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The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus . Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae , which is available at ictv.global/report/turriviridae.

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Proteins in the structural maintenance of chromosomes (SMC) superfamily play key roles in chromosome organization and are ubiquitous across all domains of life. However, SMC proteins are notably absent in the Desulfurococcales of phylum Crenarchaeota. Intrigued by this observation, we performed chromosome conformation capture experiments in the model Desulfurococcales species Aeropyrum pernix. As in other archaea, we observe chromosomal interaction domains across the chromosome. The boundaries between chromosomal interaction domains show a dependence on transcription and translation for their definition. Importantly, however, we reveal an additional higher-order, bipartite organization of the chromosome-with a small high-gene-expression and self-interacting domain that is defined by transcriptional activity and loop structures. Viewing these data in the context of the distribution of SMC superfamily proteins in the Crenarchaeota, we suggest that the organization of the Aeropyrum genome represents an evolutionary antecedent of the compartmentalized architecture observed in the Sulfolobus lineage., (© 2023. The Author(s).)

Analysis of the profile of the tRNA modifications in several Archaea allowed us to observe a novel modified uridine in the V-loop of several tRNAs from two species: Pyrococcus furiosus and Sulfolobus acidocaldarius Recently, Ohira and colleagues characterized 2'-phosphouridine (U p ) at position 47 in tRNAs of thermophilic Sulfurisphaera tokodaii , as well as in several other archaea and thermophilic bacteria. From the presence of the gene arkI corresponding to the RNA kinase responsible for U p 47 formation, they also concluded that U p 47 should be present in tRNAs of other thermophilic Archaea Reanalysis of our earlier data confirms that the unidentified residue in tRNAs of both P. furiosus and S. acidocaldarius is indeed 2'-phosphouridine followed by m 5 C48. Moreover, we find this modification in several tRNAs of other Archaea and of the hyperthermophilic bacterium Aquifex aeolicus ., (© 2023 Wolff et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0-9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg 2+ . Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs., Competing Interests: Declaration of competing interest All authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cell cycle regulation is of paramount importance for all forms of life. Here, we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifesting as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-fold), including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters and 5' UTRs of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg 2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Anti-Clustered regularly interspaced small palindromic repeat (CRISPR) (Acr) phages cooperate to establish a successful infection in CRISPR-containing host. We report here the selective advantage provided by a replication initiator, Rep, toward cooperative host immunosuppression by viruses encoding Acrs. A rep knockout mutant (Δ gp16 ) of Sulfolobus islandicus rod-shaped virus 2 produced around fourfold less virus in a CRISPR-null host, suggesting that Rep is the major replication initiator. In addition to Rep-dependent replication initiation from the viral genomic termini, we detected Rep-independent replication initiation from nonterminal sites. Intriguingly, despite the presence of Acrs, lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus ( rep -containing) outcompeted the Δ gp16 mutant much more quickly in the CRISPR-containing host than in CRISPR-null host. Despite the nonessentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales.

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.

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  o C and at pH 8.0 ∼ 9.0. Furthermore, Sis-UDGV displays a substrate preference for uracil-containing ssDNA over uracil-containing dsDNA, but has no activity and weak activity for excising hypoxanthine from ssDNA and dsDNA, respectively. Importantly, we dissected the roles of seven conserved residues in Sis-UDGV by mutational analyses, demonstrating that residues D91, E117, E128, H167 and R192 are essential for catalysis. To our knowledge, it is the first report on the novel Family V UDG from Archaea with bi-functionality that harbors glycosylase/AP lyase activity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.

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 m values of proteins are mainly distributed around the optimum growth temperature (OGT). The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. The protein thermal stability of S. islandicus cultured under 65 °C and 85 °C was higher than that of 75 °C. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time. SIGNIFICANCE: The molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. Our proteomics study identified 477 differentially expressed proteins of S. islandicus at different temperatures, suggesting that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are affected by temperature. Meanwhile, we found that methylation modification of some proteins changed with temperature. To evaluate the thermal stability of the proteome, we performed thermal proteome profiling to analyze the Tm of proteins under 65 °C-85 °C growth conditions. T m values of proteins are mainly distributed around the optimum growth temperature. The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 Saccharolobus islandicus , and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2α HD and Cmr2α Palm mutants, but the inhibition was diminished in Cmr2α HD,Palm . Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

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'.

