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All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~10(2)-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.

Transposon insertion mutagenesis is a forward genetic approach that has been widely utilized for genetic characterization of bacteria and single-celled eukaryotes, and its applications are being rapidly expanded into a few archaeal model organisms for gene function analysis. Previously, we developed a Tn5-based in vivo transposon insertion mutagenesis system in the hyperthermophilic crenarchaeon S. islandicucs M.16.4 and defined the essential gene set under laboratory growth conditions. In this chapter, we will mainly focus on presenting details regarding the generation of a near-saturating transposon insertion mutant library in this crenarchaeal model. We envision that the traditional transposon-based forward mutagenesis screening paired with next generation sequencing will greatly speed up the exploration of archaeal genomic features.

Homologous recombination-based gene targeting is a powerful and classic reverse genetics approach to precisely elucidate in vivo gene functions in the organisms across all three domains of life. Gene function studies in Archaea, particularly for those flourishing in inhospitable natural environments that are anaerobic, usually hot, and acidic, have been a great challenge; however, this situation was recently overturned with the increasing availability of genetic manipulation systems in several cultivable archaeal species. In the present chapter, we describe a detailed procedure to rapidly generate gene disruption mutants in the hyperthermophilic crenarchaeon Sulfolobus islandicus via a recently developed Microhomology-Mediated Gene Inactivation (MMGI) approach. We highlight crucial experimental details required to be carefully considered when using the MMGI approach for genetic manipulations. We hope this highly reproducible procedure can supplement existing genetic tools for studying the biology of archaeal order Sulfolobales.

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the

Archaeal viruses with a spindle-shaped virion are abundant and widespread in extremely diverse environments. However, efforts to obtain the high-resolution structure of a spindle-shaped virus have been unsuccessful. Here, we present the structure of SSV19, a spindle-shaped virus infecting the hyperthermophilic archaeonSulfolobussp. E11-6. Our near-atomic structure reveals an unusual sevenfold symmetrical virus tail consisting of the tailspike, nozzle, and adaptor proteins. The spindle-shaped capsid shell is formed by seven left-handed helical strands, constructed of the hydrophobic major capsid protein, emanating from the highly glycosylated tail assembly. Sliding between adjacent strands is responsible for the variation of a virion in size. Ultrathin sections of the SSV19-infected cells show that SSV19 virions adsorb to the host cell membrane through the tail after penetrating the S-layer. The tailspike harbors a putative endo-mannanase domain, which shares structural similarity to aBacteroides thetaiotaomicroendo-mannanase. Molecules of glycerol dibiphytanyl glycerol tetraether lipid were observed in hydrophobic clefts between the tail and the capsid shell. The nozzle protein resembles the stem and clip domains of the portals of herpesviruses and bacteriophages, implying an evolutionary relationship among the archaeal, bacterial, and eukaryotic viruses.

Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in

Sulfolobus islandicus is thermophilic archaea that live in an extreme environment of 75 °C-80 °C and pH 2-3. Currently, the molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. This study utilizes proteomics to analyze the differential expression of S. islandicus proteins at different temperatures. We found that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are all affected by temperature. Methylation modification of some proteins changed with temperature. Thermal proteome profiling (TPP) was used to analyze the thermal stability of proteins under 65 °C-85 °C growth conditions. It is suggested that the T

Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.

The crenarchaeon Sulfolobus acidocaldarius, growing optimally at temperatures between 75 and 80 °C, thrives in volcanic hot spring habitats that are typified by large temperature gradients, which impose frequent temperature stresses on the cells. Heat shock response is characterized by an upregulation of heat shock proteins, but similar to most (hyper-)thermophilic archaea, S. acidocaldarius seems to be able to bear supra-optimal temperatures with a restricted repertoire of chaperones. Here, we study the physiological consequences of continuous high-temperature stress and rapid heat shock for S. acidocaldarius. Growth experiments and cell viability assays demonstrate that temperatures of 85 °C and higher result in a decreased growth rate and, when the cells are rapidly subjected to a heat shock, a dynamic increase in mRNA levels of all relevant heat shock proteins and a subset of transcription regulators is observed. When exponentially growing cultures are exposed to a heat shock, the survival tipping point is situated around 90 °C, and the rate of heating determines whether cells are able to cope with this stress or whether the defense mechanism immediately fails, leading to extensive cell death. In conclusion, S. acidocaldarius does not seem to be better equipped to handle sudden supra-optimal temperature stress than mesophilic organisms.

