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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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Analysis of reverse TetR chimeras in M. smegmatis. (A) β-galactosidase activities (β-gal). Expressed TetR variants are specified left of the bar graph. In the schemas on the left, gray and black rectangles indicate sequences derived from tetR(B) and tetR(D) respectively. Red stars indicate the location of mutation causing the reverse phenotype of TetR r1.7. Blue stars indicate TetR(B) to TetR(D) mutations. β-Gal 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. (B) β-galactosidase activities (β-gal). As described for (A). (C) Western blots. Protein lysates were prepared from M. smegmatis expressing TetR variants encoded by episomally replicating plasmids. TetR was detected by a monoclonal anti-TetR antibody (lower panel) and the dihydrolipoamide acyltransferase (DlaT) signal was used as a loading control and detected by a polyclonal anti-DlaT antibody (upper panel). (D) Structure of tetO-bound TetR(D) (22). The two monomers are shown in light and dark gray. The side chains of the amino acids that are mutated in and cause the reverse phenotype of TetR r1.7 are shown in red. In the dark gray monomer helices 4 to 7 are drawn in yellow. Side chains that are different in TetR(B) and TetR(D) and within a radius of 15 Å to positions 15, 17 and 25 are shown in light blue (for clarity sake only within one monomer).
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Figure 5: Analysis of reverse TetR chimeras in M. smegmatis. (A) β-galactosidase activities (β-gal). Expressed TetR variants are specified left of the bar graph. In the schemas on the left, gray and black rectangles indicate sequences derived from tetR(B) and tetR(D) respectively. Red stars indicate the location of mutation causing the reverse phenotype of TetR r1.7. Blue stars indicate TetR(B) to TetR(D) mutations. β-Gal 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. (B) β-galactosidase activities (β-gal). As described for (A). (C) Western blots. Protein lysates were prepared from M. smegmatis expressing TetR variants encoded by episomally replicating plasmids. TetR was detected by a monoclonal anti-TetR antibody (lower panel) and the dihydrolipoamide acyltransferase (DlaT) signal was used as a loading control and detected by a polyclonal anti-DlaT antibody (upper panel). (D) Structure of tetO-bound TetR(D) (22). The two monomers are shown in light and dark gray. The side chains of the amino acids that are mutated in and cause the reverse phenotype of TetR r1.7 are shown in red. In the dark gray monomer helices 4 to 7 are drawn in yellow. Side chains that are different in TetR(B) and TetR(D) and within a radius of 15 Å to positions 15, 17 and 25 are shown in light blue (for clarity sake only within one monomer).

Mentions: We first examined if a reverse TetR(B) could be constructed by introducing the amino acid exchanges present in TetR r1.7. Expression of this repressor using an episomally replicating plasmid containing Pimyc-tetR(Bsyn1–207)E15A-L17G-L25V did not affect the β-galactosidase activity of M. smegmatis Pmyc1tetO-lacZL5 with or without atc (Figure 5A). When expressed by the strong promoter Psmyc, this repressor reduced β-galactosidase activity ∼5-fold in the presence of atc (data not shown). No repression was observed in the absence of atc. TetR(Bsyn1-207)E15A-L17G-L25V thus encoded a less efficient reverse TetR than the corresponding tetR(BDsyn1–208) gene (tetR(BDsyn1–208)E15A-L17G-L25V, Figure 3B) even though the steady-state level of the TetR(B) protein was higher than that of the TetR(BD) protein (Figure 5C). This suggested that additional TetR(B) amino acids needed to be replaced by the corresponding TetR(D) amino acids to not only achieve an increase in protein expression but also generate an efficient reverse phenotype.Figure 5.


Improved tetracycline repressors for gene silencing in mycobacteria.

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

Analysis of reverse TetR chimeras in M. smegmatis. (A) β-galactosidase activities (β-gal). Expressed TetR variants are specified left of the bar graph. In the schemas on the left, gray and black rectangles indicate sequences derived from tetR(B) and tetR(D) respectively. Red stars indicate the location of mutation causing the reverse phenotype of TetR r1.7. Blue stars indicate TetR(B) to TetR(D) mutations. β-Gal 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. (B) β-galactosidase activities (β-gal). As described for (A). (C) Western blots. Protein lysates were prepared from M. smegmatis expressing TetR variants encoded by episomally replicating plasmids. TetR was detected by a monoclonal anti-TetR antibody (lower panel) and the dihydrolipoamide acyltransferase (DlaT) signal was used as a loading control and detected by a polyclonal anti-DlaT antibody (upper panel). (D) Structure of tetO-bound TetR(D) (22). The two monomers are shown in light and dark gray. The side chains of the amino acids that are mutated in and cause the reverse phenotype of TetR r1.7 are shown in red. In the dark gray monomer helices 4 to 7 are drawn in yellow. Side chains that are different in TetR(B) and TetR(D) and within a radius of 15 Å to positions 15, 17 and 25 are shown in light blue (for clarity sake only within one monomer).
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Figure 5: Analysis of reverse TetR chimeras in M. smegmatis. (A) β-galactosidase activities (β-gal). Expressed TetR variants are specified left of the bar graph. In the schemas on the left, gray and black rectangles indicate sequences derived from tetR(B) and tetR(D) respectively. Red stars indicate the location of mutation causing the reverse phenotype of TetR r1.7. Blue stars indicate TetR(B) to TetR(D) mutations. β-Gal 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. (B) β-galactosidase activities (β-gal). As described for (A). (C) Western blots. Protein lysates were prepared from M. smegmatis expressing TetR variants encoded by episomally replicating plasmids. TetR was detected by a monoclonal anti-TetR antibody (lower panel) and the dihydrolipoamide acyltransferase (DlaT) signal was used as a loading control and detected by a polyclonal anti-DlaT antibody (upper panel). (D) Structure of tetO-bound TetR(D) (22). The two monomers are shown in light and dark gray. The side chains of the amino acids that are mutated in and cause the reverse phenotype of TetR r1.7 are shown in red. In the dark gray monomer helices 4 to 7 are drawn in yellow. Side chains that are different in TetR(B) and TetR(D) and within a radius of 15 Å to positions 15, 17 and 25 are shown in light blue (for clarity sake only within one monomer).
Mentions: We first examined if a reverse TetR(B) could be constructed by introducing the amino acid exchanges present in TetR r1.7. Expression of this repressor using an episomally replicating plasmid containing Pimyc-tetR(Bsyn1–207)E15A-L17G-L25V did not affect the β-galactosidase activity of M. smegmatis Pmyc1tetO-lacZL5 with or without atc (Figure 5A). When expressed by the strong promoter Psmyc, this repressor reduced β-galactosidase activity ∼5-fold in the presence of atc (data not shown). No repression was observed in the absence of atc. TetR(Bsyn1-207)E15A-L17G-L25V thus encoded a less efficient reverse TetR than the corresponding tetR(BDsyn1–208) gene (tetR(BDsyn1–208)E15A-L17G-L25V, Figure 3B) even though the steady-state level of the TetR(B) protein was higher than that of the TetR(BD) protein (Figure 5C). This suggested that additional TetR(B) amino acids needed to be replaced by the corresponding TetR(D) amino acids to not only achieve an increase in protein expression but also generate an efficient reverse phenotype.Figure 5.

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