Archaeal roots of intramembrane aspartyl protease siblings signal peptide peptidase and presenilin.

Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD…GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.

Deep-sea hydrothermal vent metagenome-assembled genomes provide insight into the phylum Nanoarchaeota.

Ectosymbiotic Nanoarchaeota live on the surface of diverse archaeal hosts. Despite being broadly distributed in global geothermal systems, only three Nanoarchaeota have been successfully co-cultivated with their hosts, and until now no nanoarchaeotal cultures or genomes have been described from deep-sea hydrothermal vents. We recovered three nanoarchaeotal metagenome-assembled genomes (MAGs) from deep-sea hydrothermal vent sites at the Eastern Lau Spreading Center (M10-121), Guaymas Basin (Gua-46) and the Mid-Cayman Rise (MC-1). Based on average amino acid identity analysis, M10-121 is a novel species in the candidate genus Nanoclepta, while the other two MAGs represent novel genera in the Nanoarchaeota. Like previously sequenced Nanoarchaeota, each MAG encodes at least one split protein-coding gene. The MAGs also contain a mosaic of key nanoarchaeotal features, including CRISPR repeat regions and marker genes for gluconeogenesis and archaeal flagella. MC-1 also encodes the pentose bisphosphate pathway, which may allow the nanoarchaeote to bypass several steps in glycolysis and produce ATP.

Single-cell genomics of co-sorted Nanoarchaeota suggests novel putative host associations and diversification of proteins involved in symbiosis.

BACKGROUND: Nanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity. RESULTS: From 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs. CONCLUSIONS: This population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota ('Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of 'Nanopusillus' are among the smallest known cellular organisms (100-300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

Nanoarchaeota, Their Sulfolobales Host, and Nanoarchaeota Virus Distribution across Yellowstone National Park Hot Springs.

Nanoarchaeota are obligate symbionts with reduced genomes first described from marine thermal vent environments. Here, both community metagenomics and single-cell analysis revealed the presence of Nanoarchaeota in high-temperature (∼90°C), acidic (pH ≈ 2.5 to 3.0) hot springs in Yellowstone National Park (YNP) (United States). Single-cell genome analysis of two cells resulted in two nearly identical genomes, with an estimated full length of 650 kbp. Genome comparison showed that these two cells are more closely related to the recently proposed Nanobsidianus stetteri from a more neutral YNP hot spring than to the marine Nanoarchaeum equitans. Single-cell and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis of environmental hot spring samples identified the host of the YNP Nanoarchaeota as a Sulfolobales species known to inhabit the hot springs. Furthermore, we demonstrate that Nanoarchaeota are widespread in acidic to near neutral hot springs in YNP. An integrated viral sequence was also found within one Nanoarchaeota single-cell genome and further analysis of the purified viral fraction from environmental samples indicates that this is likely a virus replicating within the YNP Nanoarchaeota.

Formal proof that the split genes of tRNAs of Nanoarchaeum equitans are an ancestral character.

A proof is given that the genes of the tRNA molecule of Nanoarchaeum equitans split into the 5' and 3' halves are an ancestral trait. First, the existence of a natural succession of evolutionary stages will be proven, formed in the order of the three gene structures of tRNAs known today: (i) the split genes of tRNAs, (ii) the genes of tRNAs with introns, and (iii) the genes of tRNAs continuously codifying for the tRNA molecule. This succession of evolutionary stages identifies the split genes of tRNAs as a pleisiomorphic character. The proof that this succession of evolutionary stages is, moreover, true is performed by proving that all the possible remaining five successions of evolutionary stages are false. Indeed, the succession of evolutionary stages considering split genes as a derived character turns out to be false in that the increase in complexity inherent to this succession cannot be justified by the split genes of tRNAs because these could not have conferred any selective advantage justifying this increase in complexity. Furthermore, genetic drift is unable to explain the evolution of split genes of tRNAs because of the enormous genetic effective size of the population observed in these organisms. The remaining four successions of evolutionary stages are also false because: (i) they are not natural successions of evolutionary stages, (ii) the absolute observed frequencies of these evolutionary stages are such as to exclude categorically that they might be natural successions of evolutionary stages, and also (iii) two of these are falsified by the fact that they do not place the evolutionary stage of genes of tRNAs with introns in a close evolutionary relationship with that of the split genes of tRNAs which can, instead, be proven to have a close evolutionary link. Therefore, there remains only the succession of evolutionary stages considering the split genes of tRNAs codifying for the 5' and 3' halves, as a pleisiomorphic character, as the only succession compatible with all the arguments presented in this article and as the one that actually operated during the evolution of the tRNA molecule. This proof has two very important implications. One regards how the tRNA molecule originated; considering how tRNA originated as the union of two hairpin-like structures, the split genes of tRNAs might be the transition stage through which the evolution of this molecule passed. The other regards when the genes of tRNAs originated, reaching the conclusion that the origin of these genes is polyphyletic, i.e. not monophyletic and hence contrary to the assumptions of the current paradigm.

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments.

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.

The basal phylogenetic position of Nanoarchaeum equitans (Nanoarchaeota).

Using sequences of ribosomal RNA from organisms belonging exclusively to the Archaea domain and by means of two methods to remove the phylogenetic noise, we investigate the phylogenetic position of Nanoarchaeum equitans. The results obtained are compatible with the hypothesis that N. equitans represents a new phylum within the Archaea domain because the characteristic long branch of N. equitans in phylogenetic trees is conserved even after most of the phylogenetic noise has been removed, thus implying that its rRNA might indeed be singular. However, our analysis is unable to be equally as clear on the phylogenetic position of Methanopyrus kandleri.

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

BACKGROUND: Cultivable archaeal species are assigned to two phyla -- the Crenarchaeota and the Euryarchaeota -- by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum -- the Nanoarchaeota -- that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain. RESULTS: We tested the placement of N. equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes. We indicate that the placement of N. equitans in archaeal phylogenies on the basis of ribosomal protein concatenation may be strongly biased by the coupled effect of its above-average evolutionary rate and lateral gene transfers. Indeed, we show that different subsets of ribosomal proteins harbor a conflicting phylogenetic signal for the placement of N. equitans. A BLASTP-based survey of the phylogenetic pattern of all open reading frames (ORFs) in the genome of N. equitans revealed a surprisingly high fraction of close hits with Euryarchaeota, notably Thermococcales. Strikingly, a specific affinity of N. equitans and Thermococcales was strongly supported by phylogenies based on a subset of ribosomal proteins, and on a number of unrelated molecular markers. CONCLUSION: We suggest that N. equitans may more probably be the representative of a fast-evolving euryarchaeal lineage (possibly related to Thermococcales) than the representative of a novel and early diverging archaeal phylum.