Limits...
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.

Show MeSH

Related in: MedlinePlus

Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded wt TetRs. (A) Genetic organization of the assay strains. The Pmyc1tetO-lacZ reporter gene cassette was integrated into the mycobacteriophage L5 attachment site. The Pimyc-tetR genes were located on episomally replicating plasmids. Binding of TetR to tetOs in the absence of atc caused repression of Pmyc1tetO. Symbols: TetRs, gray ovals; tetOs, black boxes; atc, black hexagons. (B) β-Galactosidase activities (β-gal). Expressed TetR variants are identified underneath the graph. Values were normalized to the β-galactosidase activity measured in the absence of TetR, which was set to 100%. Bars represent averages of three measurements and are representative of at least two independent experiments. Error bars indicate standard deviations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665214&req=5

Figure 1: Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded wt TetRs. (A) Genetic organization of the assay strains. The Pmyc1tetO-lacZ reporter gene cassette was integrated into the mycobacteriophage L5 attachment site. The Pimyc-tetR genes were located on episomally replicating plasmids. Binding of TetR to tetOs in the absence of atc caused repression of Pmyc1tetO. Symbols: TetRs, gray ovals; tetOs, black boxes; atc, black hexagons. (B) β-Galactosidase activities (β-gal). Expressed TetR variants are identified underneath the graph. Values were normalized to the β-galactosidase activity measured in the absence of TetR, which was set to 100%. Bars represent averages of three measurements and are representative of at least two independent experiments. Error bars indicate standard deviations.

Mentions: We first analyzed the tetR(B) genes in M. smegmatis Pmyc1tetO-lacZL5, which contains the Pmyc1tetO-lacZ expression cassette (7) integrated into the chromosomal attachment site of the mycobacteriophage L5. Mycobacterium smegmatis Pmyc1tetO-lacZL5 displayed a β-galactosidase activity of ∼8000 relative fluorescence units (RFUs). The limit of β-galactosidase detection was at ∼70 RFUs. Episomally replicating plasmids containing tetR downstream of the constitutive promoter Pimyc (23) were used to express each TetR variant (Figure 1A). Transformation of M. smegmatis Pmyc1tetO-lacZL5 with the Pimyc-tetR(B) plasmid reduced β-galactosidase activity to ∼3% of that of M. smegmatis Pmyc1tetO-lacZL5 without tetR (Figure 1B). Strains containing the two synthetic tetR(B) genes, Pimyc-tetR(Bsyn1–50) or Pimyc-tetR(Bsyn1–207) displayed β-galactosidase activities of ∼1%. In M. bovis BCG Pmyc1tetO-lacZL5 the original tetR(B) gene reduced β-galactosidase activity to 17% of the activity of M. bovis BCG Pmyc1tetO-lacZL5 without tetR. In contrast, the two synthetic tetR(B) genes both caused repression to <1%.Figure 1.


Improved tetracycline repressors for gene silencing in mycobacteria.

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

Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded wt TetRs. (A) Genetic organization of the assay strains. The Pmyc1tetO-lacZ reporter gene cassette was integrated into the mycobacteriophage L5 attachment site. The Pimyc-tetR genes were located on episomally replicating plasmids. Binding of TetR to tetOs in the absence of atc caused repression of Pmyc1tetO. Symbols: TetRs, gray ovals; tetOs, black boxes; atc, black hexagons. (B) β-Galactosidase activities (β-gal). Expressed TetR variants are identified underneath the graph. Values were normalized to the β-galactosidase activity measured in the absence of TetR, which was set to 100%. Bars represent averages of three measurements and are representative of at least two independent experiments. Error bars indicate standard deviations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded wt TetRs. (A) Genetic organization of the assay strains. The Pmyc1tetO-lacZ reporter gene cassette was integrated into the mycobacteriophage L5 attachment site. The Pimyc-tetR genes were located on episomally replicating plasmids. Binding of TetR to tetOs in the absence of atc caused repression of Pmyc1tetO. Symbols: TetRs, gray ovals; tetOs, black boxes; atc, black hexagons. (B) β-Galactosidase activities (β-gal). Expressed TetR variants are identified underneath the graph. Values were normalized to the β-galactosidase activity measured in the absence of TetR, which was set to 100%. Bars represent averages of three measurements and are representative of at least two independent experiments. Error bars indicate standard deviations.
Mentions: We first analyzed the tetR(B) genes in M. smegmatis Pmyc1tetO-lacZL5, which contains the Pmyc1tetO-lacZ expression cassette (7) integrated into the chromosomal attachment site of the mycobacteriophage L5. Mycobacterium smegmatis Pmyc1tetO-lacZL5 displayed a β-galactosidase activity of ∼8000 relative fluorescence units (RFUs). The limit of β-galactosidase detection was at ∼70 RFUs. Episomally replicating plasmids containing tetR downstream of the constitutive promoter Pimyc (23) were used to express each TetR variant (Figure 1A). Transformation of M. smegmatis Pmyc1tetO-lacZL5 with the Pimyc-tetR(B) plasmid reduced β-galactosidase activity to ∼3% of that of M. smegmatis Pmyc1tetO-lacZL5 without tetR (Figure 1B). Strains containing the two synthetic tetR(B) genes, Pimyc-tetR(Bsyn1–50) or Pimyc-tetR(Bsyn1–207) displayed β-galactosidase activities of ∼1%. In M. bovis BCG Pmyc1tetO-lacZL5 the original tetR(B) gene reduced β-galactosidase activity to 17% of the activity of M. bovis BCG Pmyc1tetO-lacZL5 without tetR. In contrast, the two synthetic tetR(B) genes both caused repression to <1%.Figure 1.

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