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

The knowledge of antibiotics produced by Archaea (archaeocins) is still limited. So far, only two types of archaeocins are known: (i) sulfolobicins, produced by the extremely thermophilic Sulfolobus spp. and (ii) haloarcheocins, produced by halophilic archaea. Haloarcheocins were first discovered in the 1980s, but most of their characterisation was solely based on supernatant-based assays. Only a few were successfully purified and sequenced, and even fewer have a proposed biosynthetic model. Furthermore, their mode of action, ecological role and biotechnological potential are still to be explored. Haloarcheocin C8 (HalC8) is the best well-characterised haloarcheocin. We applied an approach of comparative genomics in order to go a step further in the knowledge of their biosynthetic clusters as well as the clusters encoding HalC8-like peptides. These peptides can be classified, at least, into 4 different clades, and there is low gene conservation between them. However, the putative function of some proteins is conserved. These include uncharacterized major facilitator superfamily proteins, transmembrane peptides, DNA-binding transcriptional regulators and proteins with extracellular domains. Our analysis reinforces the association of these proteins with HalC8/HalC8-like biosynthesis. Their functionality is unknown, and, in an era where it is known that haloarchaea are not confined to high salt habitats, the advance in the knowledge of their specialised metabolites will be imperative.

The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)30 over oligo(dC)30 or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)30. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three β-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.

Uracil DNA glycosylase (UDG) can excise uracil from DNA, thus playing an essential role in counteracting mutations. The genome of the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A encodes one putative Family V UDG (Sis-UDGV). Herein, we provide evidence that Sis-UDGV is a bi-functional glycosylase that can not only excise uracil from DNA, but cleave the generated apurinic/apyrimidinic (AP) site, which differs from other reported mono-functional Family V UDG homologs. Intriguingly, the enzyme can cleave DNA containing an AP site, thus suggesting that it might be involved in AP site repair. Biochemical data demonstrate that Sis-UDGV displays maximum activity for uracil removal at 45 °C ∼ 65

Virus–host interactions evolve along a symbiosis continuum from antagonism to mutualism. Long-term associations between virus and host, such as those in chronic infection, will select for traits that drive the interaction towards mutualism, especially when susceptible hosts are rare in the population. Virus–host mutualism has been demonstrated in thermophilic archaeal populations where Sulfolobus spindle-shaped viruses (SSVs) provide a competitive advantage to their host Sulfolobus islandicus by producing a toxin that kills uninfected strains. Here, we determine the genetic basis of this killing phenotype by identifying highly transcribed genes in cells that are chronically infected with a diversity of SSVs. We demonstrate that these genes alone confer growth inhibition by being expressed in uninfected cells via a Sulfolobus expression plasmid. Challenge of chronically infected strains with vector-expressed toxins revealed a nested network of cross-toxicity among divergent SSVs, with both broad and specific toxin efficacies. This suggests that competition between viruses and/or their hosts could maintain toxin diversity. We propose that competitive interactions among chronic viruses to promote their host fitness form the basis of virus–host mutualism. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Virus–host interactions evolve along a symbiosis continuum from antagonism to mutualism. Long-term associations between virus and host, such as those in chronic infection, will select for traits that drive the interaction towards mutualism, especially when susceptible hosts are rare in the population. Virus–host mutualism has been demonstrated in thermophilic archaeal populations whereSulfolobusspindle-shaped viruses (SSVs) provide a competitive advantage to their hostSulfolobus islandicusby producing a toxin that kills uninfected strains. Here, we determine the genetic basis of this killing phenotype by identifying highly transcribed genes in cells that are chronically infected with a diversity of SSVs. We demonstrate that these genes alone confer growth inhibition by being expressed in uninfected cells via aSulfolobusexpression plasmid. Challenge of chronically infected strains with vector-expressed toxins revealed a nested network of cross-toxicity among divergent SSVs, with both broad and specific toxin efficacies. This suggests that competition between viruses and/or their hosts could maintain toxin diversity. We propose that competitive interactions among chronic viruses to promote their host fitness form the basis of virus–host mutualism.This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Since its invention, recombinant protein expression has greatly facilitated our understanding of various cellular processes in different biological systems because theoretically this technique renders any gene to be expressed in a mesophilic host like Escherichia coli, thus allowing functional characterizations of proteins of interest. However, such a practice has only yielded a limited success for proteins encoded in thermophilic archaea since thermophilic proteins are often present in an insoluble form when expressed in E. coli. As a result, it is advantageous to express recombinant proteins of thermophilic archaea in a homologous host, allowing a native form of recombinant protein to be purified and characterized. Here we present a detailed protocol for the homologous expression and purification of proteins in the thermophilic archaeon, Sulfolobus islandicus Rey15A.

