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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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Disruption of the mce3R gene of M. tuberculosis H37Rv. (A) Southern blot analysis of chromosomal DNA from sucR counterselected tuberculosis clone (lane 3) and parental strain (lane 2). Genomic DNA was digested with EcoRI and hybridized to the mce3R probe. Arrows indicate position of hybridizing fragments. MWM 1 kb Promega is shown on the left (lane 1). (B) Restriction map of mutant and wild type strains. The insertion of hygromycin-resistant cassettes is indicated (hyg). Arrows represent the length of expected bands after digestion with EcoRI (E). Value on each arrow indicates the molecular weight of expected bands expressed in base pairs (bp).
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Figure 1: Disruption of the mce3R gene of M. tuberculosis H37Rv. (A) Southern blot analysis of chromosomal DNA from sucR counterselected tuberculosis clone (lane 3) and parental strain (lane 2). Genomic DNA was digested with EcoRI and hybridized to the mce3R probe. Arrows indicate position of hybridizing fragments. MWM 1 kb Promega is shown on the left (lane 1). (B) Restriction map of mutant and wild type strains. The insertion of hygromycin-resistant cassettes is indicated (hyg). Arrows represent the length of expected bands after digestion with EcoRI (E). Value on each arrow indicates the molecular weight of expected bands expressed in base pairs (bp).

Mentions: As a first step to assess the mce3 operon expression in the absence of Mce3R, we obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The site-directed mutant strain of M. tuberculosis was obtained by two-step mutagenesis strategy by using the p2NIL shuttle plasmid [21], which carries the lacZ gene and the counter selectable marker sacB. Allelic exchange was confirmed in the selected clones (HyR, KmS, and SacR) by Southern blotting (Fig. 1A), since the mutant showed a hybridizing fragment of about 1.5 kb absent in the wild-type strain. This polymorphism is due to the introduction of an extra EcoRI site present in the hygromycin cassette (Fig. 1B). The mutant strain was designated Δmce3R. The mutation was complemented by transforming the plasmid pSummce3R into the mutant.


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)

Disruption of the mce3R gene of M. tuberculosis H37Rv. (A) Southern blot analysis of chromosomal DNA from sucR counterselected tuberculosis clone (lane 3) and parental strain (lane 2). Genomic DNA was digested with EcoRI and hybridized to the mce3R probe. Arrows indicate position of hybridizing fragments. MWM 1 kb Promega is shown on the left (lane 1). (B) Restriction map of mutant and wild type strains. The insertion of hygromycin-resistant cassettes is indicated (hyg). Arrows represent the length of expected bands after digestion with EcoRI (E). Value on each arrow indicates the molecular weight of expected bands expressed in base pairs (bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Disruption of the mce3R gene of M. tuberculosis H37Rv. (A) Southern blot analysis of chromosomal DNA from sucR counterselected tuberculosis clone (lane 3) and parental strain (lane 2). Genomic DNA was digested with EcoRI and hybridized to the mce3R probe. Arrows indicate position of hybridizing fragments. MWM 1 kb Promega is shown on the left (lane 1). (B) Restriction map of mutant and wild type strains. The insertion of hygromycin-resistant cassettes is indicated (hyg). Arrows represent the length of expected bands after digestion with EcoRI (E). Value on each arrow indicates the molecular weight of expected bands expressed in base pairs (bp).
Mentions: As a first step to assess the mce3 operon expression in the absence of Mce3R, we obtained a knockout mutant strain of M. tuberculosis H37Rv by inserting a hygromycin cassette into the mce3R gene. The site-directed mutant strain of M. tuberculosis was obtained by two-step mutagenesis strategy by using the p2NIL shuttle plasmid [21], which carries the lacZ gene and the counter selectable marker sacB. Allelic exchange was confirmed in the selected clones (HyR, KmS, and SacR) by Southern blotting (Fig. 1A), since the mutant showed a hybridizing fragment of about 1.5 kb absent in the wild-type strain. This polymorphism is due to the introduction of an extra EcoRI site present in the hygromycin cassette (Fig. 1B). The mutant strain was designated Δmce3R. The mutation was complemented by transforming the plasmid pSummce3R into the mutant.

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