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [ 31 P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [ 31 P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg 2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The V max value was 850 μmol/min/mg and the K m for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC 50 of 2.26 mM, compared to a IC 50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [ 31 P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chromosome conformation capture (3C) techniques are emerging as promising approaches to study genome organization in Archaea, the least understood domain of life in terms of chromosome biology. Here, we describe a 3C technique combined with deep sequencing for the hyperthermophilic archaeal genus Sulfolobus . Instead of using restriction enzymes compatible with fill-in labeling, this protocol uses the 4-bp blunt cutter AluI to generate high-resolution (up to 2 kb) contact maps from Sulfolobus species. For complete details on the use and execution of this protocol, please refer to Takemata and Bell (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites. In this study, we explored mutable conserved sites and immutable non-conserved sites by testing random mutations of two thermostable proteins, an esterase from Sulfolobus tokodaii (Sto-Est) and a subtilisin from Thermococcus kodakarensis (Tko-Sub). The subtilisin domain of Tko-Sub needs Ca 2+ ions and the propeptide domain for stability, folding and maturation. The results from the two proteins showed that about one-third of the mutable sites were detected in conserved sites and some non-conserved sites lost enzymatic activity at high temperatures due to mutation. Of the conserved sites in Sto-Est, the sites on the loop, on the surface, and far from the active site are more resistant to mutation. In Tko-Sub, the sites flanking Ca 2+ -binding sites and propeptide were undesirable for mutation. The results presented here serve as an index for selecting mutation sites and contribute to the expansion of available sequence range by introducing mutations at conserved sites., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus , membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Over the last couple of decades there has been considerable progress in the identification and understanding of the mobile genetic elements that are exchanged between microbes in extremely acidic environments, and of the genes piggybacking on them. Numerous plasmid families, unique viruses of bizarre morphologies and lyfe cycles, as well as plasmid-virus chimeras, have been isolated from acidophiles and characterized to varying degrees. Growing evidence provided by omic-studies have shown that the mobile elements repertoire is not restricted to plasmids and viruses, but that a plethora of integrative elements ranging from miniature inverted repeat transposable elements to large integrative conjugative elements populate the genomes of acidophilic bacteria and archaea. This article reviews the diversity of elements that have been found to constitute the flexible genome of acidophiles. Special emphasis is put on the knowledge generated for Sulfolobus (archaea) and species of the bacterial genera Acidithiobacillus and Leptospirillum . Also, recent knowledge on the strategies used by acidophiles to contain deletereous exchanges while allowing innovation, and the emerging details of the molecular biology of these systems, are discussed. Major lacunae in our understanding of the mobilome of acidophilic prokaryotes and topics for further investigations are identified.

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

CRISPR-Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of Interests G.M. and S.S. are co-founders and board members of Twelve Bio., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered λ DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea. IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota., (Copyright © 2020 Zhang et al.)

Sulfolobus spindle-shaped virus 1 is the only UV-inducible member of the virus family Fuselloviridae. Originally isolated from Saccharolobus shibatae B12, it can also infect Saccharolobus solfataricus. Like the CI repressor of the bacteriophage λ, the SSV1-encoded F55 transcription repressor acts as a key regulator for the maintenance of the SSV1 carrier state. In particular, F55 binds to tandem repeat sequences located within the promoters of the early and UV-inducible transcripts. Upon exposure to UV light, a temporally coordinated pattern of gene expression is triggered. In the case of the better characterized bacteriophage λ, the switch from lysogenic to lytic development is regulated by a crosstalk between the virus encoded CI repressor and the host RecA, which regulates also the SOS response. For SSV1, instead, the regulatory mechanisms governing the switch from the carrier to the induced state have not been completely unravelled. In this study we have applied an integrated biochemical approach based on a variant of the EMSA assay coupled to mass spectrometry analyses to identify the proteins associated with F55 when bound to its specific DNA promoter sequences. Among the putative F55 interactors, we identified RadA and showed that the archaeal molecular components F55 and RadA are functional homologs of bacteriophage λ (factor CI) and Escherichia coli (RecA) system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity. IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long host-virus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPR-Cas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair., (Copyright © 2020 DeWerff et al.)