Archaea are preferred hosts for CRISPR-Cas systems. This adaptive immune system is not only widespread in archaeal organisms, but different types of CRISPR-Cas also co-exist in the same organism. Sulfolobus islandicus provides a good model for CRISPR research as genetic assays have been developed for revealing CRISPR immunity for the crenarchaeal model, and native ribonucleoprotein effector complexes have been expressed in this crenarchaeon and purified for characterization. Here we report a detailed protocol of purification and characterization of the Sulfolobus islandicus Cmr-β, the largest CRISPR effector known to date. The method can readily be applied to the purification of effectors encoded by other CRISPR-Cas systems in this organism, with the possibility to extend the application to other Sulfolobales.

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition., Understanding the structural basis for the inhibition of archaeal eukaryotic-like RNA polymerases (RNAPs) during virus infection is of interest for drug design. Here, the authors present the cryo-EM structures of apo Sulfolobus acidocaldarius RNAP and the RNAP complex structures with two regulatory factors, RIP and TFS4 that inhibit transcription and discuss their inhibitory mechanisms.

The COVID‐19 pandemic, caused by the SARS‐CoV‐2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin‐like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS‐CoV‐2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS‐CoV‐2 receptor binding domain (RBD) coupled to Dps (RBD‐S‐Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD‐S‐Dps completely protected hACE2‐expressing mice from serious illness and led to viral clearance from the lungs upon SARS‐CoV‐2 infection. Our data highlight that multimerised SARS‐CoV‐2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra‐stable scaffold., Preparation and quality control of coupled antigen–Dps complexes (Ag‐S‐Dps). (A) SDS/PAGE of the three expressed and purified antigens as introduced in Fig. 1C, Coomassie stained. Glycosylation of Spike leads to a fuzzy appearance of its band. RBD‐SpyT2 and Spike‐SpyT2 were expressed in mammalian cells, and SpyT2‐NP was expressed in bacteria, as was the SpyC‐Dps scaffold. (B) Size exclusion chromatography to separate excess antigens after the SpyCatcher/Spytag2 coupling reactions; Superose 6 Increase in PBS. (C) SDS/PAGE of the coupled and purified Ag‐S‐Dps complexes. ‘RT’, no heating; ‘99’, heated to 99 °C. The SpyC‐Dps scaffold alone, as well as all the three coupled complexes show high‐molecular weight complexes, presumably dodecameric, that disappear only after heating of the samples in SDS loading buffer (Coomassie stained). (D) Negative‐stain electron microscopy analyses of the three multimeric Ag‐S‐Dps complexes, showing that all samples form defined and monodisperse spheres that display the antigens on their surface, leading to particles of different sizes for the three differently sized antigens.

Endonuclease III (EndoIII), which is ubiquitous in bacteria, Archaea and eukaryotes, plays an important role in excising thymine glycol (Tg) from DNA. Herein, we present evidence that an EndoIII from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-EndoIII) is capable of removing Tg from DNA at high temperature. Biochemical data show that the optimal temperature and pH of Sis-EndoIII are ca.70 °C and ca.7.0–8.0, respectively. Furthermore, the recombinant Sis-EndoIII retains relative weak activity without a divalent metal ion, and displays maximum activity in the presence of Mg2+ or Ca2+. Additionally, we first revealed the activation energy (Ea) of 39.7 ± 4.2 kcal/mol for Sis-EndoIII to remove Tg from dsDNA. As a bifunctional glycosylase, Sis-EndoIII possesses AP lyase activity in addition to glycosylase activity. Additionally, a covalent intermediate is formed between Sis-EndoIII and Tg-containing dsDNA. Mutational studies demonstrate that residues D50, K133 and D151 in Sis-EndoIII are responsible for removal of Tg from dsDNA and K133 and D151 are essential for formation of the covalent intermediate. To our knowledge, it is the first report of Tg excision by crenarchaeal EndoIII, thus augmenting our understanding on archaeal EndoIII function.

In all organisms, the DNA sequence and the structural organization of chromosomes affect gene expression. The extremely thermophilic crenarchaeon Sulfolobus has one circular chromosome with three origins of replication. We previously revealed that this chromosome has defined A and B compartments that have high and low gene expression, respectively. As well as higher levels of gene expression, the A compartment contains the origins of replication. To evaluate the impact of three-dimensional organization on genome evolution, we characterized the effect of replication origins and compartmentalization on primary sequence evolution in eleven Sulfolobus species. Using single-nucleotide polymorphism analyses, we found that distance from an origin of replication was associated with increased mutation rates in the B but not in the A compartment. The enhanced polymorphisms distal to replication origins suggest that replication termination may have a causal role in their generation. Further mutational analyses revealed that the sequences in the A compartment are less likely to be mutated, and that there is stronger purifying selection than in the B compartment. Finally, we applied the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to show that the B compartment is less accessible than the A compartment. Taken together, our data suggest that compartmentalization of chromosomal DNA can influence chromosome evolution in Sulfolobus. We propose that the A compartment serves as a haven for stable maintenance of gene sequences, while sequences in the B compartment can be diversified.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius. Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications.

