Nucleic Acids Research Advance Access originally published online on June 18, 2007
Nucleic Acids Research 2007 35(12):e88; doi:10.1093/nar/gkm449
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Nucleic Acids Research, 2007, Vol. 35, No. 12 e88
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea
1Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany, 2Department of Biological Sciences, University of Cincinnati, OH 45221-0006, USA and 3Department of Molecular Microbiology, University of Groningen, 9751 NN Haren, The Netherlands
*To whom correspondence should be addressed: Tel: +49 921 552433, Fax: +49 921 552432, Email: Georg.Lipps{at}uni-bayreuth.de
Received March 20, 2007. Revised May 14, 2007. Accepted May 18, 2007.
The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the ß-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this ß-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.