We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus . The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P _sac7d -hmg ), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P _sac7d -hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His ₆ -tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers ( pyrEF and hmg ) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (Δ herA ) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus . To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

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. Significance Studies on bacterial and eukaryotic viruses have revealed a range of strategies used by viruses to subdue host cells for efficient virus replication. How archaeal viruses interact with their hosts remains largely unknown. We characterize a new strategy employed by a nonlytic archaeal virus STSV2 to transform its host into a giant virion-producing factory, whereby the virus infection blocks normal cell division by binary fission, leading to gradual cell growth to unprecedented sizes. The giant infected cells divide asymmetrically by budding, replenishing the pool of susceptible hosts. Thus, although tinkering with the cell cycle is a common practice among evolutionarily unrelated viruses from different domains of life, the mechanisms and manifestation of these actions can be highly diverse and unexpected.

Hjm and Hel308a are novel, RecQ-like DNA helicases recently identified in the euryarchaeotesPyrococcus furiosusandMethanothermobacter thermautotrophicus, respectively. In this study, an Hjm/Hel308 homologue (designated StoHjm) fromSulfolobus tokodaii, a hyperthermophilic archaeon belonging to theCrenarchaeotasubdomain of archaea, was cloned, purified, and characterized. Unlike Hjm and Hel308a, which unwind DNA in a 3′-to-5′ direction, StoHjm unwound DNA in both 3′-to-5′ and 5′-to-3′ directions. Remarkably, StoHjm exhibited structure-specific single-stranded-DNA-annealing and fork regression activities in vitro. In addition, gel filtration, affinity pulldown, and yeast two-hybrid analyses revealed that StoHjm physically interacted with StoHjc, the Holliday junction-specific endonuclease fromS. tokodaii. This interaction may have functional significance, because the unwinding activity of StoHjm was inhibited by StoHjc in vitro. These results may suggest that the Hjm/Hel308 family helicases, in association with Hjc endonucleases, are involved in processing of stalled replication forks.

ATPase/Helicases and nucleases play important roles in homologous recombination repair (HRR). Many of the mechanistic details relating to these enzymes and their function in this fundamental and complicated DNA repair process remain poorly understood in archaea. Here we employed Sulfolobus islandicus, a hyperthermophilic archaeon, as a model to investigate the in vivo functions of the ATPase/helicase HerA, the nuclease NurA, and their associated proteins Mre11 and Rad50. We revealed that each of the four genes in the same operon, mre11, rad50, herA, and nurA, are essential for cell viability by a mutant propagation assay. A genetic complementation assay with mutant proteins was combined with biochemical characterization demonstrating that the ATPase activity of HerA, the interaction between HerA and NurA, and the efficient 5′-3′ DNA end resection activity of the HerA-NurA complex are essential for cell viability. NurA and two other putative HRR proteins: a PIN (PilT N-terminal)-domain containing ATPase and the Holliday junction resolvase Hjc, were co-purified with a chromosomally encoded N-His-HerA in vivo. The interactions of HerA with the ATPase and Hjc were further confirmed by in vitro pull down. Efficient 5′-3′ DNA end resection activity of the HerA-NurA complex contributes to necessity of HerA and NurA in Sulfolobus, which is crucial to yield a 3′-overhang in HRR. HerA may have additional binding partners in cells besides NurA.

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.