In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection.

Type III CRISPR-Cas systems code for a multi-subunit ribonucleoprotein (RNP) complex that mediates DNA cleavage and synthesizes cyclic oligoadenylate (cOA) second messenger to confer anti-viral immunity. Both immune activities are to be activated upon binding to target RNA transcripts by their complementarity to crRNA, and autoimmunity avoidance is determined by extended complementarity between the 5ʹ-repeat tag of crRNA and 3ʹ-flanking sequences of target transcripts (anti-tag). However, as to how the strategy could achieve stringent autoimmunity avoidance remained elusive. In this study, we systematically investigated how the complementarity of the crRNA 5ʹ-tag and anti-tag (i.e., tag complementarity) could affect the interference activities (DNA cleavage activity and cOA synthesis activity) of Cmr-α, a type III-B system in Sulfolobus islandicus Rey15A. The results revealed an increasing suppression on both activities by increasing degrees of tag complementarity and a critical function of the 7(th) nucleotide of crRNA in avoiding autoimmunity. More importantly, mutagenesis of Cmr3α exerts either positive or negative effects on the cOA synthesis activity depending on the degrees of tag complementarity, suggesting that the subunit, coupling with the interaction between crRNA tag and anti-tag, function in facilitating immunity and avoiding autoimmunity in Type III-B systems.

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Å crystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency. ispartof: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY vol:174 pages:252-264 ispartof: location:France status: published

The thermo-acidophilic archaeon, Sulfolobus tokodaii was utilized for the production of20 Pd(0) bionanoparticles from acidic Pd(II) solution. Use of active cells was essential to form well- dispersed Pd(0) nanoparticles located on the cell surface. The particle size could be manipulated22 by modifying the concentration of formate (as electron donor; e-donor) and by addition of23 enzymatic inhibitor (Cu2+) in the range of 14-63 nm mean size. Since robust Pd(II) reduction24 progressed in pre-grown S. tokodaii cells even in the presence of up to 500 mM Cl-, it was possible25 to conversely utilize the effect of Cl- to produce even finer and denser particles in the range of 8.7-26 15 nm mean size. This effect likely resulted from the increasing stability of anionic Pd(II)-chloride27 complex at elevated Cl- concentrations, eventually allowing involvement of greater number of28 initial Pd(0) crystal nucleation sites (enzymatic sites). The catalytic activity (evaluated based on29 Cr(VI) reduction reaction) of Pd(0) bionanoparticles of varying particle size formed under30 different conditions were compared. The finest Pd(0) bionanoparticles obtained at 50 mM Cl-31 (mean 8.7 nm; median 5.6 nm) exhibited the greatest specific Cr(VI) reduction rate, with 4-times32 higher catalytic activity compared to commercial Pd/C. The potential applicability of S. tokodaii33 cells in the recovery of highly catalytic Pd(0) nanoparticles from actual acidic chloride leachate34 was thus suggested.

The crystal structure of the 1-Cys peroxiredoxin from S. islandicus is presented; it assembles into a ring-shaped decamer composed of five homodimers. It is concluded that this Prx6-like protein undergoes decamerization independently of arm-domain cysteines., Aerobic thermoacidophilic archaea belonging to the genus Sulfolobus harbor peroxiredoxins, thiol-dependent peroxidases that assist in protecting the cells from oxidative damage. Here, the crystal structure of the 1-Cys peroxiredoxin from Sulfolobus islandicus, named 1-Cys SiPrx, is presented. A 2.75 Å resolution data set was collected from a crystal belonging to space group P212121, with unit-cell parameters a = 86.8, b = 159.1, c = 189.3 Å, α = β = γ = 90°. The structure was solved by molecular replacement using the homologous Aeropyrum pernix peroxiredoxin (ApPrx) structure as a search model. In the crystal structure, 1-Cys SiPrx assembles into a ring-shaped decamer composed of five homodimers. This quaternary structure corresponds to the oligomeric state of the protein in solution, as observed by size-exclusion chromatography. 1-Cys SiPrx harbors only a single cysteine, which is the peroxidatic cysteine, and lacks both of the cysteines that are highly conserved in the C-terminal arm domain in other archaeal Prx6-subfamily proteins such as ApPrx and that are involved in the association of dimers into higher-molecular-weight decamers and dodecamers. It is thus concluded that the Sulfolobus Prx6-subfamily protein undergoes decamerization independently of arm-domain cysteines.

