Inactivation of Target RNA Cleavage of a III-B CRISPR-Cas System Induces Robust Autoimmunity in Saccharolobus islandicus.

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.

A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.

Pararcticibacter amylolyticus gen. nov., sp. nov., Isolated from a Rotten Hemp Rope, and Reclassification of Pedobacter tournemirensis as Pararcticibacter tournemirensis comb. nov.

A Gram-stain-negative, rod-shaped, non-motile, facultatively anaerobic bacterium, designated FJ4-8T, was isolated from a rotten hemp rope in Chongqing City, PR China. Phylogenetic analysis of 16S rRNA gene sequences indicated that the isolate was closely related to members of the family Sphingobacteriaceae, with the highest similarity to Pedobacter tournemirensis TF5-37.2-LB10T (95.3%) and low similarities to all other species of the genus Pedobacter (90.4-93.9%). Phylogenetic analyses demonstrated that strain FJ4-8T formed a stable subclade with Pedobacter tournemirensis TF5-37.2-LB10T. The clade with these two strains branched adjacent to a clade containing three species of the genus Arcticibacter. MK-7 was detected as the only respiratory quinone. The major fatty acids composed iso-C15:0, iso-C17:0 3-OH and summed feature three. Phosphatidylethanolamine, three aminophospholipids and one unidentified lipid were found as the major polar lipids. The major polyamine was identified as sym-homospermidine. The DNA-DNA hybridization value between strain FJ4-8T and Pedobacter tournemirensis TF5-37.2-LB10T was 42.0 ± 2.5%. Based on its phylogenetic, chemotaxonomic and phenotypic characteristics, the novel strain and TF5-37.2-LB10T were found to be different from members of genera Pedobacter and Arcticibacter. FJ4-8T and TF5-37.2-LB10T represented different species. Therefore, FJ4-8T should be classified as a novel species of a novel genus in the family Sphingobacteriaceae, for which the name Pararcticibacter amylolyticus gen. nov., sp. nov. is proposed. The type strain is FJ4-8T (= KCTC 62640T = CCTCC AB 2018052T). The draft genome sequence is 6290, 449 bp in length, the genomic DNA G+C content was 44.4 mol%. Pedobacter tournemirensis TF5-37.2-LB10T should be transferred to the novel genus as Pararcticibacter tournemirensis comb. nov. (The type strain is TF5-37.2-LB10T (= DSM 23085T = CIP 110085T = MOLA 820T).

An Orc1/Cdc6 ortholog functions as a key regulator in the DNA damage response in Archaea.

While bacteria and eukaryotes show distinct mechanisms of DNA damage response (DDR) regulation, investigation of ultraviolet (UV)-responsive expression in a few archaea did not yield any conclusive evidence for an archaeal DDR regulatory network. Nevertheless, expression of Orc1-2, an ortholog of the archaeal origin recognition complex 1/cell division control protein 6 (Orc1/Cdc6) superfamily proteins was strongly activated in Sulfolobus solfataricus and Sulfolobus acidocaldarius upon UV irradiation. Here, a series of experiments were conducted to investigate the possible functions of Orc1-2 in DNA damage repair in Sulfolobus islandicus. Study of DDR in Δorc1-2 revealed that Orc1-2 deficiency abolishes DNA damage-induced differential expression of a large number of genes and the mutant showed hypersensitivity to DNA damage treatment. Reporter gene and DNase I footprinting assays demonstrated that Orc1-2 interacts with a conserved hexanucleotide motif present in several DDR gene promoters and regulates their expression. Manipulation of orc1-2 expression by promoter substitution in this archaeon revealed that a high level of orc1-2 expression is essential but not sufficient to trigger DDR. Together, these results have placed Orc1-2 in the heart of the archaeal DDR regulation, and the resulting Orc1-2-centered regulatory circuit represents the first DDR network identified in Archaea, the third domain of life.

A transcriptional factor B paralog functions as an activator to DNA damage-responsive expression in archaea.

