by mantle degassing. Key organisms belonging to uncultivated Candidatus Altiarchaeum show a global biogeographic pattern and site-specific adaptations shaped by gene loss and inter-kingdom horizontal gene transfer. Comparison of the geyser community to 16 other publicly available deep subsurface sites demonstrate a conservation of chemolithoautotrophic metabolism across sites. In silico replication measures suggest a linear relationship of bacterial replication with ecosystems depth with the exception of impacted sites, which show near surface characteristics. Our results suggest that subsurface ecosystems affected by geological degassing are hotspots for microbial life in the deep biosphere.

Leave no stone unturned: individually adapted xerotolerant Thaumarchaeota sheltered below the boulders of the Atacama Desert hyperarid core.

BACKGROUND: The hyperarid core of the Atacama Desert is an extremely harsh environment thought to be colonized by only a few heterotrophic bacterial species. Current concepts for understanding this extreme ecosystem are mainly based on the diversity of these few species, yet a substantial area of the Atacama Desert hyperarid topsoil is covered by expansive boulder accumulations, whose underlying microbiomes have not been investigated so far. With the hypothesis that these sheltered soils harbor uniquely adapted microbiomes, we compared metagenomes and geochemistry between soils below and beside boulders across three distantly located boulder accumulations in the Atacama Desert hyperarid core. RESULTS: Genome-resolved metagenomics of eleven samples revealed substantially different microbial communities in soils below and beside boulders, despite the presence of shared species. Archaea were found in significantly higher relative abundance below the boulders across all samples within distances of up to 205 km. These key taxa belong to a novel genus of ammonia-oxidizing Thaumarchaeota, Candidatus Nitrosodeserticola. We resolved eight mid-to-high quality genomes of this genus and used comparative genomics to analyze its pangenome and site-specific adaptations. Ca. Nitrosodeserticola genomes contain genes for ammonia oxidation, the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation pathway, and acetate utilization indicating a chemolithoautotrophic and mixotrophic lifestyle. They also possess the capacity for tolerating extreme environmental conditions as highlighted by the presence of genes against oxidative stress and DNA damage. Site-specific adaptations of the genomes included the presence of additional genes for heavy metal transporters, multiple types of ATP synthases, and divergent genes for aquaporins. CONCLUSION: We provide the first genomic characterization of hyperarid soil microbiomes below the boulders in the Atacama Desert, and report abundant and highly adapted Thaumarchaeaota with ammonia oxidation and carbon fixation potential. Ca. Nitrosodeserticola genomes provide the first metabolic and physiological insight into a thaumarchaeal lineage found in globally distributed terrestrial habitats characterized by various environmental stresses. We consequently expand not only the known genetic repertoire of Thaumarchaeota but also the diversity and microbiome functioning in hyperarid ecosystems. Video Abstract.

Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition.

The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

Saccharibacteria as Organic Carbon Sinks in Hydrocarbon-Fueled Communities.

Organisms of the candidate phylum Saccharibacteria have frequently been detected as active members of hydrocarbon degrading communities, yet their actual role in hydrocarbon degradation remained unclear. Here, we analyzed three enrichment cultures of hydrocarbon-amended groundwater samples using genome-resolved metagenomics to unravel the metabolic potential of indigenous Saccharibacteria. Community profiling based on ribosomal proteins revealed high variation in the enrichment cultures suggesting little reproducibility although identical cultivation conditions were applied. Only 17.5 and 12.5% of the community members were shared between the three enrichment cultures based on ribosomal protein clustering and read mapping of reconstructed genomes, respectively. In one enrichment, two Saccharibacteria strains dominated the community with 16.6% in relative abundance and we were able to recover near-complete genomes for each of them. A detailed analysis of their limited metabolism revealed the capacity for peptide degradation, lactate fermentation from various hexoses, and suggests a scavenging lifestyle with external retrieval of molecular building blocks. In contrast to previous studies suggesting that Saccharibacteria are directly involved in hydrocarbon degradation, our analyses provide evidence that these organisms can be highly abundant scavengers acting rather as organic carbon sinks than hydrocarbon degraders in these communities.

An archaeal origin of the Wood-Ljungdahl H4MPT branch and the emergence of bacterial methylotrophy.

