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Journal of Bacteriology, February 2003, p. 772-778, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.772-778.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development of a Gene Knockout System for the Halophilic Archaeon Haloferax volcanii by Use of the pyrE Gene

Gili Bitan-Banin, Ron Ortenberg,{dagger} and Moshe Mevarech*

Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

Received 12 July 2002/ Accepted 30 October 2002

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A {Delta}pyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-6408715. Fax: 972-3-6409407. E-mail: mevarech{at}post.tau.ac.il.

{dagger} Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

Journal of Bacteriology, February 2003, p. 772-778, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.772-778.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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