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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 its own transcription during in vitro growth of M.tuberculosis. Comparison of mce3R promoter activity during the growth of Δmce3R (pPR3-lacZ) (white bars) and H37Rv (pPR3-lacZ- Mce3R endogenous) (grey 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 (pPR3-lacZ) (circle) and H37Rv (pPR3-lacZ- Mce3R endogenous) (rhombus) strains are shown and the OD 600nm values are indicated on the right. Results represent one of three independent experiments.
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Figure 5: Effect of Mce3R on its own transcription during in vitro growth of M.tuberculosis. Comparison of mce3R promoter activity during the growth of Δmce3R (pPR3-lacZ) (white bars) and H37Rv (pPR3-lacZ- Mce3R endogenous) (grey 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 (pPR3-lacZ) (circle) and H37Rv (pPR3-lacZ- Mce3R endogenous) (rhombus) strains are shown and the OD 600nm values are indicated on the right. Results represent one of three independent experiments.

Mentions: To investigate whether mce3R is subject to transcriptional autoregulation, a transcriptional fusion was constructed between the mce3R promoter and the lacZ reporter. Since mce3R is located adjacent to mce3 operon and divergently oriented, the mce3R promoter is situated in mce3R-yrbE3A intergenic region. The entire intergenic region was fused to the lacZ gene within pYUB178-lacZ to create plasmid pPR3-lacZ. The pPR3-lacZ plasmid was transformed into the wild type M. tuberculosis H37Rv and the M. tuberculosis Δmce3R mutant, and β-galactosidase activity was measured to assess the levels of mce3R promoter activity with and without Mce3R regulator. As shown in figure 5, in the presence of Mce3R (wild type H37Rv strain) the activity of mce3R promoter is steadily and significantly reduced as compared with that in the absence of the Mce3R regulator (mutant Δmce3R strain). This reduction in mce3R promoter activity was more evident during the stationary growth phase. These experiments demonstrate that the Mce3R protein is able to transcriptionally repress expression of the mce3R promoter in M. tuberculosis during the in vitro culture condition tested.


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 its own transcription during in vitro growth of M.tuberculosis. Comparison of mce3R promoter activity during the growth of Δmce3R (pPR3-lacZ) (white bars) and H37Rv (pPR3-lacZ- Mce3R endogenous) (grey 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 (pPR3-lacZ) (circle) and H37Rv (pPR3-lacZ- Mce3R endogenous) (rhombus) strains are shown and the OD 600nm values are indicated on the right. Results represent one of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of Mce3R on its own transcription during in vitro growth of M.tuberculosis. Comparison of mce3R promoter activity during the growth of Δmce3R (pPR3-lacZ) (white bars) and H37Rv (pPR3-lacZ- Mce3R endogenous) (grey 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 (pPR3-lacZ) (circle) and H37Rv (pPR3-lacZ- Mce3R endogenous) (rhombus) strains are shown and the OD 600nm values are indicated on the right. Results represent one of three independent experiments.
Mentions: To investigate whether mce3R is subject to transcriptional autoregulation, a transcriptional fusion was constructed between the mce3R promoter and the lacZ reporter. Since mce3R is located adjacent to mce3 operon and divergently oriented, the mce3R promoter is situated in mce3R-yrbE3A intergenic region. The entire intergenic region was fused to the lacZ gene within pYUB178-lacZ to create plasmid pPR3-lacZ. The pPR3-lacZ plasmid was transformed into the wild type M. tuberculosis H37Rv and the M. tuberculosis Δmce3R mutant, and β-galactosidase activity was measured to assess the levels of mce3R promoter activity with and without Mce3R regulator. As shown in figure 5, in the presence of Mce3R (wild type H37Rv strain) the activity of mce3R promoter is steadily and significantly reduced as compared with that in the absence of the Mce3R regulator (mutant Δmce3R strain). This reduction in mce3R promoter activity was more evident during the stationary growth phase. These experiments demonstrate that the Mce3R protein is able to transcriptionally repress expression of the mce3R promoter in M. tuberculosis during the in vitro culture condition tested.

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