The tetrahydromethanopterin (H4MPT) methyl branch of the Wood-Ljungdahl pathway is shared by archaeal and bacterial metabolisms that greatly contribute to the global carbon budget and greenhouse gas fluxes: methanogenesis and methylotrophy, including methanotrophy1-3. It has been proposed that the H4MPT branch dates back to the last universal common ancestor4-6. Interestingly, it has been identified in numerous recently sequenced and mostly uncultured non-methanogenic and non-methylotrophic archaeal and bacterial lineages, where its function remains unclear5,7. Here, we have examined the distribution and phylogeny of the enzymes involved in the H4MPT branch and the biosynthesis of its cofactors in over 6,400 archaeal and bacterial genomes. We find that a full Wood-Ljungdahl H4MPT pathway is widespread in Archaea and is likely ancestral to this domain, whereas this is not the case for Bacteria. Moreover, the inclusion of recently sequenced lineages leads to an important shortening of the branch separating Archaea and Bacteria with respect to previous phylogenies of the H4MPT branch. Finally, the genes for the pathway are colocalized in many of the recently sequenced archaeal lineages, similar to bacteria. Together, these results weaken the last universal common ancestor hypothesis and rather favour an origin of the H4MPT branch in Archaea and its subsequent transfer to Bacteria. We propose a scenario for its potential initial role in the first bacterial recipients and its evolution up to the emergence of aerobic methylotrophy. Finally, we discuss how an ancient horizontal transfer not only triggered the emergence of key metabolic processes but also important transitions in Earth's history.

Wide diversity of methane and short-chain alkane metabolisms in uncultured archaea.

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth's climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes (MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea. Five of these MAGs represent under-sampled (Verstraetearchaeota, Methanonatronarchaeia, ANME-1 and GoM-Arc1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation substantially expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea ('Candidatus Methanoliparia'). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.

Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a five-subunit enzyme complex responsible for the carbonyl branch of the Wood-Ljungdahl (WL) pathway, considered one of the most ancient metabolisms for anaerobic carbon fixation, but its origin and evolutionary history have been unclear. While traditionally associated with methanogens and acetogens, the presence of CODH/ACS homologs has been reported in a large number of uncultured anaerobic lineages. Here, we have carried out an exhaustive phylogenomic study of CODH/ACS in over 6,400 archaeal and bacterial genomes. The identification of complete and likely functional CODH/ACS complexes in these genomes significantly expands its distribution in microbial lineages. The CODH/ACS complex displays astounding conservation and vertical inheritance over geological times. Rare intradomain and interdomain transfer events might tie into important functional transitions, including the acquisition of CODH/ACS in some archaeal methanogens not known to fix carbon, the tinkering of the complex in a clade of model bacterial acetogens, or emergence of archaeal-bacterial hybrid complexes. Once these transfers were clearly identified, our results allowed us to infer the presence of a CODH/ACS complex with at least four subunits in the last universal common ancestor (LUCA). Different scenarios on the possible role of ancestral CODH/ACS are discussed. Despite common assumptions, all are equally compatible with an autotrophic, mixotrophic, or heterotrophic LUCA. Functional characterization of CODH/ACS from a larger spectrum of bacterial and archaeal lineages and detailed evolutionary analysis of the WL methyl branch will help resolve this issue.

The growing tree of Archaea: new perspectives on their diversity, evolution and ecology.

The Archaea occupy a key position in the Tree of Life, and are a major fraction of microbial diversity. Abundant in soils, ocean sediments and the water column, they have crucial roles in processes mediating global carbon and nutrient fluxes. Moreover, they represent an important component of the human microbiome, where their role in health and disease is still unclear. The development of culture-independent sequencing techniques has provided unprecedented access to genomic data from a large number of so far inaccessible archaeal lineages. This is revolutionizing our view of the diversity and metabolic potential of the Archaea in a wide variety of environments, an important step toward understanding their ecological role. The archaeal tree is being rapidly filled up with new branches constituting phyla, classes and orders, generating novel challenges for high-rank systematics, and providing key information for dissecting the origin of this domain, the evolutionary trajectories that have shaped its current diversity, and its relationships with Bacteria and Eukarya. The present picture is that of a huge diversity of the Archaea, which we are only starting to explore.

HU histone-like DNA-binding protein from Thermus thermophilus: structural and evolutionary analyses.

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus-Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.

Methanogenesis and the Wood-Ljungdahl Pathway: An Ancient, Versatile, and Fragile Association.

Methanogenesis coupled to the Wood-Ljungdahl pathway is one of the most ancient metabolisms for energy generation and carbon fixation in the Archaea. Recent results are sensibly changing our view on the diversity of methane-cycling capabilities in this Domain of Life. The availability of genomic sequences from uncharted branches of the archaeal tree has highlighted the existence of novel methanogenic lineages phylogenetically distant to previously known ones, such as the Methanomassiliicoccales. At the same time, phylogenomic analyses have suggested a methanogenic ancestor for all Archaea, implying multiple independent losses of this metabolism during archaeal diversification. This prediction has been strengthened by the report of genes involved in methane cycling in members of the Bathyarchaeota (a lineage belonging to the TACK clade), representing the first indication of the presence of methanogenesis outside of the Euryarchaeota. In light of these new data, we discuss how the association between methanogenesis and the Wood-Ljungdahl pathway appears to be much more flexible than previously thought, and might provide information on the processes that led to loss of this metabolism in many archaeal lineages. The combination of environmental microbiology, experimental characterization and phylogenomics opens up exciting avenues of research to unravel the diversity and evolutionary history of fundamental metabolic pathways.