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain., (© 2019 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Fuselloviruses are among the most widespread and best-characterized archaeal viruses. They exhibit remarkable diversity, as the list of members of this family is rapidly growing. However, it has yet to be shown how a fuselloviral genome may undergo variation at the levels of both single nucleotides and sequence stretches. Here, we report the isolation and characterization of four novel spindle-shaped viruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22), from a hot spring in the Philippines. SSV19 is a member of the genus Alphafusellovirus , whereas SSV20-22 belong to the genus Betafusellovirus The genomes of SSV20-SSV22 are identical except for the presence of two large variable regions, as well as numerous sites of single-nucleotide polymorphisms (SNPs) unevenly distributed throughout the genomes and enriched in certain regions, including the gene encoding the putative end filament protein VP4. We show that coinfection of the host with SSV20 and SSV22 led to the formation of an SSV21-like virus, presumably through homologous recombination. In addition, large numbers of SNPs were identified in DNA sequences retrieved by PCR amplification targeting the SSV20-22 vp4 gene from the original enrichment culture, indicating the enormous diversity of SSV20-22-like viruses in the environment. The high variability of VP4 is consistent with its potential role in host recognition and binding by the virus. IMPORTANCE How a virus survives in the arms race with its host is an intriguing question. In this study, we isolated and characterized four novel fuselloviruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22). Interestingly, SSV20-22 differ primarily in two genomic regions and are apparently convertible through homologous recombination during coinfection. Moreover, sites of single-nucleotide polymorphism (SNP) were identified throughout the genomes of SSV20-22 and, notably, enriched in certain regions, including the gene encoding the putative end filament protein VP4, which is believed to be involved in host recognition and binding by the virus., (Copyright © 2020 American Society for Microbiology.)

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA) 1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP) 3-5 . Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain 6 , sculpting a powerful antiviral response 7-10 that can drive viruses to extinction 7,8 . Cyclic nucleotides are increasingly implicated in host-pathogen interactions 11-13 . Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA 4 ). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA 4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA 4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

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 5'-repeat tag of crRNA and 3'-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 5'-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.

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., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Lonely Guy (LOG) proteins are important enzymes in cellular organisms, which catalyze the final step in the production of biologically active cytokinins via dephosphoribosylation. LOG proteins are vital enzymes in plants for the activation of cytokinin precursors, which is crucial for plant growth and development. In fungi and bacteria, LOGs are implicated in pathogenic or nonpathogenic interactions with their plant hosts. However, LOGs have also been identified in the human pathogen Mycobacterium tuberculosis , and the accumulation of cytokinin-degraded products, aldehydes, within bacterial cells is lethal to the bacterium in the presence of nitric oxide, suggesting diverse roles of LOGs in various species. In this study, we conducted biochemical and genetic analysis of a LOG homologue, SiRe_0427, from the hyperthermophilic archaeon Sulfolobus islandicus REY15A. The protein possessed the LOG motif GGGxGTxxE and exhibited phosphoribohydrolase activity on adenosine-5-monophosphate (AMP), similar to LOGs from eukaryotes and bacteria. Alanine mutants at either catalytic residues or substrate binding sites lost their activity, resembling other known LOGs. SiRe_0427 is probably a homotetramer, as revealed by size exclusion chromatography and chemical cross-linking. We found that the gene encoding SiRe_0427 could be knocked out; however, the Δ sire_0427 strain exhibited no apparent difference in growth compared to the wild type, nor did it show any difference in sensitivity to UV irradiation under our laboratory growth conditions. Overall, these findings indicate that archaeal LOG homologue is active as a phosphoribohydrolase. IMPORTANCE Lonely Guy (LOG) is an essential enzyme for the final biosynthesis of cytokinins, which regulate almost every aspect of growth and development in plants. LOG protein was originally discovered 12 years ago in a strain of Oryza sativa with a distinct floral phenotype of a single stamen. Recently, the presence of LOG homologues has been reported in Mycobacterium tuberculosis , an obligate human pathogen. To date, active LOG proteins have been reported in plants, pathogenic and nonpathogenic fungi, and bacteria, but there have been no experimental reports of LOG protein from archaea. In the current work, we report the identification of a LOG homologue active on AMP from Sulfolobus islandicus REY15A, a thermophilic archaeon. The protein likely forms a tetramer in solution and represents a novel evolutionary lineage. The results presented here expand our knowledge regarding proteins with phosphoribohydrolase activities and open an avenue for studying signal transduction networks of archaea and potential applications of LOG enzymes in agriculture and industry., (Copyright © 2019 American Society for Microbiology.)

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA 4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA 4 binding site and a ssRNA catalytic pocket. cOA 4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA 4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA 4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin.", (Copyright © 2019 Elsevier Inc. All rights reserved.)

CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo , we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.

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 Δ slaB 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 Δ slaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The Δ slaA 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., (Copyright © 2019 Zhang et al.)