Several species of Sulfolobales have been used as model organisms in the study of response mechanisms to ultraviolet (UV) irradiation in hyperthermophilic crenarchaea. To date, the transcriptional responses of genes involved in the initiation of DNA replication, transcriptional regulation, protein phosphorylation, and hypothetical function have been observed in Sulfolobales species after UV irradiation. However, due to the absence of knockout experiments, the functions of these genes under in situ UV irradiation have not yet been demonstrated. In the present study, we constructed five gene knockout strains (cdc6-2, tfb3, rio1, and two genes encoding the hypothetical proteins, Saci_0951 and Saci_1302) of Sulfolobus acidocaldarius and examined their sensitivities to UV irradiation. The knockout strains exhibited significant sensitivities to UV-B irradiation, indicating that the five UV-regulated genes play an important role in responses to UV irradiation in vivo. Furthermore, Δcdc6-2, Δrio1, ΔSaci_0951, and Δtfb3 were sensitive to a wide variety of helix-distorting DNA lesions, including UV-induced DNA damage, an intra-strand crosslink, and bulky adducts. These results reveal that cdc6-2, tfb3, rio1, and Saci_0951 are play more important roles in broad responses to helix-distorting DNA damage than in specific responses to UV irradiation.

A small subset of acidic hot springs sampled in Yellowstone National Park yielded rod-shaped viruses which lysed liquid host cultures and formed clear plaques on lawns of host cells. Three isolates chosen for detailed analysis were found to be genetically related to previously described isolates of the Sulfolobus islandicus rod-shaped virus (SIRV), but distinct from them and from each other. Functional stability of the new isolates was assessed in a series of inactivation experiments. UV-C radiation inactivated one of the isolates somewhat faster than bacteriophage λ, suggesting that encapsidation in the SIRV-like virion did not confer unusual protection of the DNA from UV damage. With respect to high temperature, the new isolates were extremely, but not equally, stable. Several chemical treatments were found to inactivate the virions and, in some cases, to reveal apparent differences in virion stability among the isolates. Screening a larger set of isolates identified greater variation of these stability properties but found few correlations among the resulting profiles. The majority of host cells infected by the new isolates were killed, but survivors exhibited heritable resistance, which could not be attributed to CRISPR spacer acquisition or the loss of the pilus-related genes identified by earlier studies. Virus-resistant host variants arose at high frequency and most were resistant to multiple viral strains; conversely, resistant host clones generated virus-sensitive variants, also at high frequency. Virus-resistant cells lacked the ability of virus-sensitive cells to bind virions in liquid suspensions. Rapid interconversion of sensitive and resistant forms of a host strain suggests the operation of a yet-unidentified mechanism that acts to allow both the lytic virus and its host to propagate in highly localized natural populations, whereas variation of virion-stability phenotypes among the new viral isolates suggests that multiple molecular features contribute to the biological durability of these viruses.

The text-book picture of a perfect, well organised metabolism with highly specific enzymes, is challenged by non-enzymatic reactions and promiscuous enzymes. This, so-called ‘underground metabolism’, is a special challenge for hyperthermophilic Archaea that thrive at temperatures above 80 °C and possess modified central metabolic pathways often with promiscuous enzymes. Hence, the question arises how extremely thermophilic Archaea can operate their unusual metabolism at temperatures where many pathway intermediates are unstable? We herein discuss current insights in the underground metabolism and metabolic thermoadaptation of (hyper)thermophilic Archaea. So far, only a few repair enzymes and salvaging pathways have been investigated in Archaea. Studies of the central carbohydrate metabolism indicate that a number of different strategies have evolved: 1) reduction of the concentration of unstable metabolites, 2) different pathway topologies are used with newly induced enzymes, and 3) damaged metabolites are removed via new metabolic pathways.

