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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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Related in: MedlinePlus

Effect of the mce3R mutation on in vitro growth of M. tuberculosis. Cultures of the mutant [Δmce3R, square], the complemented [Δmce3R (pSummce3R), triangle] and the parental wild-type [H37Rv, rhombus] strains were grown to stationary phase and inoculated into fresh Dubos medium supplemented with 0.4% glucose at OD600nm 0.005 and the OD600nm was measured at various time points. It is shown a representative experiment from triplicate.
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Figure 2: Effect of the mce3R mutation on in vitro growth of M. tuberculosis. Cultures of the mutant [Δmce3R, square], the complemented [Δmce3R (pSummce3R), triangle] and the parental wild-type [H37Rv, rhombus] strains were grown to stationary phase and inoculated into fresh Dubos medium supplemented with 0.4% glucose at OD600nm 0.005 and the OD600nm was measured at various time points. It is shown a representative experiment from triplicate.

Mentions: To determine whether mce3R disruption introduces alterations during in vitro growth, growth curves of the Δmce3R mutant, complemented, and parental strains were compared under standard culture conditions. All assayed strains showed similar doubling time and growth characteristics throughout the culture period (Fig. 2). This result indicates that the mutation does not affect the in vitro growth of M. tuberculosis.


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 the mce3R mutation on in vitro growth of M. tuberculosis. Cultures of the mutant [Δmce3R, square], the complemented [Δmce3R (pSummce3R), triangle] and the parental wild-type [H37Rv, rhombus] strains were grown to stationary phase and inoculated into fresh Dubos medium supplemented with 0.4% glucose at OD600nm 0.005 and the OD600nm was measured at various time points. It is shown a representative experiment from triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of the mce3R mutation on in vitro growth of M. tuberculosis. Cultures of the mutant [Δmce3R, square], the complemented [Δmce3R (pSummce3R), triangle] and the parental wild-type [H37Rv, rhombus] strains were grown to stationary phase and inoculated into fresh Dubos medium supplemented with 0.4% glucose at OD600nm 0.005 and the OD600nm was measured at various time points. It is shown a representative experiment from triplicate.
Mentions: To determine whether mce3R disruption introduces alterations during in vitro growth, growth curves of the Δmce3R mutant, complemented, and parental strains were compared under standard culture conditions. All assayed strains showed similar doubling time and growth characteristics throughout the culture period (Fig. 2). This result indicates that the mutation does not affect the in vitro growth of M. tuberculosis.

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