Vector Development for Thermophilic Archaea
Kenneth STEDMAN*, Christa SCHLEPER, Hans-Peter ARNOLD, Ingelore HOLZ, Evelyn RUMPF, Wolfram ZILLIG
Max Planck Institute for Biochemistry, D-82152 Martinsried, GERMANY
No suitable genetic system has yet been described for any of the generally thermophilic Crenarcheota which has greatly hindered research in this area. SSV1, Sulfolobus Spindle Shaped Virus-1, a virus infecting the extremely thermophilic, heterotrophic and aerobic archaeon Sulfolobus, was chosen as a candidate for vector development. SSV1 integrates stably into the genome of Sulfolobus, has a genome of 15,465 basepairs, can be induced by ultraviolet irradiation and is "self-selecting" (1). The complete sequence of the virus genome is known (2), but 24 of the 34 open reading frames have no known function or known utility. Three approaches have been taken to test potential sites for integration of foreign DNA into the SSV1 genome: 1) Single restriction endonuclease sites have been used to both clone E. coli DNA into the SSV1 genome and disrupt open reading frames. 2) A PCR based technique has been used to specifically delete open reading frames. 3) A technique based on partial enzyme digestion and dual selection in E. coli and Sulfolobus is being used to genetically select functional shuttle vectors. Essential regions for virus function in the SSV1 genome have been determined as well as potential insertion sites for foreign DNA.
1. Schleper, C., Kubo, K. and Zillig, W. (1992) Proc. Natl. Acad. Sci., U.S.A. 89, 7645-7649
2. Palm, P., Schleper, C., Grampp, B., Yeats, S., McWilliam, P., Reiter, W.-D. and Zillig, W. (1991) Virology 185, 242-250
