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Nucleic Acids Research Advance Access originally published online on January 27, 2009
Nucleic Acids Research 2009 37(6):1778-1788; doi:10.1093/nar/gkp015
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Nucleic Acids Research, 2009, Vol. 37, No. 6 1778-1788
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Improved tetracycline repressors for gene silencing in mycobacteria

Marcus Klotzsche, Sabine Ehrt and Dirk Schnappinger*

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA

*To whom correspondence should be addressed. Tel: +212 746 3788; Fax: +212 746 8587; Email: dis2003{at}med.cornell.edu

Received November 17, 2008. Revised December 29, 2008. Accepted January 7, 2009.

Tetracycline repressor (TetR)-controlled expression systems have recently been developed for mycobacteria and proven useful for the construction of conditional knockdown mutants and their analysis in vitro and during infections. However, even though these systems allowed tight regulation of some mycobacterial genes, they only showed limited or no phenotypic regulation for others. By adapting their codon usage to that of the Mycobacterium tuberculosis genome, we created tetR genes that mediate up to ~50-fold better repression of reporter gene activities in Mycobacterium smegmatis and Mycobacterium bovis BCG. In addition to these repressors, for which anhydrotetracycline (atc) functions as an inducer of gene expression, we used codon-usage adaption and structure-based design to develop improved reverse TetRs, for which atc functions as a corepressor. The previously described reverse repressor TetR only functioned when expressed from a strong promoter on a multicopy plasmid. The new reverse TetRs silence target genes more efficiently and allowed complete phenotypic silencing of M. smegmatis secA1 with chromosomally integrated tetR genes.

Present address: Marcus Klotzsche, Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen-Nürnberg, Staudstrasse 5, 91058 Erlangen


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