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Applied and Environmental Microbiology, July 2005, p. 3889-3899, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3889-3899.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon Thermococcus kodakaraensis

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Received 23 November 2004/ Accepted 27 January 2005

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (pyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (pyrF trpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (pyrF trpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (pyrF trpE hisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.

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