Cargando…

Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycoba...

Descripción completa

Detalles Bibliográficos
Autores principales: Ehrt, Sabine, Guo, Xinzheng V., Hickey, Christopher M., Ryou, Marvin, Monteleone, Mercedes, Riley, Lee W., Schnappinger, Dirk
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548372/
https://www.ncbi.nlm.nih.gov/pubmed/15687379
http://dx.doi.org/10.1093/nar/gni013
_version_ 1782122353888591872
author Ehrt, Sabine
Guo, Xinzheng V.
Hickey, Christopher M.
Ryou, Marvin
Monteleone, Mercedes
Riley, Lee W.
Schnappinger, Dirk
author_facet Ehrt, Sabine
Guo, Xinzheng V.
Hickey, Christopher M.
Ryou, Marvin
Monteleone, Mercedes
Riley, Lee W.
Schnappinger, Dirk
author_sort Ehrt, Sabine
collection PubMed
description Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.
format Text
id pubmed-548372
institution National Center for Biotechnology Information
language English
publishDate 2005
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-5483722005-02-10 Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor Ehrt, Sabine Guo, Xinzheng V. Hickey, Christopher M. Ryou, Marvin Monteleone, Mercedes Riley, Lee W. Schnappinger, Dirk Nucleic Acids Res Methods Online Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome. Oxford University Press 2005 2005-02-01 /pmc/articles/PMC548372/ /pubmed/15687379 http://dx.doi.org/10.1093/nar/gni013 Text en © The Author 2005. Published by Oxford University Press. All rights reserved
spellingShingle Methods Online
Ehrt, Sabine
Guo, Xinzheng V.
Hickey, Christopher M.
Ryou, Marvin
Monteleone, Mercedes
Riley, Lee W.
Schnappinger, Dirk
Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title_full Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title_fullStr Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title_full_unstemmed Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title_short Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
title_sort controlling gene expression in mycobacteria with anhydrotetracycline and tet repressor
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548372/
https://www.ncbi.nlm.nih.gov/pubmed/15687379
http://dx.doi.org/10.1093/nar/gni013
work_keys_str_mv AT ehrtsabine controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT guoxinzhengv controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT hickeychristopherm controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT ryoumarvin controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT monteleonemercedes controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT rileyleew controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor
AT schnappingerdirk controllinggeneexpressioninmycobacteriawithanhydrotetracyclineandtetrepressor