Search

Abstract TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

Abstract Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1‐based vectors consistently failed to yield any stable host–vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1‐based and pRN2‐based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

Abstract CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together ‘chimeric’ chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

Abstract Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Diverse prokaryotic Agos (pAgos) play potential functions in microbial defense. The functions and mechanisms of a group of full-length yet catalytically inactive pAgos, long-B pAgos, remain unclear. Here, we show that most long-B pAgos are functionally connected with distinct associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. The long-B pAgo-nuclease system (BPAN) is activated by guide RNA-directed target DNA recognition and performs collateral DNA degradation in vitro. In vivo, the system mediates genomic DNA degradation after sensing invading plasmid, which kills the infected cells and results in the depletion of the invader from the cell population. Together, the BPAN system provides immunoprotection via abortive infection. Our data also suggest that the defense strategy is employed by other long-B pAgos equipped with distinct associated proteins.

Summary: Single-stranded DNA-binding proteins (SSBs) have been regarded as indispensable replication factors. Herein, we report that the genes encoding the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in Saccharolobus islandicus REY15A are not essential for cell viability. Interestingly, at a lower temperature (55°C), the protein level of SisSSB increases and the growth of ΔSisssb and ΔSisssbΔSisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and is able to melt RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of cold-shock proteins in E. coli. Importantly, these activities are conserved in the canonical SSBs from Crenarchaeota species that lack bacterial Csp homologs. Overall, our study has clarified the function of the archaeal canonical SSBs which do not function as a DNA-processing factor, but play a role in the processes requiring melting of dsRNA or DNA/RNA hybrid.

Type III CRISPR-Cas10 systems employ multiple immune activities to defend their hosts against invasion from mobile genetic elements (MGEs), including DNase and cyclic oligoadenylates (cOA) synthesis both of which are hosted by the type-specific protein Cas10. Extensive investigations conducted for the activation of Cas accessory proteins by cOAs have revealed their functions in the type III immunity, but the function of the Cas10 DNase in the same process remains elusive. Here, Lactobacillus delbrueckii subsp. Bulgaricus type III-A (Ld) Csm system, a type III CRISPR system that solely relies on its Cas10 DNase for providing immunity, was employed as a model to investigate the DNase function. Interference assay was conducted in Escherichia coli using two plasmids: pCas carrying the LdCsm system and pTarget producing target RNAs. The former functioned as a de facto “CRISPR host element” while the latter, mimicking an invading MGE. We found that, upon induction of immune responses, the fate of each genetic element was determined by their copy numbers: plasmid of a low copy number was selectively eliminated from the E. coli cells regardless whether it represents a de facto CRISPR host or an invader. Together, we reveal, for the first time, that the immune mechanisms of Cas10 DNases are of two folds: the DNase activity is capable of removing low-copy invaders from infected cells, but it also leads to abortive infection when the invader copy number is high.

Abstract Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N6‐threonylcarbamoyl adenosine (t6A) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1‐like (or Gon7‐like), just like eukaryotic KEOPS. Pcc1‐like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1‐Pcc1‐Pcc1‐Kae1), suggesting that Pcc1‐like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1‐like, can be deleted in the wild type and in a t6A modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock‐down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular t6A modification level. In addition, archaeal Cgi121 possesses dsDNA‐binding activity that relies on its tRNA 3ʹ CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t6A modification and the additional function, presumably HR.

ABSTRACT Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

ABSTRACT B-family DNA polymerases (PolBs) of different groups are widespread in Archaea, and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that is poorly characterized is PolB2, whose members occur in many archaea but are predicted to be inactivated forms of DNA polymerase. Here, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme, was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase possesses a robust nucleotide incorporation activity but is devoid of the 3′–5′ exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. This extender polymerase represents a novel type of prokaryotic PolB specialized for DNA damage repair in Archaea. IMPORTANCE In this work, we report that Sulfolobus islandicus Dpo2, a B-family DNA polymerase once predicted to be an inactive form, is a bona fide DNA polymerase functioning in translesion synthesis. S. islandicus Dpo2 is a member of a large group of B-family DNA polymerases (PolB2) that are present in many archaea and some bacteria, and they carry variations in well-conserved amino acids in the functional domains responsible for polymerization and proofreading. However, we found that this prokaryotic B-family DNA polymerase not only replicates undamaged DNA with high fidelity but also extends mismatch and DNA lesion-containing substrates with high efficiencies. With these data, we propose this enzyme functions as an extender polymerase, the first prokaryotic enzyme of this type. Our data also suggest this PolB2 enzyme represents a functional counterpart of the eukaryotic DNA polymerase Pol zeta, an enzyme that is devoted to DNA damage repair.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5′-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Sulfolobuss islandicus Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4’s activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5–6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an “extender” to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Saccharolobus spindle-shaped virus 1 (SSV1) was one of the first viruses identified in the archaeal kingdom. Originally isolated from a Japanese species of Saccharolobus back in 1984, it has been extensively used as a model system for genomic, transcriptomic, and proteomic studies, as well as to unveil the molecular mechanisms governing the host–virus interaction. The purpose of this mini review is to supply a compendium of four decades of research on the SSV1 virus.

Type III CRISPR-Cas RNases from the Csm and Csx families are activated by cyclic oligoadenylates (cOA). Here the authors present the cOA bound Sulfolobus islandicus Csx1 structure, which forms a hexamer and reveal an allosteric mechanism for the activation of Csx1 RNase.