Archaea have many unique physiological features of which the lipid composition of their cellular membranes is the most striking. Archaeal ether-linked isoprenoidal membranes can occur as bilayers or monolayers, possess diverse polar head groups, and a multiplicity of ring structures in the isoprenoidal cores. These lipid structures are proposed to provide protection from the extreme temperature, pH, salinity, and nutrient-starved conditions that many archaea inhabit. However, many questions remain regarding the synthesis and physiological role of some of the more complex archaeal lipids. In this study, we identify a radical S-adenosylmethionine (SAM) protein in Sulfolobus acidocaldarius required for the synthesis of a unique cyclopentyl head group, known as calditol. Calditol-linked glycerol dibiphytanyl glycerol tetraethers (GDGTs) are membrane spanning lipids in which calditol is ether bonded to the glycerol backbone and whose production is restricted to a subset of thermoacidophilic archaea of the Sulfolobales order within the Crenarchaeota phylum. Several studies have focused on the enzymatic mechanism for the synthesis of the calditol moiety, but to date no protein that catalyzes this reaction has been discovered. Phylogenetic analyses of this putative calditol synthase (Cds) reveal the genetic potential for calditol–GDGT synthesis in phyla other than the Crenarchaeota, including the Korarchaeota and Marsarchaeota. In addition, we identify Cds homologs in metagenomes predominantly from acidic ecosystems. Finally, we demonstrate that deletion of calditol synthesis renders S. acidocaldarius sensitive to extremely low pH, indicating that calditol plays a critical role in protecting archaeal cells from acidic stress.

Significant technical challenges have limited the study of extremophile cell biology. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius with unprecedented resolution. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.

Three-dimensional structure of chromosomes displays diverse patterns across the tree of life, with compartments, interaction domains and loops being quite universally observed. The archaeal kingdom remains understudied to this extent so far, despite representing an interesting area from evolutionary and other perspectives.Here we describe the spatial chromosomal organization of a hyperthermophilic crenarchaeon Thermofilum adornatum strain 1910b based on high-throughput chromosome conformation capture (3C-seq) approach. The chromosome contact map showed a curved secondary diagonal almost orthogonal to the main one. No evidence of chromosome loops was present. We were able to identify boundaries of different strengths between chromosome interaction domains (CIDs) albeit moderate. The plaid-like patterns previously reported for Sulfolobus archaea were not observed. However, the calculation of A/B compartments divided the genome into 2 domains that were different by the density of predicted highly expressed genes and location of origins.Further comparison of these domains with whole-genome gene expression profiles will allow to test whether these domains represent expression-associated compartments. If so, it is possible that they represent primitive compartments evolutionarily older than the plaid patterns of Sulfolobus and higher eukaryotes. Further exploration of 3D chromatin in all branches of archaeal diversity will elucidate the evolution of the links between structural and functional organization in live organisms.

International audience; Archaeal viruses represent one of the most mysterious parts of the global virosphere, with many virus groups sharing no evolutionary relationship to viruses of bacteria or eukaryotes. How these viruses interact with their hosts remains largely unexplored. Here we show that nonlytic lemon-shaped virus STSV2 interferes with the cell cycle control of its host, hyperthermophilic and acidophilic archaeon Sulfolobus islandicus, arresting the cell cycle in the S phase. STSV2 infection leads to transcriptional repression of the cell division machinery, which is homologous to the eukaryotic endosomal sorting complexes required for transport (ESCRT) system. The infected cells grow up to 20-fold larger in size, have 8,000-fold larger volume compared to noninfected cells, and accumulate massive amounts of viral and cellular DNA. Whereas noninfected Sulfolobus cells divide symmetrically by binary fission, the STSV2-infected cells undergo asymmetric division, whereby giant cells release normal-sized cells by budding, resembling the division of budding yeast. Reinfection of the normal-sized cells produces a new generation of giant cells. If the CRISPR-Cas system is present, the giant cells acquire virus-derived spacers and terminate the virus spread, whereas in its absence, the cycle continues, suggesting that CRISPR-Cas is the primary defense system in Sulfolobus against STSV2. Collectively, our results show how an archaeal virus manipulates the cell cycle, transforming the cell into a giant virion-producing factory.

Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus . This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae.

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C(1) compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea. IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea. Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10−3) than that of the wild type (4.6 × 10−6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

The Sulfolobus Spindle-shaped Virus (SSV) system is a model for studying thermophilic archaeal virus biology. Several factors make the SSV system amenable to studying archaeal genetics and virus-host interactions in extreme environments. It has been shown that populations of Sulfolobus, the natural host, exhibit biogeographic structure. The acidic (pH

Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.

Over the past decade, advances in methodologies for the determination of chromosome conformation have provided remarkable insight into the local and higher-order organization of bacterial and eukaryotic chromosomes. Locally folded domains are found in both bacterial and eukaryotic genomes, although they vary in size. Importantly, genomes of metazoans also possess higher-order organization into A- and B-type compartments, regions of transcriptionally active and inactive chromatin, respectively. Until recently, nothing was known about the organization of genomes of organisms in the third domain of life – the archaea. However, despite archaea possessing simple circular genomes that are morphologically reminiscent of those seen in many bacteria, a recent study of archaea of the genus Sulfolobus has revealed that it organizes its genome into large-scale domains. These domains further interact to form defined A- and B-type compartments. The interplay of transcription and localization of a novel structural maintenance of chromosomes (SMC) superfamily protein, termed coalescin, defines compartment identity. In this Review, we discuss the mechanistic and evolutionary implications of these findings.

