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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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Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded reverse 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 revTetR to tetOs in the presence 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 bar 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.
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Figure 3: Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded reverse 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 revTetR to tetOs in the presence 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 bar 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: Reverse TetRs allow gene silencing by adding atc instead of removing atc (Figure 3A), which is advantageous under conditions were atc cannot be easily depleted (e.g. during hypoxia). TetR r1.7, a reverse TetR(BD) variant that contained three mutations in its DNA-binding domain (E15A, L17G and L25V), allowed atc-induced gene silencing in both, M. smegmatis (7) and M. tuberculosis (5). However, repression by TetR r1.7 was less efficient than repression by wt TetR (7). To improve gene silencing by reverse TetRs we first mutated the tetR(BDsyn1–208) gene to encode TetR r1.7; the resulting gene was named tetR(BDsyn1–208)E15A-L17G-L25V (in contrast to tetR(BD)E15A-L17G-L25V, the noncodon usage adapted gene encoding TetR r1.7) Transformation of M. smegmatis Pmyc1tetO-lacZL5 with an episomally replicating plasmid containing Pimyc-tetR(BDsyn1–208)E15A-L17G-L25V reduced β-galactosidase activity to ∼6% of the strain without tetR in the presence of atc (Figure 3B). No repression was observed in the absence of atc. As had been observed previously (7), Pimyc-tetR(BD)E15A-L17G-L25V did not change β-galactosidase of M. smegmatis. We also constructed and analyzed three additional tetR(BDsyn1–208) derivates which all encoded reverse TetRs that function efficiently in E. coli (19). However, the most efficient of these repressors reduced β-galactosidase activity only to 22% in M. smegmatis (Figure 3B) and none of them caused any repression if expressed by the original, noncodon usage adapted, tetR(BD) genes. These experiments demonstrated that codon-usage adaptation improved gene silencing by TetR r1.7 and that among the four TetRs evaluated, TetR r1.7 was most efficient in M. smegmatis.Figure 3.


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 reverse 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 revTetR to tetOs in the presence 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 bar 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.
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Figure 3: Impact of tetR codon usage adaptation on repression of chromosomally encoded β-galactosidase activities by episomally encoded reverse 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 revTetR to tetOs in the presence 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 bar 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: Reverse TetRs allow gene silencing by adding atc instead of removing atc (Figure 3A), which is advantageous under conditions were atc cannot be easily depleted (e.g. during hypoxia). TetR r1.7, a reverse TetR(BD) variant that contained three mutations in its DNA-binding domain (E15A, L17G and L25V), allowed atc-induced gene silencing in both, M. smegmatis (7) and M. tuberculosis (5). However, repression by TetR r1.7 was less efficient than repression by wt TetR (7). To improve gene silencing by reverse TetRs we first mutated the tetR(BDsyn1–208) gene to encode TetR r1.7; the resulting gene was named tetR(BDsyn1–208)E15A-L17G-L25V (in contrast to tetR(BD)E15A-L17G-L25V, the noncodon usage adapted gene encoding TetR r1.7) Transformation of M. smegmatis Pmyc1tetO-lacZL5 with an episomally replicating plasmid containing Pimyc-tetR(BDsyn1–208)E15A-L17G-L25V reduced β-galactosidase activity to ∼6% of the strain without tetR in the presence of atc (Figure 3B). No repression was observed in the absence of atc. As had been observed previously (7), Pimyc-tetR(BD)E15A-L17G-L25V did not change β-galactosidase of M. smegmatis. We also constructed and analyzed three additional tetR(BDsyn1–208) derivates which all encoded reverse TetRs that function efficiently in E. coli (19). However, the most efficient of these repressors reduced β-galactosidase activity only to 22% in M. smegmatis (Figure 3B) and none of them caused any repression if expressed by the original, noncodon usage adapted, tetR(BD) genes. These experiments demonstrated that codon-usage adaptation improved gene silencing by TetR r1.7 and that among the four TetRs evaluated, TetR r1.7 was most efficient in M. smegmatis.Figure 3.

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