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Applied and Environmental Microbiology, February 2004, p. 943-953, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.943-953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes

Thorsten Allers,1* Hien-Ping Ngo,1 Moshe Mevarech,2 and Robert G. Lloyd1

Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom,1 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel2

Received 14 July 2003/ Accepted 13 November 2003

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. {Delta}leuB and {Delta}trpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.

* Corresponding author. Mailing address: Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone: 44-(0)115-970-9404. Fax: 44-(0)115-970-9906. E-mail: thorsten.allers{at}nottingham.ac.uk.

Applied and Environmental Microbiology, February 2004, p. 943-953, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.943-953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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