The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.

The Sulfolobus Spindle-shaped Virus (SSV) system has become a model for studying thermophilic virus biology, including archaeal host-virus interactions and biogeography. Several factors make the SSV system amenable to studying archaeal genetic mechanisms (e.g., CRISPRs) as well as virus-host interactions in high temperature acidic environments. First, it has been shown that endemic populations of Sulfolobus, the reported SSV host, exhibit biogeographic structure. Second, the acidic (pHhost switching. Third, SSVs and their hosts are readily cultured in liquid media and on gellan gum plates. Fourth, given the wide geographic separation between the various SSV-Sulfolobus habitats, the system is amenable for studying allopatric versus sympatric virus-host interactions. Previously, we reported that SSVs exhibit differential infectivity on allopatric and sympatric hosts. We also noticed a wide host range for virus strain SSV9 (a.k.a., SSVK1). For decades, SSVs have been described as “non-lytic” dsDNA viruses that infect species of the genus Sulfolobus and release virions via “blebbing” or “budding” as a preferred strategy over host lysis. Here, we show that SSVs infect more than one genus of the family Sulfolobaceae and, in allopatric hosts, SSV9 does not appear to release virions by blebbing. Instead, SSV9 appears to lyse all susceptible allopatric hosts tested, while exhibiting canonical non-lytic viral release via “blebbing” (historically reported for all other SSVs), on a single sympatric host. Lytic versus non-lytic virion release does not appear to be driven by multiplicity of infection (MOI). Greater relative stability of SSV9 compared to other SSVs (i.e., SSV1) in high temperature, low pH environments may contribute to higher transmission rates. However, neither higher transmission rate nor relative virulence in SSV9 infection drives replication profile (i.e., lytic versus non-lytic) in susceptible hosts. Although it is known that CRISPR-Cas systems offer protection against viral infection in prokaryotes, CRISPRS are not reported to be a determinant virus replication strategy. Thus, the genetic/molecular mechanisms underlying SSV9-induced lysis are unknown. These results suggest that there are unknown genetic elements, resulting from allopatric evolution, that drive virion release strategy in specific host strain-SSV strain pairings.

A gene encoding a dye-linked d -amino acid dehydrogenase (Dye-DADH) homologue was found in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. The predicted amino acid sequence suggested that the gene product is a membrane-bound type enzyme. The gene was overexpressed in Escherichia coli, but the recombinant protein was exclusively produced as an inclusion body. In order to avoid production of the inclusion body, an expression system using the thermoacidophilic archaeon Sulfolobus acidocaldarius instead of E. coli as the host cell was constructed. The gene was successfully expressed in Sulfolobus acidocaldarius, and its product was purified to homogeneity and characterized. The purified enzyme catalyzed the dehydrogenation of various d -amino acids, with d -phenylalanine being the most preferred substrate. The enzyme retained its full activity after incubation at 90 °C for 30 min and after incubation at pH 4.0–11.0 for 30 min at 50 °C. This is the first report on membrane-bound Dye-DADH from thermophilic archaea that was successfully expressed in an archaeal host.

Tuberculosis is the most dangerous disease causing maximum deaths than any other, caused by single infectious agent. Due to multidrug resistant of Mycobacterium tuberculosis strains, there is a need of new drugs and drug targets. In this work, we have selected RmlD (α-dTDP-6-deoxy-lyxo-4-hexulose reductase) in the dTDP Rhamnose pathway as drug target to control tuberculosis using Rhodanine analogues. In order to study interaction of RmlD with Rhodanine analogues, a three-dimensional model based on crystal structures such as 1VLO from Clostridium, 1KBZ from Salmonella typhimurium, and 2GGS from Sulfolobus was generated using Modeller 9v7. The modeled structure reliability has been checked using programs such as Procheck, What if, Prosa, Verify 3D, and Errat. In an attempt to find new inhibitors for RmlD enzyme, docking studies were done with a series of Rhodanine and its analogues. Detailed analysis of enzyme-inhibitor interactions identified specific key residues, SER5, VAL9, ILE51, HIS54, and GLY55 which were important in forming hydrogen bonds in binding affinity. Homology modeling and docking studies on RmlD model provided valuable insight information for designing better inhibitors as novel anti-tuberculosis drugs by rational method.