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 Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,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.

Chromosome replication is an essential process for cell division. The mode of chromosome replication has important impacts on the structure of the chromosome and replication speed.

Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage.

Abstract Background Vibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. Raw seafood is often subjected to post-harvest processing and low-temperature storage. To date, very little information is available regarding the biological functions of cold shock proteins (CSPs) in the low-temperature survival of the bacterium. In this study, we determined the complete genome sequence of V. parahaemolyticus CHN25 (serotype: O5:KUT). The two main CSP-encoding genes (VpacspA and VpacspD) were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by V. parahaemolyticus. Results The 5,443,401-bp V. parahaemolyticus CHN25 genome (45.2% G + C) consisted of two circular chromosomes and three plasmids with 4,724 predicted protein-encoding genes. One dual-gene and two single-gene deletion mutants were generated for VpacspA and VpacspD by homologous recombination. The growth of the ΔVpacspA mutant was strongly inhibited at 10 °C, whereas the VpacspD gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. The complementary phenotypes were observed in the reverse mutants (ΔVpacspA-com, and ΔVpacspD-com). The transcriptome data revealed that 12.4% of the expressed genes in V. parahaemolyticus CHN25 were significantly altered in the ΔVpacspA mutant when it was grown at 10 °C. These included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with ATP-binding cassette transporters. However, a low temperature elicited significant expression changes for 10.0% of the genes in the ΔVpacspD mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. The major metabolic pathways that were altered by the dual-gene deletion mutant (ΔVpacspAD) radically differed from those that were altered by single-gene mutants. Comparison of the transcriptome profiles further revealed numerous differentially expressed genes that were shared among the three mutants and regulators that were specifically, coordinately or antagonistically modulated by VpaCspA and VpaCspD. Our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium. Conclusions This study is the first to describe the complete genome sequence of V. parahaemolyticus (serotype: O5:KUT). The gene deletions, complementary insertions, and comparative transcriptomics demonstrate that VpaCspA is a primary CSP in the bacterium, while VpaCspD functions as a growth inhibitor at 10 °C. These results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide.

Arid and semi-arid regions comprise nearly one-fifth of the earth's terrestrial surface. However, the diversities and functions of their soil microbial communities are not well understood, despite microbial ecological importance in driving biogeochemical cycling. Here, we analyzed the geochemistry and microbial communities of the desert soils from Tarim Basin, northwestern China. Our geochemical data indicated half of these soils are saline. Metagenomic analysis showed that bacterial phylotypes (89.72% on average) dominated the community, with relatively small proportions of Archaea (7.36%) and Eukaryota (2.21%). Proteobacteria, Firmicutes, Actinobacteria, and Euryarchaeota were most abundant based on metagenomic data, whereas genes attributed to Proteobacteria, Actinobacteria, Euryarchaeota, and Thaumarchaeota most actively transcribed. The most abundant phylotypes (Halobacterium, Halomonas, Burkholderia, Lactococcus, Clavibacter, Cellulomonas, Actinomycetospora, Beutenbergia, Pseudomonas, and Marinobacter) in each soil sample, based on metagenomic data, contributed marginally to the population of all microbial communities, whereas the putative halophiles, which contributed the most abundant transcripts, were in the majority of the active microbial population and is consistent with the soil salinity. Sample correlation analyses according to the detected and active genotypes showed significant differences, indicating high diversity of microbial communities among the Tarim soil samples. Regarding ecological functions based on the metatranscriptomic data, transcription of genes involved in various steps of nitrogen cycling, as well as carbon fixation, were observed in the tested soil samples. Metatranscriptomic data also indicated that Thaumarchaeota are crucial for ammonia oxidation and Proteobacteria play the most important role in other steps of nitrogen cycle. The reductive TCA pathway and dicarboxylate-hydroxybutyrate cycle attributed to Proteobacteria and Crenarchaeota, respectively, were highly represented in carbon fixation. Our study reveals that the microbial communities could provide carbon and nitrogen nutrients for higher plants in the sandy saline soils of Tarim Basin.

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed.

To investigate DNA damage response in the model crenarchaeon Sulfolobus islandicus, four different DNA damage agents were tested for their effects on cell death of this archaeon, including UV irradiation, methyl methanesulfonate, cisplatin, and 4-nitroquinoline 1-oxide (NQO). Cell death featured with DNA-less cell formation was revealed in DNA damage treatment with each agent. Cellular responses upon NQO treatment were characterized in details, and following sequential events were revealed, including: a modest accumulation of G1/S phase cells, membrane depolarization, proteolytic degradation of chromatin proteins, and chromosomal DNA degradation. Further insights into the process were gained from studying drugs that affect the archaeal ATP synthase, including a proton gradient uncoupler and an ATP synthase inhibitor. Whereas the proton uncoupler-mediated excess proton influx yielded cell death as observed for the NQO treatment, inhibition of ATP synthase attenuated NQO-induced membrane depolarization and DNA-less cell formation. In conclusion, the NQO-induced cell death in S. islandicus is characterized by proteolytic degradation of chromatin protein, and chromosomal DNA degradation, which probably represents a common feature for the cell death induced by different DNA damage agents.

Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H2S. Two large clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins.

Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme was reduced in S. islandicus cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which topR1 mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the S. islandicus CRISPRi cells and that the cells underwent accelerated genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo.

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin–antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus–host interactions and the CRISPR/Cas defence mechanism in Archaea.