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Improved tetracycline repressors for gene silencing in mycobacteria.

TitleImproved tetracycline repressors for gene silencing in mycobacteria.
Publication TypeJournal Article
Year of Publication2009
AuthorsKlotzsche M, Ehrt S, Schnappinger D
JournalNucleic Acids Res
Volume37
Issue6
Pagination1778-88
Date Published2009 Apr
ISSN1362-4962
KeywordsChromosomes, Bacterial, Codon, Gene Expression Regulation, Bacterial, Gene Silencing, Genes, Reporter, Mutation, Mycobacterium, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Repressor Proteins
Abstract

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 approximately 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.

DOI10.1093/nar/gkp015
Alternate JournalNucleic Acids Res
PubMed ID19174563
PubMed Central IDPMC2665214
Grant ListAI063446 / AI / NIAID NIH HHS / United States
/ / Wellcome Trust / United Kingdom

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