Motility structures are vital in all three domains of life. In Archaea, motility is mediated by the archaellum, a rotating type IV pilus-like structure that is a unique nanomachine for swimming motility in nature. Whereas periplasmic FlaF binds the surface layer (S-layer), the structure, assembly and roles of other periplasmic components remain enigmatic, limiting our knowledge of the archaellum’s functional interactions. Here, we find that the periplasmic protein FlaG and the association with its paralogue FlaF are essential for archaellation and motility. Therefore, we determine the crystal structure of Sulfolobus acidocaldarius soluble FlaG (sFlaG), which reveals a β-sandwich fold resembling the S-layer-interacting FlaF soluble domain (sFlaF). Furthermore, we solve the sFlaG(2)–sFlaF(2) co-crystal structure, define its heterotetrameric complex in solution by small-angle X-ray scattering and find that mutations that disrupt the complex abolish motility. Interestingly, the sFlaF and sFlaG of Pyrococcus furiosus form a globular complex, whereas sFlaG alone forms a filament, indicating that FlaF can regulate FlaG filament assembly. Strikingly, Sulfolobus cells that lack the S-layer component bound by FlaF assemble archaella but cannot swim. These collective results support a model where a FlaG filament capped by a FlaG–FlaF complex anchors the archaellum to the S-layer to allow motility.

Summary Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. Although similar techniques have been applied to the study of halophilic archaea [1, 2, 3, 4, 5], our ability to explore the cell biology of thermophilic archaea has been limited by the technical challenges of imaging at high temperatures. Sulfolobus are the most intensively studied members of TACK archaea and have well-established molecular genetics [6, 7, 8, 9]. Additionally, studies using Sulfolobus were among the first to reveal striking similarities between the cell biology of eukaryotes and archaea [10, 11, 12, 13, 14, 15]. However, to date, it has not been possible to image Sulfolobus cells as they grow and divide. Here, we report the construction of the Sulfoscope, a heated chamber on an inverted fluorescent microscope that enables live-cell imaging of thermophiles. By using thermostable fluorescent probes together with this system, we were able to image Sulfolobus acidocaldarius cells live to reveal tight coupling between changes in DNA condensation, segregation, and cell division. Furthermore, by imaging deletion mutants, we observed functional differences between the two ESCRT-III proteins implicated in cytokinesis, CdvB1 and CdvB2. The deletion of cdvB1 compromised cell division, causing occasional division failures, whereas the ΔcdvB2 exhibited a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRT-III polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division., Graphical Abstract, Highlights • Development of a heating chamber for live imaging of hyperthermophiles • Live imaging of cell division in the archaeon Sulfolobus acidocaldarius • DNA reorganization is tightly coupled to cell division in Sulfolobus acidocaldarius • ESCRT-III homologs play distinct roles in ensuring robust and symmetric division, Pulschen et al. describe the “Sulfoscope,” a microscopy platform that allows live imaging of hyperthermophiles. By imaging cell division in the archaeon Sulfolobus, they observe tight coupling between DNA reorganization and cytokinesis and complementary roles for two ESCRT-III homologs in ensuring robust and symmetric cytokinesis.

We have determined the cryo-electron microscopic (cryo-EM) structures of two archaeal type IV pili (T4P), from Pyrobaculum arsenaticum and Saccharolobus solfataricus, at 3.8 Å and 3.4 Å resolution, respectively. This triples the number of high resolution archaeal T4P structures, and allows us to pinpoint the evolutionary divergence of bacterial T4P, archaeal T4P and archaeal flagellar filaments. We suggest that extensive glycosylation previously observed in T4P of Sulfolobus islandicus is a response to an acidic environment, as at even higher temperatures in a neutral environment much less glycosylation is present for Pyrobaculum than for Sulfolobus and Saccharolobus pili. Consequently, the Pyrobaculum filaments do not display the remarkable stability of the Sulfolobus filaments in vitro. We identify the Saccharolobus and Pyrobaculum T4P as host receptors recognized by rudivirus SSRV1 and tristromavirus PFV2, respectively. Our results illuminate the evolutionary relationships among bacterial and archaeal T4P filaments and provide insights into archaeal virus-host interactions., Archaeal type IV pili (T4P) mediate adhesion to surfaces and are receptors for hyperthermophilic archaeal viruses. Here, the authors present the cryo-EM structures of two archaeal T4P from Pyrobaculum arsenaticum and Saccharolobus solfataricus and discuss evolutionary relationships between bacterial T4P, archaeal T4P and archaeal flagellar filaments.