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

We investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood.

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger 1-3 , activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction 4,5 . Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 6,7 . Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.Type III CRISPR-Cas systems synthesise the signalling molecule cyclic oligoadenylate (cOA) from ATP 1,2 when they detect viral RNA. cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain. CARF domains are found fused to a variety of effector domains that are known or predicted to cleave RNA, DNA, or function as transcription factors 11 . The best characterised CARF protein family is the Csx1/Csm6 family of HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) ribonucleases, which are activated by cOA binding and cleave RNA with minimal sequence dependence 1-3 . A number of studies have demonstrated that the cOA signalling component of type III systems is crucial for effective immunity against viruses 4,12-15 , highlighting the importance of this facet of CRISPR immunity.Recently, we identified a cellular enzyme in Sulfolobus solfataricus, hereafter referred to as the Crn1 family (for "CRISPR associated ring nuclease 1"), that degrades cA4 molecules and thus deactivates the Csx1 ribonuclease in vitro 16 . These enzymes exhibit very slow kinetics, and are thought to act by mopping up cA4 molecules in the cell without compromising the immunity provided by the type III CRISPR system. Unsurprisingly, viruses have responded to the threat of the CRISPR system by evolving a range of anti-CRISPR (Acr) proteins, which are used to inhibit and overcome the cell's CRISPR defences (reviewed in 17 ). Acr's have been identified for the type I-D 18 , I-F , II-A and V-A effector complexes (reviewed in 17,19,20 ), numbering over 40 families 21 , but importantly not for type III systems. We focussed on one of the protein families, DUF1874, conserved and widespread ...

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. Type III CRISPR systems detect viral RNA, resulting in activation of a HD nuclease domain for DNA degradation 1,2 and a Cyclase domain that synthesises cyclic oligoadenylate (cOA) from ATP 3-5. cOA activates defence enzymes with a CARF (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 as playing an important role 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 proviruses. The enzyme uses a novel fold to bind cA 4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise 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. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying their phenotype. We found that this archaeon may not encode a eukarya-type of NER (nucleotide excision repair) pathway because depleting each of the eukaryal NER homologues XPD, XPB and XPF did not impair the DNA repair capacity in their mutants. However, among seven homologous recombination proteins, including RadA, Hel308/Hjm, Rad50, Mre11, HerA, NurA and Hjc, only the Hjc nuclease is dispensable for cell viability. Sulfolobus encodes redundant BER (base excision repair) enzymes such as two uracil DNA glycosylases and two putative apurinic/apyrimidinic lyases, but inactivation of one of the redundant enzymes already impaired cell growth, highlighting their important roles in archaeal DNA repair. Systematically characterizing these mutants and generating mutants lacking two or more DNA repair genes will yield further insights into the genetic mechanisms of DNA repair in this model organism.

Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMIDpcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Dpcna1 or Dpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.

Sulfolobus belongs to the hyperthermophilic archaea and it serves as a model organism to study archaeal molecular biology and evolution. In the last few years, we have focused on developing genetic systems for Sulfolobus islandicus using pyrEF as a selection marker and versatile genetic tools have been developed, including methods for constructing gene knockouts and for identifying essential genes. These genetic tools enable us to conduct genetic analysis on the functions of the genes involved in DNA replication and repair processes in S. islandicus and they should also facilitate in vivo analysis of functions of other genes in this model organism.

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