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Applied and Environmental Microbiology, February 2005, p. 728-733, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.728-733.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis

Annette Kamionka, Ralph Bertram, and Wolfgang Hillen*

Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

Received 19 August 2004/ Accepted 20 September 2004

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 µM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany. Phone: 49 9131 85-28081. Fax: 49 9131 85-28082. E-mail: whillen{at}biologie.uni-erlangen.de.

Applied and Environmental Microbiology, February 2005, p. 728-733, Vol. 71, No. 2
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.2.728-733.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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