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Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

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Abstract

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 β-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 ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

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Acknowledgments

We thank Roger Garrett for providing the draft genome sequence of S. islandicus HVE10/4 in the early stage of this work and for critical reading of the manuscript. Mariana Awayez and Maria Dreibøl (University of Copenhagen) are thanked for their technical assistance. This work was supported by grants of Danish Research Council of Technology and Production (274-07-0116), Danish Science Research Council (272-05-400) and a special grant from the Huazhong Agricultural University, China.

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, Huazhong Agricultural University, 430070, Wuhan, People’s Republic of China

    Ling Deng, Zhengjun Chen & Yun Xiang Liang

  2. Danish Archaea Centre, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen Biocenter, 2200, København N, Denmark

    Ling Deng, Haojun Zhu, Zhengjun Chen & Qunxin She

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  1. Ling Deng
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Correspondence to Qunxin She.

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Communicated by T. Matsunaga.

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Deng, L., Zhu, H., Chen, Z. et al. Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus . Extremophiles 13, 735–746 (2009). https://doi.org/10.1007/s00792-009-0254-2

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