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Tetracycline-inducible gene regulation in mycobacteria

A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetra...

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Detalles Bibliográficos
Autores principales: Blokpoel, Marian C. J., Murphy, Helen N., O'Toole, Ronan, Wiles, Siouxsie, Runn, Ellen S. C., Stewart, Graham R., Young, Douglas B., Robertson, Brian D.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548381/
https://www.ncbi.nlm.nih.gov/pubmed/15687380
http://dx.doi.org/10.1093/nar/gni023
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author Blokpoel, Marian C. J.
Murphy, Helen N.
O'Toole, Ronan
Wiles, Siouxsie
Runn, Ellen S. C.
Stewart, Graham R.
Young, Douglas B.
Robertson, Brian D.
author_facet Blokpoel, Marian C. J.
Murphy, Helen N.
O'Toole, Ronan
Wiles, Siouxsie
Runn, Ellen S. C.
Stewart, Graham R.
Young, Douglas B.
Robertson, Brian D.
author_sort Blokpoel, Marian C. J.
collection PubMed
description A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(−1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.
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spelling pubmed-5483812005-02-10 Tetracycline-inducible gene regulation in mycobacteria Blokpoel, Marian C. J. Murphy, Helen N. O'Toole, Ronan Wiles, Siouxsie Runn, Ellen S. C. Stewart, Graham R. Young, Douglas B. Robertson, Brian D. Nucleic Acids Res Methods Online A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(−1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells. Oxford University Press 2005 2005-02-01 /pmc/articles/PMC548381/ /pubmed/15687380 http://dx.doi.org/10.1093/nar/gni023 Text en © The Author 2005. Published by Oxford University Press. All rights reserved
spellingShingle Methods Online
Blokpoel, Marian C. J.
Murphy, Helen N.
O'Toole, Ronan
Wiles, Siouxsie
Runn, Ellen S. C.
Stewart, Graham R.
Young, Douglas B.
Robertson, Brian D.
Tetracycline-inducible gene regulation in mycobacteria
title Tetracycline-inducible gene regulation in mycobacteria
title_full Tetracycline-inducible gene regulation in mycobacteria
title_fullStr Tetracycline-inducible gene regulation in mycobacteria
title_full_unstemmed Tetracycline-inducible gene regulation in mycobacteria
title_short Tetracycline-inducible gene regulation in mycobacteria
title_sort tetracycline-inducible gene regulation in mycobacteria
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548381/
https://www.ncbi.nlm.nih.gov/pubmed/15687380
http://dx.doi.org/10.1093/nar/gni023
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