Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. ...Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only a diverse proteome, enriched in membrane proteins, but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results indicate that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. We find that all components of the ESCRT apparatus are encapsidated into EVs. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. In particular, the active EV production appears to coincide with the expression patterns of ESCRT-III-1 and ESCRT-III-2, rather than ESCRT-III, suggesting a prime role of these proteins in EV budding. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.
Summary
Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. ...It serves as a natural host to the extensively studied spindle‐shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus–host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus‐to‐plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.
•Many archaeal viruses display unique virion morphologies and distinct gene contents.•Cosmopolitan fraction of the archaeal virosphere shares history with bacterial and eukaryotic viruses.•Functional ...genomics studies uncovered intricacies of the archaeal virus-host interactions.•Metagenomics shed light on the unknown virosphere associated with uncultivated archaea.•Many archaeal virus groups could evolve from non-viral mobile genetic elements.
Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among viruses of bacteria or eukaryotes. The uniqueness of the virion morphologies is matched by the distinctiveness of the genomes of these viruses, with ∼75% of genes encoding unique proteins, refractory to functional annotation based on sequence analyses. In this review, we summarize the state-of-the-art knowledge on various aspects of archaeal virus genomics. First, we outline how structural and functional genomics efforts provided valuable insights into the functions of viral proteins and revealed intricate details of the archaeal virus-host interactions. We then highlight recent metagenomics studies, which provided a glimpse at the diversity of uncultivated viruses associated with the ubiquitous archaea in the oceans, including Thaumarchaeota, Marine Group II Euryarchaeota, and others. These findings, combined with the recent discovery that archaeal viruses mediate a rapid turnover of thaumarchaea in the deep sea ecosystems, illuminate the prominent role of these viruses in the biosphere. Finally, we discuss the origins and evolution of archaeal viruses and emphasize the evolutionary relationships between viruses and non-viral mobile genetic elements. Further exploration of the archaeal virus diversity as well as functional studies on diverse virus-host systems are bound to uncover novel, unexpected facets of the archaeal virome.
Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we ...investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.
Cells from the three domains of life produce extracellular membrane vesicles (MVs), suggesting that MV production is a fundamental aspect of cellular physiology. We have recently shown that MVs ...produced by the hyperthermophilic archaeon Thermococcus kodakaraensis can be used as vehicles to transfer exogenous recombinant plasmid DNA from cell to cell. Here, we show that Thermococcus nautilus, which harbours three plasmids, pTN1, pTN2 and pTN3, produces MVs, and that some of them selectively incorporate pTN1 and pTN3. Interestingly, pTN3 represents the genome of a defective virus, which encodes signature proteins common to a large group of viruses infecting hosts from all three cellular domains. However, preparations of MVs produced by T. nautilus have a protein composition similar to that of classical MVs from Thermococcales and do not contain the viral major capsid protein encoded by pTN3. Our results suggest that MVs can serve as vehicles for the intercellular transport of viral genomes and facilitate recombination between viral, plasmid and/or cellular chromosomes in the absence of viral infection.
In this article, we – the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) – summarise the results of our activities ...for the period March 2020 – March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).
Abstract Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve ...diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.
Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient ...cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been 'domesticated', that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.
Ammonia-oxidizing archaea (AOA) fromthe phylum Thaumarchaeota are ubiquitous in marine ecosystems and play a prominent role in carbon and nitrogen cycling. Previous studies have suggested that, like ...allmicrobes, thaumarchaea are infected by viruses and that viral predation has a profound impact on thaumarchaeal functioning and mortality, thereby regulating global biogeochemical cycles. However, not a single virus capable of infecting thaumarchaea has been reported thus far. Here we describe the isolation and characterization of three Nitrosopumilus spindle-shaped viruses (NSVs) that infect AOA and are distinct from other known marine viruses. Although NSVs have a narrow host range, they efficiently infect autochthonous Nitrosopumilus strains and display high rates of adsorption to their host cells. The NSVs have linear double-stranded DNA genomes of ∼28 kb that do not display appreciable sequence similarity to genomes of other known archaeal or bacterial viruses and could be considered as representatives of a new virus family, the “Thaspiviridae.” Upon infection, NSV replication leads to inhibition of AOA growth, accompanied by severe reduction in the rate of ammonia oxidation and nitrite reduction. Nevertheless, unlike in the case of lytic bacteriophages, NSV propagation is not associated with detectable degradation of the host chromosome or a decrease in cell counts. The broad distribution of NSVs in AOA-dominated marine environments suggests that NSV predation might regulate the diversity and dynamics of AOA communities. Collectively, our results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important mesophilic archaea.
While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the biofilms formed by archaea. It has been known that ...extracellular filaments in archaea, such as type IV pili, hami, and cannulae, play a part in the formation of archaeal biofilms. We have used cryo-electron microscopy to determine the atomic structure of a previously uncharacterized class of archaeal surface filaments from hyperthermophilic
These filaments, which we call archaeal bundling pili (ABP), assemble into highly ordered bipolar bundles. The bipolar nature of these bundles most likely arises from the association of filaments from at least two different cells. The component protein, AbpA, shows homology, both at the sequence and structural level, to the bacterial protein TasA, a major component of the extracellular matrix in bacterial biofilms, contributing to biofilm stability. We show that AbpA forms very stable filaments in a manner similar to the donor-strand exchange of bacterial TasA fibers and chaperone-usher pathway pili where a β-strand from one subunit is incorporated into a β-sheet of the next subunit. Our results reveal likely mechanistic similarities and evolutionary connection between bacterial and archaeal biofilms, and suggest that there could be many other archaeal surface filaments that are as yet uncharacterized.
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