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Research

At an early stage, the archaea group became involved in whole genome sequencing of selected hyperthermophilic crenarchaea with a view to gaining a deeper insight into the biochemistry, cellular biology and evolution of the Archaea. The early genome projects were supported by EU grants and involved European networks and, initially, a Canadian network. Some of these projects have been completed and we have started post-genomic studies which involve different approaches to archaeal genome evolution and cellular biology.

Many of the basic cellular molecular processes of archaea exhibit either archaeal-specific of eukaryal-like features. This applies to the basic informational processes, replication, transcription and translation, as well as cell division, intron splicing from tRNAs, rRNAs and mRNAs, and many other cellular processes. It is also true of many cellular structures including the ether-linked lipid membranes, and chromatin folding proteins which can be either archaeal-specific or eukaryal-like (histones) (Garrett and Klenk, 2007).

In addition, some of the processes involved in DNA transfer show archaeal-specific features. For example, the gene products implicated in the conjugal transfer of both plasmid and chromosomal DNA show no sequence similarity to proteins which facilitate bacterial conjugation (Greve et al., 2004; Chen et al., 2005). Moreover, one of the basic mechanisms of integration into archaeal chromosomes involved partitioning of the integrase gene such that it can no longer express an intact integrase. This can lead to the genetic element being encaptured, and eventually spread, in the chromosome, if the cell is cured of the free integrative genetic element (She et al., 2001; She et al., 2005).

Archaeal genomics

DAC has contributed to several whole genome sequencing projects of extreme/hyper-thermophilic crenarchaea. It coordinated the EU Consortium (Project BIO4CT-960270) in collaboration with a Canadian Consortium (Dalhousie University and Institute for Marine Biosciences, Halifax), headed by W. Ford Doolittle, to complete the genome sequence of the hyperthermophilic Sulfolobus solfataricus P2 (She et al., 2001). Moreover, within the EU-SCREEN project (QLK3CT-2000-00649), together with Hans-Peter Klenk's laboratory (e-gene, Biotechnologie, Munich), we completed the genomes of the extreme thermophile Sulfolobus acidocaldarius (Chen et al., 2005) and hyperthermophile Hyperthermus butylicus (Brügger et al., 2007). We have recently completed the genome of Sulfolobus islandicus HVE10/4, in house, which is a good host for several of the novel crenarchaeal viruses and some conjugative plasmids. Furthermore, the sequence of Sulfolobus islandicus REY15A, which is being used for genetic studies, was completed together with Li Huang (Chinese Academy of Sciences, Beijing) (see below).

Many of the archaeal genomes are very rich in mobile elements, IS elements and MITEs (Miniature Inverted-repeat Transposable Elements) which facilitate continual rearrangements and other major genomic changes (Redder & Garrett, 2006). S. solfataricus P2 constitutes an extreme example, with at least 343 potentially mobile elements, although there is strong evidence to suggest that transpositional activity is regulated by anti-sense RNAs (Tang et al., 2005).

Archaeal global regulatory mechanisms

At present, very little is known about global regulation in archaea and research is greatly hindered by the lack of good genetic systems, especially for the crenarchaea. Therefore, the main effort of Q. She's laboratory has been to develop tools for genetic and functional genomics analyses of Sulfolobus. A basic genetic system has been developed based on uracil drop-out selection in S. islandicus REY15A and used for developing allelic gene replacement and unmarked gene deletion methods (Deng et al. 2008). Other genetic tools such as shuttle vectors for over-expression, reporter gene system and genetic complementation have also been tested (She et al. 2008). Currently, these tools have been used for genetic analyses of the functions of DNA replication and repair genes, as well as of the virus life cycle of the Sulfolobus fusellovirus SSV2, and the virus satellites pSSVx and pSSVi. Qunxin She is a partner in the international Sulfolobus consortium which produced a DNA chip for transcriptome analyses of whole genome expression in the S. solfataricus P2 host and genetic elements. Furthermore, in collaboration with Li Huang at Institute of Microbiology, Chinese Academy of Sciences, China, we have sequenced the host S. islandicus REY15A, for genetic studies, and used the sequence to construct a genome microarray. With these recently developed genetic and functional genomics tools, we are currently investigating mechanisms of global gene regulation and host-genetic element interactions using S. islandicus and S. solfataricus as models.

