Limits...
A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

Torres-Puig S, Broto A, Querol E, Piñol J, Pich OQ - Nucleic Acids Res. (2015)

Bottom Line: Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function.Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor.Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

Show MeSH

Related in: MedlinePlus

Analysis of protein expression by western blotting. (A) Immunoblot analysis of MG428 expression in the WT strain and several representative mutants. Lane 1, WT; lane 2, ΔMG_428; lane 3, Tn::MG_390-1; lane 4, Tn::MG_281-1; lane 5, Tn::recA-2; lane 6, Tn::MG_220-1; lane 7, Tn::MG_191-2 and lane 8, Tn::MG_192-1. (B) Immunoblot analysis of mCherry expression. Lane 1, WT; lane 2, Cat:Ch; lane 3, RecA:Ch; lane 4, MG_428:Ch; lane 5, ΔMG_428-RecA:Ch; lane 6, RecA:Ch-10; lane 7, RecA:Ch-22 and lane 8, RecA:Ch-35. Because the Cat-mCherry fusion is expressed at very high levels compared to the RecA-mCherry fusion, the amount of total protein loaded for the Cat:Ch mutant was reduced 20 times. HsdS protein was detected with a monoclonal antibody and used as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4446450&req=5

Figure 2: Analysis of protein expression by western blotting. (A) Immunoblot analysis of MG428 expression in the WT strain and several representative mutants. Lane 1, WT; lane 2, ΔMG_428; lane 3, Tn::MG_390-1; lane 4, Tn::MG_281-1; lane 5, Tn::recA-2; lane 6, Tn::MG_220-1; lane 7, Tn::MG_191-2 and lane 8, Tn::MG_192-1. (B) Immunoblot analysis of mCherry expression. Lane 1, WT; lane 2, Cat:Ch; lane 3, RecA:Ch; lane 4, MG_428:Ch; lane 5, ΔMG_428-RecA:Ch; lane 6, RecA:Ch-10; lane 7, RecA:Ch-22 and lane 8, RecA:Ch-35. Because the Cat-mCherry fusion is expressed at very high levels compared to the RecA-mCherry fusion, the amount of total protein loaded for the Cat:Ch mutant was reduced 20 times. HsdS protein was detected with a monoclonal antibody and used as a loading control.

Mentions: Expression of the MG428 protein was analyzed by Western blot using anti-MG428 polyclonal antibodies (Figure 2A). The MG428 protein was not detected in lysates of the WT strain, indicating that it was expressed at very low levels in M. genitalium. Likewise, MG428 expression was not detected in lysates of the ΔMG_428 mutant. In contrast, a band of the predicted molecular mass of the MG428 full-length protein (17 kDa), was clearly detected in the complemented mutants. This result indicates that transcriptional fusion of the MG_428 gene to its own promoter, located upstream of the MG_427 gene, leads to increased levels of MG428 expression as compared to the WT strain. In light of this finding, we analyzed the intergenic region between the MG_427 and MG_428 genes and we identified a possible Rho-independent terminator (Supplementary Figure S4). The presence of this putative terminator, which is supported by several transcription terminator prediction softwares (34–36), could explain the reduced levels of MG428 expression observed in the WT strain. On the other hand, MG428 levels differed considerably among the complemented strains, indicating that the genetic context of the transposon insertion had a significant impact on the expression of the MG_428 ectopic copy. Finally, in agreement with the presence of a truncated copy of the MG_428 gene, a 9 kDa band was detected in the Tn::MG_390-1 mutant.


A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

Torres-Puig S, Broto A, Querol E, Piñol J, Pich OQ - Nucleic Acids Res. (2015)

Analysis of protein expression by western blotting. (A) Immunoblot analysis of MG428 expression in the WT strain and several representative mutants. Lane 1, WT; lane 2, ΔMG_428; lane 3, Tn::MG_390-1; lane 4, Tn::MG_281-1; lane 5, Tn::recA-2; lane 6, Tn::MG_220-1; lane 7, Tn::MG_191-2 and lane 8, Tn::MG_192-1. (B) Immunoblot analysis of mCherry expression. Lane 1, WT; lane 2, Cat:Ch; lane 3, RecA:Ch; lane 4, MG_428:Ch; lane 5, ΔMG_428-RecA:Ch; lane 6, RecA:Ch-10; lane 7, RecA:Ch-22 and lane 8, RecA:Ch-35. Because the Cat-mCherry fusion is expressed at very high levels compared to the RecA-mCherry fusion, the amount of total protein loaded for the Cat:Ch mutant was reduced 20 times. HsdS protein was detected with a monoclonal antibody and used as a loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446450&req=5

Figure 2: Analysis of protein expression by western blotting. (A) Immunoblot analysis of MG428 expression in the WT strain and several representative mutants. Lane 1, WT; lane 2, ΔMG_428; lane 3, Tn::MG_390-1; lane 4, Tn::MG_281-1; lane 5, Tn::recA-2; lane 6, Tn::MG_220-1; lane 7, Tn::MG_191-2 and lane 8, Tn::MG_192-1. (B) Immunoblot analysis of mCherry expression. Lane 1, WT; lane 2, Cat:Ch; lane 3, RecA:Ch; lane 4, MG_428:Ch; lane 5, ΔMG_428-RecA:Ch; lane 6, RecA:Ch-10; lane 7, RecA:Ch-22 and lane 8, RecA:Ch-35. Because the Cat-mCherry fusion is expressed at very high levels compared to the RecA-mCherry fusion, the amount of total protein loaded for the Cat:Ch mutant was reduced 20 times. HsdS protein was detected with a monoclonal antibody and used as a loading control.
Mentions: Expression of the MG428 protein was analyzed by Western blot using anti-MG428 polyclonal antibodies (Figure 2A). The MG428 protein was not detected in lysates of the WT strain, indicating that it was expressed at very low levels in M. genitalium. Likewise, MG428 expression was not detected in lysates of the ΔMG_428 mutant. In contrast, a band of the predicted molecular mass of the MG428 full-length protein (17 kDa), was clearly detected in the complemented mutants. This result indicates that transcriptional fusion of the MG_428 gene to its own promoter, located upstream of the MG_427 gene, leads to increased levels of MG428 expression as compared to the WT strain. In light of this finding, we analyzed the intergenic region between the MG_427 and MG_428 genes and we identified a possible Rho-independent terminator (Supplementary Figure S4). The presence of this putative terminator, which is supported by several transcription terminator prediction softwares (34–36), could explain the reduced levels of MG428 expression observed in the WT strain. On the other hand, MG428 levels differed considerably among the complemented strains, indicating that the genetic context of the transposon insertion had a significant impact on the expression of the MG_428 ectopic copy. Finally, in agreement with the presence of a truncated copy of the MG_428 gene, a 9 kDa band was detected in the Tn::MG_390-1 mutant.

Bottom Line: Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function.Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor.Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

Show MeSH
Related in: MedlinePlus