Extremophiles

, 13:735

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

  • Ling Deng
  • Haojun Zhu
  • Zhengjun Chen
  • Yun Xiang Liang
  • Qunxin She
Method Paper

DOI: 10.1007/s00792-009-0254-2

Cite this article as:
Deng, L., Zhu, H., Chen, Z. et al. Extremophiles (2009) 13: 735. doi:10.1007/s00792-009-0254-2

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, SulfolobusE. 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.

Keywords

pyrEF marker Unmarked gene deletion S. islandicus host–vector system pyrEFlacS deletion mutant 

Copyright information

© Springer 2009

Authors and Affiliations

  • Ling Deng
    • 1
    • 2
  • Haojun Zhu
    • 2
  • Zhengjun Chen
    • 1
    • 2
  • Yun Xiang Liang
    • 1
  • Qunxin She
    • 2
  1. 1.State Key Laboratory of Agricultural Microbiology and College of Life Science and TechnologyHuazhong Agricultural UniversityWuhanPeople’s Republic of China
  2. 2.Danish Archaea Centre, Department of BiologyUniversity of CopenhagenKøbenhavn NDenmark

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