Archaeal viruses

Many novel archaeal viruses have been characterized over the past few years especially for the crenarchaea. They exhibit a wide range of morphotypes some of which are exclusive to archaea. Together with David Prangishvili's laboratory, now located at the Pasteur Institute, Paris, we have sequenced and analysed several of the viral genomes. The results reinforce the special character of archaeal viruses and most have now been classified into seven new archaeal viral families which include spindle-shaped and enveloped Fuselloviridae (SSV1, SSV2, SSV3, SSV4, SSV5, SSV6, SSV7, SSVk1, SSVrh and ASV1), lipid-containing filamentous and enveloped Lipothrixviridae (SIFV, TTV1, AFV1, AFV2, AFV3, AFV6, AFV7, AFV8, AFV9), rod-shaped unenveloped Rudiviridae (SIRV1, SIRV2, SRV, ARV1), spherical Globuloviridae (PSV, TTSV1), bearded-droplet-shaped Guttaviridae (SDNV), bottle-shaped Ampullaviridae (ABV) and two-tailed Bicaudaviridae (ATV). The novel viruses STIV and STSV1 remain unclassified. All these viruses carry double-stranded DNA genomes, either circular or linear, and infect hyperthermophilic crenarchaea of the orders Sulfolobales or Thermoproteales (Prangishvili et al., 2006, 2007). At present, we know very little about the mechanisms of host infection/extrusion, viral DNA replication and packaging, or about virus-host relationships in general. Several studies are currently underway on the molecular structures and functions of these viruses in different laboratories, and our own, with considerable progress being made on protein crystal structure analyses.

CRISPR virus-plasmid defence-regulatory system

Almost all archaeal chromosomes and some conjugative plasmids contain one or more clusters of short regularly inter-spaced repeats (CRISPR) some of which constitute over 100 repeat-spacer units. These have been implicated in inhibiting propagation of viruses and plasmids in archaea (and in many bacteria) via an intermediate RNA mechanism (Tang et al., 2002; Tang et al., 2005; Mojica et al., 2005). Our laboratory has identified the protein which specifically binds to crenarchaeal repeats (Peng et al., 2003) and the processing products of the cluster-encoded transcripts from each DNA strand (Lillestøl et al., 2006). We are currently exploiting bioinformatic approaches (Vestergaard et al., 2008; Shah et al., 2008) and genetic experiments, to understand the dynamic interactions between chromosomal clusters and extra-chromosomal elements.

References

Brügger, K., Redder, P., She, Q., Confalonieri, F., Zivanovic, Y. & Garrett, R.A. Mobile elements in archaeal genomes. FEMS Microbiol. Letts. 206, 131-141, 2002.

Brügger, K., Chen, L., Stark, M., Zibat, A, Redder, P., Ruepp, A., Awayez, M, She, Q., Garrett, R.A. & Klenk, H-P. The genome of Hyperthermus butylicus: a sulphur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108oC. Archaea, 2, 127-135, 2007.

Chen, L., Brügger, K., Skovgaard, M., Redder, P., She, Q.,Torarinsson, E., Greve, B., Awayez, M., Zibat, A., Klenk, H.-P. & Garrett, R. A. The Genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota J. Bacteriol. 187, 4992-4999, 2005.

Deng, L., Zhu, H., Chen, Z., Liang, Y.X., She, Q. (2009) Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus. Extremophiles 13: 735-746.

