Unmarked gene deletion and host–vector system for

Authors: Ling Deng Haojun Zhu Zhengjun Chen Yun Xiang Liang Qunxin She

Publish Date: 2009/06/10

Volume: 13, Issue: 4, Pages: 735-

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Abstract

Sulfolobus islandicus is being used as a model for studying archaeal biology geobiology 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 βglycosidase 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 ie 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 markersWe 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 274070116 Danish Science Research Council 27205400 and a special grant from the Huazhong Agricultural University China

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