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Improved tetracycline repressors for gene silencing in mycobacteria.

Klotzsche M, Ehrt S, Schnappinger D - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

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Comparison of TetR steady state levels in M. smegmatis and M. bovis BCG containing tetR(B)syn1–207 or tetR(BD)syn1–208. Protein lysates were from M. smegmatis (labeled ‘Msm’) and M. bovis BCG (labeled ‘BCG’) containing episomally replicating Pimyc-tetR plasmids. A monoclonal anti-TetR antibody recognizing an epitope within the DNA-binding region of TetR was used to detect purified TetR as well as TetR in protein lysates from M. smegmatis and M. bovis BCG. The DlaT signal was used as a loading control and detected by a polyclonal anti-DlaT antibody.
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Figure 4: Comparison of TetR steady state levels in M. smegmatis and M. bovis BCG containing tetR(B)syn1–207 or tetR(BD)syn1–208. Protein lysates were from M. smegmatis (labeled ‘Msm’) and M. bovis BCG (labeled ‘BCG’) containing episomally replicating Pimyc-tetR plasmids. A monoclonal anti-TetR antibody recognizing an epitope within the DNA-binding region of TetR was used to detect purified TetR as well as TetR in protein lysates from M. smegmatis and M. bovis BCG. The DlaT signal was used as a loading control and detected by a polyclonal anti-DlaT antibody.

Mentions: All reverse TetRs analyzed so far were originally isolated in genetic screens using mutants of tetR(BD) (19). Unfortunately, repression of lacZ by TetR(BD) in M. smegmatis and M. bovis BCG was less efficient than repression by TetR(B) (Figure 1B). To determine if the difference in repression by TetR(B) and TetR(BD) might be caused by different protein steady state levels, lysates from M. smegmatis and M. bovis BCG containing Pimyc-tetR(Bsyn1–207) or Pimyc-tetR(BDsyn1–208) were analyzed by Western blots. Strong signals were detected for TetR(B) in lysates from both mycobacteria but the steady state levels of TetR(BD) were below the level of detection (Figure 4). This suggested that a reverse TetR derived from TetR(B) might be more efficient in mycobacteria than those derived from TetR(BD). However, so far no reverse TetR(B) variant had been isolated.Figure 4.


Improved tetracycline repressors for gene silencing in mycobacteria.

Klotzsche M, Ehrt S, Schnappinger D - Nucleic Acids Res. (2009)

Comparison of TetR steady state levels in M. smegmatis and M. bovis BCG containing tetR(B)syn1–207 or tetR(BD)syn1–208. Protein lysates were from M. smegmatis (labeled ‘Msm’) and M. bovis BCG (labeled ‘BCG’) containing episomally replicating Pimyc-tetR plasmids. A monoclonal anti-TetR antibody recognizing an epitope within the DNA-binding region of TetR was used to detect purified TetR as well as TetR in protein lysates from M. smegmatis and M. bovis BCG. The DlaT signal was used as a loading control and detected by a polyclonal anti-DlaT antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665214&req=5

Figure 4: Comparison of TetR steady state levels in M. smegmatis and M. bovis BCG containing tetR(B)syn1–207 or tetR(BD)syn1–208. Protein lysates were from M. smegmatis (labeled ‘Msm’) and M. bovis BCG (labeled ‘BCG’) containing episomally replicating Pimyc-tetR plasmids. A monoclonal anti-TetR antibody recognizing an epitope within the DNA-binding region of TetR was used to detect purified TetR as well as TetR in protein lysates from M. smegmatis and M. bovis BCG. The DlaT signal was used as a loading control and detected by a polyclonal anti-DlaT antibody.
Mentions: All reverse TetRs analyzed so far were originally isolated in genetic screens using mutants of tetR(BD) (19). Unfortunately, repression of lacZ by TetR(BD) in M. smegmatis and M. bovis BCG was less efficient than repression by TetR(B) (Figure 1B). To determine if the difference in repression by TetR(B) and TetR(BD) might be caused by different protein steady state levels, lysates from M. smegmatis and M. bovis BCG containing Pimyc-tetR(Bsyn1–207) or Pimyc-tetR(BDsyn1–208) were analyzed by Western blots. Strong signals were detected for TetR(B) in lysates from both mycobacteria but the steady state levels of TetR(BD) were below the level of detection (Figure 4). This suggested that a reverse TetR derived from TetR(B) might be more efficient in mycobacteria than those derived from TetR(BD). However, so far no reverse TetR(B) variant had been isolated.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus