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Characterization of a conserved interaction between DNA glycosylase and ParA in Mycobacterium smegmatis and M. tuberculosis.

Huang F, He ZG - PLoS ONE (2012)

Bottom Line: Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA.Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction.In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.

ABSTRACT
The chromosome partitioning proteins, ParAB, ensure accurate segregation of genetic materials into daughter cells and most bacterial species contain their homologs. However, little is known about the regulation of ParAB proteins. In this study, we found that 3-methyladenine DNA glycosylase I MsTAG(Ms5082) regulates bacterial growth and cell morphology by directly interacting with MsParA (Ms6939) and inhibiting its ATPase activity in Mycobacterium smegmatis. Using bacterial two-hybrid and pull-down techniques in combination with co-immunoprecipitation assays, we show that MsTAG physically interacts with MsParA both in vitro and in vivo. Expression of MsTAG under conditions of DNA damage induction exhibited similar inhibition of growth as the deletion of the parA gene in M. smegmatis. Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA. Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction. Based on protein co-expression and fluorescent co-localization assays, MsParA and MsTAG were further found to co-localize in mycobacterial cells. In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis. Our findings provide important new insights into the regulatory mechanism of cell growth and division in mycobacteria.

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MsParA affects the growth and morphology of M. smegmatis.The wild-type and mutant strains were grown on the surface of solid agar medium and in the liquid 7H9 medium. (A) Strains were grown on 7H10 agar plates supplemented with 30 µg/ml Kanamycin (Kan) at 37°C for 48 hours. (B) Monitoring of growth on 7H9 medium of the M. smegmatis wild-type (Ms/pMV361), MsParA deletion strain (Msm-MsParA::hyg/pMV361) and MsParA complementation strain (Msm-MsParA::hyg/pMV361MsParA) by OD600 analysis as described under “Materials and Methods”. (C) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in the “Materials and Methods”. Representative images are shown. The images were taken at 15,000× magnification. Bars, 1 µm.
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pone-0038276-g002: MsParA affects the growth and morphology of M. smegmatis.The wild-type and mutant strains were grown on the surface of solid agar medium and in the liquid 7H9 medium. (A) Strains were grown on 7H10 agar plates supplemented with 30 µg/ml Kanamycin (Kan) at 37°C for 48 hours. (B) Monitoring of growth on 7H9 medium of the M. smegmatis wild-type (Ms/pMV361), MsParA deletion strain (Msm-MsParA::hyg/pMV361) and MsParA complementation strain (Msm-MsParA::hyg/pMV361MsParA) by OD600 analysis as described under “Materials and Methods”. (C) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in the “Materials and Methods”. Representative images are shown. The images were taken at 15,000× magnification. Bars, 1 µm.

Mentions: Next, we measured the growth of mutant and wildtype strains on the surface of solid agar medium and in liquid 7H9 medium. As shown in Figure 2A, when the mycobacterial strains were spotted on the surface of solid agar medium, a thin bacterial lawn was observed for the mutant strain in contrast to the thicker lawn for the wildtype, indicating that the parA-deleted mycobacterial strain grew at a slower rate than the wildtype. Expression of parA through a pMV361-derived vector could rescue the slow growth phenotype of the mutant strain (Fig. 2A).


Characterization of a conserved interaction between DNA glycosylase and ParA in Mycobacterium smegmatis and M. tuberculosis.

Huang F, He ZG - PLoS ONE (2012)

MsParA affects the growth and morphology of M. smegmatis.The wild-type and mutant strains were grown on the surface of solid agar medium and in the liquid 7H9 medium. (A) Strains were grown on 7H10 agar plates supplemented with 30 µg/ml Kanamycin (Kan) at 37°C for 48 hours. (B) Monitoring of growth on 7H9 medium of the M. smegmatis wild-type (Ms/pMV361), MsParA deletion strain (Msm-MsParA::hyg/pMV361) and MsParA complementation strain (Msm-MsParA::hyg/pMV361MsParA) by OD600 analysis as described under “Materials and Methods”. (C) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in the “Materials and Methods”. Representative images are shown. The images were taken at 15,000× magnification. Bars, 1 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366916&req=5

pone-0038276-g002: MsParA affects the growth and morphology of M. smegmatis.The wild-type and mutant strains were grown on the surface of solid agar medium and in the liquid 7H9 medium. (A) Strains were grown on 7H10 agar plates supplemented with 30 µg/ml Kanamycin (Kan) at 37°C for 48 hours. (B) Monitoring of growth on 7H9 medium of the M. smegmatis wild-type (Ms/pMV361), MsParA deletion strain (Msm-MsParA::hyg/pMV361) and MsParA complementation strain (Msm-MsParA::hyg/pMV361MsParA) by OD600 analysis as described under “Materials and Methods”. (C) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in the “Materials and Methods”. Representative images are shown. The images were taken at 15,000× magnification. Bars, 1 µm.
Mentions: Next, we measured the growth of mutant and wildtype strains on the surface of solid agar medium and in liquid 7H9 medium. As shown in Figure 2A, when the mycobacterial strains were spotted on the surface of solid agar medium, a thin bacterial lawn was observed for the mutant strain in contrast to the thicker lawn for the wildtype, indicating that the parA-deleted mycobacterial strain grew at a slower rate than the wildtype. Expression of parA through a pMV361-derived vector could rescue the slow growth phenotype of the mutant strain (Fig. 2A).

Bottom Line: Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA.Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction.In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.

ABSTRACT
The chromosome partitioning proteins, ParAB, ensure accurate segregation of genetic materials into daughter cells and most bacterial species contain their homologs. However, little is known about the regulation of ParAB proteins. In this study, we found that 3-methyladenine DNA glycosylase I MsTAG(Ms5082) regulates bacterial growth and cell morphology by directly interacting with MsParA (Ms6939) and inhibiting its ATPase activity in Mycobacterium smegmatis. Using bacterial two-hybrid and pull-down techniques in combination with co-immunoprecipitation assays, we show that MsTAG physically interacts with MsParA both in vitro and in vivo. Expression of MsTAG under conditions of DNA damage induction exhibited similar inhibition of growth as the deletion of the parA gene in M. smegmatis. Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA. Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction. Based on protein co-expression and fluorescent co-localization assays, MsParA and MsTAG were further found to co-localize in mycobacterial cells. In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis. Our findings provide important new insights into the regulatory mechanism of cell growth and division in mycobacteria.

Show MeSH
Related in: MedlinePlus