In vivo activity of CRISPR-mediated virus defence in a hyperthermophilic archaeon.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that - unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus- DNA is targeted in this archaeon.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili.

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.

"Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus.

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS")-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the "-omics" approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation.

The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative transmembrane protein and a protein of unknown function. Electron microscopy and image reconstruction of UV-treated cells showed straight pili with 10 nm in diameter, variable in length, not bundled or polarized and composed of three evenly spaced helices, thereby clearly being distinguishable from archaeal flagella. A deletion mutant of SSO0120, the central type II/IV secretion ATPase, did not produce pili. It could be complemented by reintroducing the gene on a plasmid vector. We have named the operon ups operon for UV-inducible pili operon of Sulfolobus. Overexpression of the pre-pilins, Ups-A/B (SSO0117/0118) in Sulfolobus resulted in production of extremely long filaments. Pronounced cellular aggregation was observed and quantified upon UV treatment. This aggregation was a UV-dose-dependent, dynamic process, not inducible by other physical stressors (such as pH or temperature shift) but stimulated by chemically induced double-strand breaks in DNA. We hypothesize that pili formation and subsequent cellular aggregation enhance DNA transfer among Sulfolobus cells to provide increased repair of damaged DNA via homologous recombination.

Astrocytes protect the CNS: antigen-specific T helper cell responses are inhibited by astrocyte-induced upregulation of CTLA-4 (CD152).

Astrocytes are the first cells that are encountered by T cells invading the central nervous system (CNS) by crossing the blood-brain barrier. We show that primary astrocytes contribute to the immune privilege of the CNS by suppressing Th1 and Th2 cell activation, proliferation and effector function. Moreover, this astrocyte-mediated inhibition of Th effector cells was effective on already activated, proliferating cells. Transforming growth factor (TGF)-beta secreted by astrocytes or T cells was not the major factor in the inhibition. The inhibition of T-cell proliferation induced by astrocytes was mainly mediated by upregulation of CTLA-4 on already activated T cells, which occurred both with and without cell-cell contact. Upregulation of the inhibitory molecule CTLA-4 on autoreactive Th cells, as mediated by astrocytes, thus represents a novel mechanism for securing the immune privilege of the CNS.