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Proteins undergo acetylation at the Nε-amino group of lysine residues and the Nα-amino group of the N terminus in Archaea as in Bacteria and Eukarya. However, the extent, pattern and roles of the modifications in Archaea remain poorly understood. Here we report the proteomic analyses of a wild-type Sulfolobus islandicus strain and its mutant derivative strains lacking either a homolog of the protein acetyltransferase Pat (Δ Sis Pat) or a homolog of the Nt-acetyltransferase Ard1 (Δ Sis Ard1). A total of 1708 Nε-acetylated lysine residues in 684 proteins (26% of the total proteins), and 158 Nt-acetylated proteins (44% of the identified proteins) were found in S. islandicus Δ Sis Ard1 grew more slowly than the parental strain, whereas Δ Sis Pat showed no significant growth defects. Only 24 out of the 1503 quantifiable Nε-acetylated lysine residues were differentially acetylated, and all but one of the 24 residues were less acetylated by >1.3 fold in Δ Sis Pat than in the parental strain, indicating the narrow substrate specificity of the enzyme. Six acyl-CoA synthetases were the preferred substrates of Sis Pat in vivo , suggesting that Nε-acetylation by the acetyltransferase is involved in maintaining metabolic balance in the cell. Acetylation of acyl-CoA synthetases by Sis Pat occurred at a sequence motif conserved among all three domains of life. On the other hand, 92% of the acetylated N termini identified were acetylated by Sis Ard1 in the cell. The enzyme exhibited broad substrate specificity and could modify nearly all types of the target N termini of human NatA-NatF. The deletion of the Sis Ard1 gene altered the cellular levels of 18% of the quantifiable proteins (1518) by >1.5 fold. Consistent with the growth phenotype of Δ Sis Ard1, the cellular levels of proteins involved in cell division and cell cycle control, DNA replication, and purine synthesis were significantly lowered in the mutant than those in the parental strain., (© 2019 Cao et al.)

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity. Recently, we demonstrated that the Csa3a regulator protein triggers CCN protospacer-adjacent motif (PAM)-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (i) in recognizing the 5' PAM and 3' nucleotide motif of protospacers and (ii) in determining both the spacer length and its orientation. Furthermore, we identify amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also that of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency, and the former proteins yielded a high incidence of PAM-dependent atypical spacer integration or of PAM-independent spacer integration. We further demonstrated that in plasmid challenge experiments, overexpressed Cas4-mediated defective spacer acquisition in turn potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivo CRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition. IMPORTANCE The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A carries two cas4 genes ( cas4 and csa1 ) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to those recently reported, were identified to be important for these functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition., (Copyright © 2019 American Society for Microbiology.)

Carrier state viral infection constitutes an equilibrium state in which a limited fraction of a cellular population is infected while the remaining cells are transiently resistant to infection. This type of infection has been characterized for several bacteriophages but not, to date, for archaeal viruses. Here we demonstrate that the rudivirus SIRV3 can produce a host-dependent carrier state infection in the model crenarchaeon Sulfolobus. SIRV3 only infected a fraction of a Sulfolobus islandicus REY15A culture over several days during which host growth was unimpaired and no chromosomal DNA degradation was observed. CRISPR spacer acquisition from SIRV3 DNA was induced by coinfecting with the monocaudavirus SMV1 and it was coincident with increased transcript levels from subtype I-A adaptation and interference cas genes. However, this response did not significantly affect the carrier state infection of SIRV3 and both viruses were maintained in the culture over 12 days during which SIRV3 anti-CRISPR genes were shown to be expressed. Transcriptome and proteome analyses demonstrated that most SIRV3 genes were expressed at varying levels over time whereas SMV1 gene expression was generally low. The study yields insights into the basis for the stable infection of SIRV3 and the resistance to the different host CRISPR-Cas interference mechanisms. It also provides a rationale for the commonly observed coinfection of archaeal cells by different viruses in natural environments.

Sulfolobus islandicus Rey15A encodes one Type I-A and two Type III-B systems, all of which are active in mediating nucleic acids interference. However, the effectiveness of each CRISPR system against virus infection was not tested in this archaeon. Here we constructed S. islandicus strains that constitutively express the antiviral immunity from either I-A, or III-B, or I-A plus III-B systems against SMV1 and tested the response of each host to SMV1 infection. We found that, although both CRISPR immunities showed a strong inhibition to viral DNA replication at an early stage of incubation, the host I-A CRISPR immunity gradually lost the control on virus proliferation, allowing accumulation of cellular viral DNA and release of a large number of viral particles. In contrast, the III-B CRISPR immunity showed a tight control on both viral DNA replication and virus particle formation. Furthermore, the SMV1 tolerance to the I-A CRISPR immunity did not result from the occurrence of escape mutations, suggesting the virus probably encodes an anti-CRISPR protein (Acr) to compromise the host I-A CRISPR immunity. Together, this suggests that the interplay between viral Acrs and CRISPR-Cas systems in thermophilic archaea could have shaped the stable virus-host relationship that is observed for many archaeal viruses.