Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography.

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.

Cell Structure Changes in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus Lacking the S-Layer.

Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the ΔslaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which the cell cycle is tightly regulated. Together, these data suggest that the lack of SlaA results in either cell fusion or irregularities in cell division. Our studies show the key physiological and cellular functions of the S-layer in this archaeal cell.IMPORTANCE The S-layer is considered to be the sole component of the cell wall in Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

The essential genome of the crenarchaeal model Sulfolobus islandicus.

Sulfolobus islandicus is a model microorganism in the TACK superphylum of the Archaea, a key lineage in the evolutionary history of cells. Here we report a genome-wide identification of the repertoire of genes essential to S. islandicus growth in culture. We confirm previous targeted gene knockouts, uncover the non-essentiality of functions assumed to be essential to the Sulfolobus cell, including the proteinaceous S-layer, and highlight essential genes whose functions are yet to be determined. Phyletic distributions illustrate the potential transitions that may have occurred during the evolution of this archaeal microorganism, and highlight sets of genes that may have been associated with each transition. We use this comparative context as a lens to focus future research on archaea-specific uncharacterized essential genes that may provide valuable insights into the evolutionary history of cells.

Novel Sulfolobus Virus with an Exceptional Capsid Architecture.

A novel archaeal virus, denoted Sulfolobus ellipsoid virus 1 (SEV1), was isolated from an acidic hot spring in Costa Rica. The morphologically unique virion of SEV1 contains a protein capsid with 16 regularly spaced striations and an 11-nm-thick envelope. The capsid exhibits an unusual architecture in which the viral DNA, probably in the form of a nucleoprotein filament, wraps around the longitudinal axis of the virion in a plane to form a multilayered disk-like structure with a central hole, and 16 of these structures are stacked to generate a spool-like capsid. SEV1 harbors a linear double-stranded DNA genome of ∼23 kb, which encodes 38 predicted open reading frames (ORFs). Among the few ORFs with a putative function is a gene encoding a protein-primed DNA polymerase. Sixfold symmetrical virus-associated pyramids (VAPs) appear on the surface of the SEV1-infected cells, which are ruptured to allow the formation of a hexagonal opening and subsequent release of the progeny virus particles. Notably, the SEV1 virions acquire the lipid membrane in the cytoplasm of the host cell. The lipid composition of the viral envelope correlates with that of the cell membrane. These results suggest the use of a unique mechanism by SEV1 in membrane biogenesis.IMPORTANCE Investigation of archaeal viruses has greatly expanded our knowledge of the virosphere and its role in the evolution of life. Here we show that Sulfolobus ellipsoid virus 1 (SEV1), an archaeal virus isolated from a hot spring in Costa Rica, exhibits a novel viral shape and an unusual capsid architecture. The SEV1 DNA wraps multiple times in a plane around the longitudinal axis of the virion to form a disk-like structure, and 16 of these structures are stacked to generate a spool-like capsid. The virus acquires its envelope intracellularly and exits the host cell by creating a hexagonal hole on the host cell surface. These results shed significant light on the diversity of viral morphogenesis.

Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus.

Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Exceptionally, a single clone S-1 isolated from an SMV1 + STSV2-infected culture, that carried STSV2-specific spacers and had lost STSV2 but not SMV1, acquired spacers from SMV1. This effect was also reproducible on reinfecting wild-type host cells with a variant SMV1 isolated from the S-1 culture. The SMV1 variant lacked a virion protein ORF114 that was shown to bind DNA. This study also provided evidence for: (i) limits on the maximum sizes of CRISPR loci; (ii) spacer uptake strongly retarding growth of infected cultures; (iii) protospacer selection being essentially random and non-directional, and (iv) the reversible uptake of spacers from STSV2 and SMV1. A hypothesis is presented to explain the interactive conflicts between SMV1 and the host CRISPR immune system.

Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission.

The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.

Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.

Potential role of cellular ESCRT proteins in the STIV life cycle.

We are examining the archaeal virus STIV (Sulfolobus turreted icosahedral virus) in order to elucidate the details of its replication cycle and its interactions with its cellular host, Sulfolobus solfataricus. Infection of Sulfolobus by STIV initiates an unusual cell lysis pathway. One component of this pathway is the formation of pyramid-like structures on the surface of infected cells. Multiple seven-sided pyramid-like structures are formed on infected cells late in the STIV replication cycle. These pyramid-like structures are formed at sites where the Sulfolobus S-layer has been disrupted and through which the cellular membrane protrudes. It is through the pyramid-like structures that virus-induced cell lysis occurs in the final stages of the STIV replication cycle. The pathway and process by which these unusual lysis structures are produced appears to be novel to archaeal viruses and are not related to the well-characterized lysis mechanisms utilized by bacterial viruses. We are interested in elucidating both the viral and cellular components involved with STIV lysis of its infected cell. In particular, we are examining the potential role that Sulfolobus ESCRT (endosomal sorting complex required for transport)-like proteins play during viral infection and lysis. We hypothesize that STIV takes advantage of the Sulfolobus ESCRT machinery for virus assembly, transport and cellular lysis.

Crenarchaeal biofilm formation under extreme conditions.

BACKGROUND: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. METHODOLOGY: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. CONCLUSION: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography.