Garrett, R.A. & Klenk, H.P. (Eds.) Archaea: Evolution, Physiology and Molecular Biology. Blackwell Publishing, Oxford, 2007

Greve, B., Jensen, S., Brügger, K., Zillig, W. & Garrett, R.A.. Genomic comparison of archaeal conjugative plasmids from Sulfolobus. Archaea, 1, 231-239, 2004.

Häring, M., Vestergaard, G., Rachel, R., Chen, L., Garrett, R.A. & Prangishvili, D. Independent virus development outside a host. Nature, 436, 1101-1102, 2005.

Lillestøl, R., Redder, P., Garrett, R.A. & Brügger, K. A putative viral defence mechanism in archaeal cells. Archaea, 2, 59-72, 2006.

Mojica, F.J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174-182, 2005.

Peng, X., Brügger, K., Shen, B., Chen, L., She, Q. & Garrett, R.A. Genus-specific protein binding to the large clusters of DNA repeats (Short Regularly Spaced Repeats) present in Sulfolobus genomes. J. Bacteriol. 185, 2410-2417, 2003.

Prangishvili, D., Forterre, P. & Garrett, R.A. Viruses of the Archaea: a unifying view. Nature Rev. Microbiol. 4, 837-848, 2006.

Prangishvili, D., Garrett, R.A. & Koonin, E. Evolutionary genomics of archaeal viruses: unique viral genomes in the third domain of life. Virus Research, 117, 52-67, 2006.

Redder, P. & Garrett, R.A. Mutations and rearrangements in the genome of Sulfolobus solfataricus P2. J. Bacteriol. 188, 4198-4206, 2006.

Shah, S.A., Hansen, N.R. & Garrett, R.A. The distributions of CRISPR spacer matches in viruses and plasmids of crenarchaeal acidothermophiles and implications for their inhibitory mechanism. Trans. Biochem. Soc., in press.

She, Q., Peng, X., Zillig, W. & Garrett, R.A. Gene capture events in archaeal chromosomes. Nature, 409, 478, 2001.

She, Q., Singh, R.K., Confalonieri, F., Zivanovic, Y., Gordon, P., Allard, G., Awayez, M.J., Chan-Weiher, C-Y., Clausen, I.G., Curtis, B., De Moors, A., Erauso, G., Fletcher, C., Gordon, P.M.K., Heikamp de Jong, I., Jeffries, A., Kozera, C.J., Medina, N., Peng, X., Phan Thi-Ngoc, H., Redder, P., Schenk, M.E., Theriault, C., Tolstrup, N., Charlebois, R. L. M., Doolittle, W. F., Duguet, M., Gaasterland, T., Garrett, R.A., Ragan, M., Sensen, C. W. & Van der Oost, J. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA, 98, 7835-7840, 2001.

She, Q., Shen, B. & Chen, L. Archaeal integrases and mechanisms of gene capture. Bioch. Soc. Trans. 32, 222-226, 2004.

She, Q. Zhang, C., Deng, L., Peng, N. Chen, Z., & Liang Y. X. Genetic analyses in hyperthermophilic archaeon Sulfolobus islandicus. Trans. Biochem. Soc., in press.

Tang, T.-H., Bachellerie, J-P., Rozhdestvensky, T., Bortolin, M-L., Huber, H., Drungowski, M., Elge, T., Brosius, J. & Hüttenhofer, A. Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA, 99:7536-7541, 2002.

Tang, T-H., Polacek, N., Zywicki, M., Huber, H., Brügger, K., Garrett, R.A., Bachellerie, J. P. & Hüttenhofer, A. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus. Molec. Microbiol. 55, 469-481, 2005.

Vestergaard, G., Shah, S.A., Bize, A., Reitberger, W., Reuter, M., Phan, H., Briegel, A., Rachel, R., Garrett, R.A. & Prangishvili, D. SRV, a new rudiviral isolate from Stygioglobus and the interplay of crenarchaeal rudiviruses with the host viral-defence CRISPR system. J. Bacteriol. 190, 6837-6845.