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Study of the role of Mce3R on the transcription of mce genes of Mycobacterium tuberculosis.

Santangelo MP, Blanco FC, Bianco MV, Klepp LI, Zabal O, Cataldi AA, Bigi F - BMC Microbiol. (2008)

Bottom Line: The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR.The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis.Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, CICVyA-INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina. psantangelo@cnia.inta.gov.ar

ABSTRACT

Background: mce3 is one of the four virulence-related mce operons of Mycobacterium tuberculosis. In a previous work we showed that the overexpression of Mce3R in Mycobacterium smegmatis and M. tuberculosis abolishes the expression of lacZ fused to the mce3 promoter, indicating that Mce3R represses mce3 transcription.

Results: We obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The mutation results in a significant increase in the expression of mce3 genes either in vitro or in a murine cell macrophages line as it was determined using promoter-lacZ fusions in M. tuberculosis. The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis.

Conclusion: Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.

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Effect of Mce3R on mce3 promoter activity during the growth of M. tuberculosis. Comparison of mce3 promoter activity during the growth of Δmce3R (pP3-mce3R-lacZ) (grey bars) and Δmce3R (pP3-lacZ) (white bars) strains in M7H9-AD-G medium. The results are presented as β-galactosidase activity expressed as Miller units ± S.D. of duplicate in three time points (1 early exponential phase, 2 exponential phase and 3 stationary phase). Growth curves of Δmce3R (pP3-lacZ) (square) and Δmce3R (pP3-mce3RlacZ) (circle) strains are shown and the OD 600nm values are indicated on the right. Results represent one of at least three independent experiments.
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Figure 3: Effect of Mce3R on mce3 promoter activity during the growth of M. tuberculosis. Comparison of mce3 promoter activity during the growth of Δmce3R (pP3-mce3R-lacZ) (grey bars) and Δmce3R (pP3-lacZ) (white bars) strains in M7H9-AD-G medium. The results are presented as β-galactosidase activity expressed as Miller units ± S.D. of duplicate in three time points (1 early exponential phase, 2 exponential phase and 3 stationary phase). Growth curves of Δmce3R (pP3-lacZ) (square) and Δmce3R (pP3-mce3RlacZ) (circle) strains are shown and the OD 600nm values are indicated on the right. Results represent one of at least three independent experiments.

Mentions: Here, in order to compare the expression the mce3 operon either in the absence or in the presence of Mce3R, DNA fusions of the mce3 promoter to lacZ reporter, containing or not containing mce3R were cloned within pYUB178-lacZ. The resulting plasmids, pP3-mce3R and pP3 respectively, were integrated into the chromosome of the Δmce3R strain. The β-galactosidase activity was measured at different points along cultures of M. tuberculosis grown in vitro and in a macrophages cell line. Since transcription of mce3 genes has previously shown to be increased when M. tuberculosis was grown in rich media [10-12] the expression of mce3 operon was assessed in both synthetic (7H9) and rich (Dubos) media (Figure 3 and data not shown).


Study of the role of Mce3R on the transcription of mce genes of Mycobacterium tuberculosis.

Santangelo MP, Blanco FC, Bianco MV, Klepp LI, Zabal O, Cataldi AA, Bigi F - BMC Microbiol. (2008)

Effect of Mce3R on mce3 promoter activity during the growth of M. tuberculosis. Comparison of mce3 promoter activity during the growth of Δmce3R (pP3-mce3R-lacZ) (grey bars) and Δmce3R (pP3-lacZ) (white bars) strains in M7H9-AD-G medium. The results are presented as β-galactosidase activity expressed as Miller units ± S.D. of duplicate in three time points (1 early exponential phase, 2 exponential phase and 3 stationary phase). Growth curves of Δmce3R (pP3-lacZ) (square) and Δmce3R (pP3-mce3RlacZ) (circle) strains are shown and the OD 600nm values are indicated on the right. Results represent one of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2277422&req=5

Figure 3: Effect of Mce3R on mce3 promoter activity during the growth of M. tuberculosis. Comparison of mce3 promoter activity during the growth of Δmce3R (pP3-mce3R-lacZ) (grey bars) and Δmce3R (pP3-lacZ) (white bars) strains in M7H9-AD-G medium. The results are presented as β-galactosidase activity expressed as Miller units ± S.D. of duplicate in three time points (1 early exponential phase, 2 exponential phase and 3 stationary phase). Growth curves of Δmce3R (pP3-lacZ) (square) and Δmce3R (pP3-mce3RlacZ) (circle) strains are shown and the OD 600nm values are indicated on the right. Results represent one of at least three independent experiments.
Mentions: Here, in order to compare the expression the mce3 operon either in the absence or in the presence of Mce3R, DNA fusions of the mce3 promoter to lacZ reporter, containing or not containing mce3R were cloned within pYUB178-lacZ. The resulting plasmids, pP3-mce3R and pP3 respectively, were integrated into the chromosome of the Δmce3R strain. The β-galactosidase activity was measured at different points along cultures of M. tuberculosis grown in vitro and in a macrophages cell line. Since transcription of mce3 genes has previously shown to be increased when M. tuberculosis was grown in rich media [10-12] the expression of mce3 operon was assessed in both synthetic (7H9) and rich (Dubos) media (Figure 3 and data not shown).

Bottom Line: The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR.The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis.Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, CICVyA-INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina. psantangelo@cnia.inta.gov.ar

ABSTRACT

Background: mce3 is one of the four virulence-related mce operons of Mycobacterium tuberculosis. In a previous work we showed that the overexpression of Mce3R in Mycobacterium smegmatis and M. tuberculosis abolishes the expression of lacZ fused to the mce3 promoter, indicating that Mce3R represses mce3 transcription.

Results: We obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The mutation results in a significant increase in the expression of mce3 genes either in vitro or in a murine cell macrophages line as it was determined using promoter-lacZ fusions in M. tuberculosis. The abundance of mce1, mce2 and mce4 mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The mce3R promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the in vitro culture of M. tuberculosis.

Conclusion: Mce3R repress the transcription of mce3 operon and self regulates its own expression but does not affect the transcription of mce1, mce2 and mce4 operons of M. tuberculosis.

Show MeSH
Related in: MedlinePlus