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.

A unique virus release mechanism in the Archaea.

Little is known about the infection cycles of viruses infecting cells from Archaea, the third domain of life. Here, we demonstrate that the virions of the archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) are released from the host cell through a mechanism, involving the formation of specific cellular structures. Large pyramidal virus-induced protrusions transect the cell envelope at several positions, rupturing the S-layer; they eventually open out, thus creating large apertures through which virions escape the cell. We also demonstrate that massive degradation of the host chromosomes occurs because of virus infection, and that virion assembly occurs in the cytoplasm. Furthermore, intracellular viral DNA is visualized by flow cytometry. The results show that SIRV2 is a lytic virus, and that the host cell dies as a consequence of elaborated mechanisms orchestrated by the virus. The generation of specific cellular structures for a distinct step of virus life cycle is known in eukaryal virus-host systems but is unprecedented in cells from other domains.

Genetics, biochemistry and structure of the archaeal virus STIV.

STIV (Sulfolobus turreted icosahedral virus) has been the subject of detailed structural, genetic, transcriptomic, proteomic and biochemical studies. STIV arguably has been investigated in more detail than any other archaeal virus. As a result, we know more about STIV than other viruses infecting members of the Archaea domain. Like most viruses isolated from crenarchaeal hosts, STIV has little in common with viruses that infect eukaryotic and bacterial hosts and should be considered the founding member of a new virus family. However, despite this lack of obvious homology with other viruses, STIV has components of gene content, replication strategy and particle structure reminiscent of viruses of the Eukarya and Bacteria domains, suggesting an evolutionary relationship between viruses from all domains of life. The present mini-review describes the current knowledge of this virus and insights it has given us into viral and cellular evolution, as well as newly developed tools for the further study of STIV-host interactions.

The flagellar filament structure of the extreme acidothermophile Sulfolobus shibatae B12 suggests that archaeabacterial flagella have a unique and common symmetry and design.

Archaea, constituting a third domain of life between Eubacteria and Eukarya, characteristically inhabit extreme environments. They swim by rotating flagellar filaments that are phenomenologically and functionally similar to those of eubacteria. However, biochemical, genetic and structural evidence has pointed to significant differences but even greater similarity to eubacterial type IV pili. Here we determined the three-dimensional symmetry and structure of the flagellar filament of the acidothermophilic archaeabacterium Sulfolobus shibatae B12 using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Processing of the cryo-negatively stained filaments included analysis of their helical symmetry and subsequent single particle reconstruction. Two filament subunit packing arrangements were identified: one has helical symmetry, similar to that of the extreme halophile Halobacterium salinarum, with ten subunits per 5.3 nm repeat and the other has helically arranged stacked disks with C(3) symmetry and 12 subunits per repeat. The two structures are related by a slight twist. The S. shibatae filament has a larger diameter compared to that of H. salinarum, at the opposite end of the archaeabacterial phylogenetic spectrum, but the basic three-start symmetry and the size and arrangement of the core domain are conserved and the filament lacks a central channel. This similarity suggests a unique and common underlying symmetry for archaeabacterial flagellar filaments.

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure.

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.

Site-specific binding of the 9.5 kilodalton DNA-binding protein ORF80 visualized by atomic force microscopy.

Atomic force microscopy (AFM) has been used to examine the binding properties of the DNA-binding protein ORF80 to DNA. ORF80 is a 9.5 kDa protein that binds site-specifically to double-stranded DNA of the sequence TTAA-N(7)-TTAA. Direct sizing of the protein complexes on DNA fragments from the plasmid pRN1 with AFM shows that the protein ORF80 binds preferentially to two positions. These positions agree well with the ORF80 binding sites determined by footprinting analysis. The measurements allow an estimate of the stoichiometry of the DNA-protein complexes. In contrast to previous results, the single-molecule experiments suggest that only a low number of ORF80 molecules bind to a DNA-binding site.

Intracellular localization of a group II chaperonin indicates a membrane-related function.

Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon Sulfolobus shibatae, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76 degrees C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92 degrees C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in S. shibatae could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C bind to liposomes made from either the bipolar tetraether lipids of Sulfolobus or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.

Single-molecule imaging by atomic force microscopy of the native chaperonin complex of the thermophilic archaeon Sulfolobus solfataricus.

The chaperonin of the extremely thermophilic archaeon Sulfolobus solfataricus has been imaged for the first time under native conditions using the atomic force microscope. This technique allows to visualize the structure of biomolecules in solution under physiological conditions providing a nanometer resolution topographic image of the sample. Single molecule studies can reveal fine structural details, providing a powerful insight into the active conformation of a macromolecule, and also allowing to detect different conformational states corresponding to functional changes.

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC).

The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150-180 micron) to a mean diameter of 42.5 micron (size fraction: 25-45 micron) enhanced the bioleaching rate from 0.05 kg m(-3) h(-1) to 0.098 kg m(-3) h(-1). The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (mu) and yield factor (Y) were 0.018-0.025 h(-1) and 0.67x10(11)-1.45x10(11) cells (g iron)(-1), respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite.

Genetic elements in the extremely thermophilic archaeon Sulfolobus.

This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons.