The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.

SummaryRecent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB. Earlier we showed that type IA activity eliminated plasmid vectors carrying matching protospacers with specific CCN PAM sequences. Here we demonstrate that interference-mediated by one type IIIB module Cmr-a, and a Csx1 protein, efficiently eliminated plasmid vectors carrying matching protospacers but lacking PAM motifs. Moreover, Cmr-a-mediated interference was dependent on directional transcription of the protospacer, in contrast to the transcription-independent activities of the type IA and type IIIA DNA interference. We infer that the interference mechanism involves transcription-dependent DNA targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S. islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers.

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.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

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.

In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers.

b Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in Sulfolobus islandicus, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6؋His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P araS-SD , an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P araS-SD directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6؋His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6؋His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6؋His tag, as shown with pSeSD-N-lacS. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon Sulfolobus can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site. C urrently, studying functions of thermophilic archaeal proteins, such as the enzymes involved in DNA replication, gene transcription, and protein translation (10), as well as clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) proteins (11, 32), relies almost exclusively on production of recombinant protein from a mesophilic bacterial host such as Escherichia coli and characterization of purified enzymes. However, there is a major drawback in this approach: many thermophilic proteins are insoluble in mesophilic bacterial cells, forming inclusion bodies. Apparently, producing recombinant thermophilic proteins in a homologous host should resolve the problem of protein solubility.For the past few decades, consistent efforts have been devoted to developing protein expression systems for hyperthermophilic organisms and to establishing genetic tools for studying functions of archaeal genes. As a result, methodologies for gene inactivation and genetic complementation have been established for several model archaeal organisms (reviewed in reference 15), including three hyperthermophilic archaea, the euryarchaea Thermococcus kodakarensis (22) and Pyrococcus furiosus (30), and the crenarchaeon Sulfolobus. While both T. kodakarensis and P. furiosus require an anaerobic condition for growth, Sulfolobus species grow aerobically, which is advantageous for laboratory manipulation. Currently, three Sulfolobus species have be...

CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems are presented.