Previously it was shown that UV irradiation induces a strong upregulation of tfb3 coding for a paralog of the archaeal transcriptional factor B (TFB) in Sulfolobus solfataricus, a crenarchaea. To investigate the function of this gene in DNA damage response (DDR), tfb3 was inactivated by gene deletion in Sulfolobus islandicus and the resulting Δtfb3 was more sensitive to DNA damage agents than the original strain. Transcriptome analysis revealed that a large set of genes show TFB3-dependent activation, including genes of the ups operon and ced system. Furthermore, the TFB3 protein was found to be associated with DDR gene promoters and functional dissection of TFB3 showed that the conserved Zn-ribbon and coiled-coil motif are essential for the activation. Together, the results indicated that TFB3 activates the expression of DDR genes by interaction with other transcriptional factors at the promoter regions of DDR genes to facilitate the formation of transcription initiation complex. Strikingly, TFB3 and Ced systems are present in a wide range of crenarchaea, suggesting that the Ced system function as a primary DNA damage repair mechanism in Crenarchaeota. Our findings further suggest that TFB3 and the concurrent TFB1 form a TFB3-dependent DNA damage-responsive circuit with their target genes, which is evolutionarily conserved in the major lineage of Archaea.

Cmr1 enables efficient RNA and DNA interference of a III-B CRISPR-Cas system by binding to target RNA and crRNA.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems provide adaptive immunity against invasive nucleic acids guided by CRISPR RNAs (crRNAs) in archaea and bacteria. Type III CRISPR-Cas effector complexes show RNA cleavage and RNA-activated DNA cleavage activity, representing the only known system of dual nucleic acid interference. Here, we investigated the function of Cmr1 by genetic assays of DNA and RNA interference activity in the mutants and biochemical characterization of their mutated Cmr complexes. Three cmr1α mutants were constructed including ΔßΔ1α, Δß1α-M1 and Δß1α-M2 among which the last two mutants carried a double and a quadruple mutation in the first α-helix region of Cmr1α. Whereas the double mutation of Cmr1α (W58A and F59A) greatly influenced target RNA capture, the quadruple mutation almost abolished crRNA binding to Cmr1α. We found that Cmr2α-6α formed a stable core complex that is active in both RNA and DNA cleavage and that Cmr1α strongly enhances the basal activity of the core complex upon incorporation into the ribonucleoprotein complex. Therefore, Cmr1 functions as an integral activation module in III-B systems, and the unique occurrence of Cmr1 in III-B systems may reflect the adaptive evolution of type III CRISPR-Cas systems in thermophiles.

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.

Harnessing Type I and Type III CRISPR-Cas systems for genome editing.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.

Antifungal activity of chitooligosaccharides against the dermatophyte Trichophyton rubrum.

Antifungal activity against the dermatophytic fungus Trichophyton rubrum by a well-characterized chitooligosaccharides (COS) sample, hydrolyzed using a recombinant chitosanase, was investigated in vitro and in vivo. The minimum inhibitory concentration (MIC) of COS ranged between 0.25 and 0.50%, which was measured using a microdilution method. Analysis of inhibition rates using an agar diffusion method showed that treatment with 0.5% and 1% COS significantly suppressed T. rubrum cell growth (p<0.05 and p<0.01, respectively, in comparison with untreated control). Morphological changes and structural alterations of cells were observed by TEM. In vivo efficacy of COS in treatment of T. rubrum dermatophytosis was evaluated using a guinea pig model. Skin lesion scores revealed a strong, dose-dependent therapeutic effect of COS. The 5% COS group showed a reduction of skin lesions even greater than that of the positive control group treated with 1% fluconazole (FCZ). Histological analysis revealed no inflammation or tissue destruction in the groups treated with 5% COS or 1% FCZ. Hyperkeratosis was also observed, perhaps resulting from a defensive response of the tissue cells to COS. The findings indicate that COS has excellent potential for development of novel antifungal drugs for clinical treatment/remission of dermatophytoses.

An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference.

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-ß) in Sulfolobus islandicus, a genetic assay was developed using plasmids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis identified a trinucleotide sequence in the 3'-region of crRNA that was crucial for RNA interference. Studying mutants lacking Cmr-α or Cmr-ß system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed distinctive interference features. Whereas Cmr-ß complexes targeted transcripts and could be recycled in RNA cleavage, Cmr-α complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr-ß exhibited much stronger RNA cleavage activity than Cmr-α. Since we previously showed that S. islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA.

Isolation, characterization, and antitumor activity of a novel heteroglycan from cultured mycelia of Cordyceps sinensis.

A novel heteroglycan, Cordyceps sinensis polysaccharide 1 (molecular weight 1 17 × 10(5) Da), was isolated and purified from mycelia of the fungus C. sinensis obtained by solid-state culture. Structural characterization by chemical analysis, GC-MS, FTIR, and NMR spectroscopy showed that C. sinensis polysaccharide 1 was mainly composed of (1 → 6)-linked α-D-Glc and α-D-Gal, with minor ß-(1 → 4)-D-Xyl and ß-(1 → 4)-D-Man residues probably located in the side chains with a trace amount of α-(1 → 3)-L-Rha residue. In biological assays, C. sinensis polysaccharide 1 significantly inhibited proliferation of sarcoma 180 cells and induced apoptosis in a dose-dependent manner. Further studies will elucidate the antitumor mechanism of C. sinensis polysaccharide 1 and promote its utilization for the development of novel, effective anticancer drugs.

Protective effect of chitooligosaccharides against cyclophosphamide-induced immunosuppression in mice.

Chitooligosaccharides (COS) display a variety of important biological activities, including antimicrobial, antitumor, anti-inflammatory, and immunoenhancing. In the present study, a COS sample (degree of polymerization 4-11, analyzed by FTIR and MALDI-TOF-MS) prepared by hydrolysis with a recombinant chitosanase was orally administered to mice for evaluation of its effect on cyclophosphamide (Cy)-induced immunosuppression. Thymus and spleen indices, delayed-type hypersensitivity (DTH) reaction, macrophage phagocytosis, and certain enzyme activities were significantly higher in mice treated with COS+Cy than in mice treated with Cy alone. ELISA experiments showed that COS treatment enhanced production of the cytokines IL-2, IL-12, and IFN-γ but decreased production of IL-10 in sera of Cy-treated mice. The well-defined COS product studied displayed strong immunoenhancing activity and a protective effect against Cy-induced immunosuppression. COS-derived products should be further investigated for possible immunostimulatory applications in the food and pharmaceutical industries.

Specificity and function of archaeal DNA replication initiator proteins.

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.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus.

Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-α and Cmr-ß. To study the genetic determinants of protospacer-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show that Cas7 and Cas5 are essential for stabilizing the processing intermediates and mature crRNAs, respectively, and that depleting the helicase or nuclease domain of Cas3 leads to the accumulation of processing intermediates. This demonstrates that in addition to Cas6, other Cas proteins of an archaeal type IA system also contribute to crRNA processing.

An archaeal protein evolutionarily conserved in prokaryotes is a zinc-dependent metalloprotease.

A putative protease gene (tldD) was previously identified from studying tolerance of letD encoding the CcdB toxin of a toxin-antidote system of the F plasmid in Escherichia coli. While this gene is evolutionarily conserved in archaea and bacteria, the proteolytic activity of encoded proteins remained to be demonstrated experimentally. Here we studied Sso0660, an archaeal TldD homologue encoded in Sulfolobus solfataricus by overexpression of the recombinant protein and characterization of the purified enzyme. We found that the enzyme is active in degrading azocasein and FITC-BSA substrates. Protease inhibitor studies showed that EDTA and o-phenanthroline, two well-known metalloprotease inhibitors, either abolished completely or strongly inhibited the enzyme activity, and flame spectrometric analysis showed that a zinc ion is a cofactor of the protease. Furthermore, the protein forms disulfide bond via the Cys416 residue, yielding protein dimer that is the active form of the enzyme. These results establish for the first time that tidD genes encode zinc-containing proteases, classifying them as a family in the metalloprotease class.

Archaeal promoter architecture and mechanism of gene activation.

Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression.

An upstream activation element exerting differential transcriptional activation on an archaeal promoter.

Microorganisms can utilize different sugars as energy and carbon sources and the genes involved in sugar metabolism often exhibit highly regulated expression. To study cis-acting elements controlling arabinose-responsive expression in archaea, the promoter of the Sulfolobus solfataricus araS gene encoding an arabinose binding protein was characterized using an Sulfolobus islandicus reporter gene system. The minimal active araS promoter (P(araS)) was found to be 59 nucleotides long and harboured four promoter elements: an ara-box, an upstream transcription factor B-responsive element (BRE), a TATA-box and a proximal promoter element, each of which contained important nucleotides that either greatly decreased or completely abolished promoter activity upon mutagenesis. The basal araS promoter was virtually inactive due to intrinsically weak BRE element, and the upstream activating sequence (UAS) ara-box activated the basal promoter by recruiting transcription factor B to its BRE. While this UAS ensured a general expression from an inactive or weak basal promoter in the presence of other tested carbon resources, it exhibited a strong arabinose-responsive transcriptional activation. To our knowledge, this represents the first example of an archaeal UAS that exhibits differential activation to the expression on the same promoter in the presence of different carbon sources.

Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus.

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

Genetic analyses in the hyperthermophilic archaeon Sulfolobus islandicus.

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.