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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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Physical interaction of MsTAG (Ms5082) with MsParA and its effect on mycobacterial growth in response to DNA damage induction.(A) Bacterial two-hybrid assays for the interaction of MsTAG with MsParA performed as described in ‘Materials and Methods’. (B) Co-IP assays. Exponentially growing cells of recombinant M. smegmatis containing MsTAG-expression plasmid were harvested, resuspended and lysed. Co-IP assays were performed as described under ‘Materials and Methods’. Right panel shows a negative control using an unrelated anti-Ms3759 anti-serum. (C) MMS sensitivity assays for the MsTAG-overexpressing M. smegmatis strains. Growth of the recombinant mycobacterial strains were examined in the presence or absence of 0.012% MMS. Aliquots were taken at the indicated times and the CFU was measured. Each analysis was performed in triplicate. Representative growth curves are shown. The recombinant mycobacterial strains are indicated above the panels. (D) Scanning electron microscopy assay of cell morphology. The recombinant mycobacterial strains were grown in the presence of 0.012% MMS and SEM observation was carried out as described in ‘Materials and Methods’. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.
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pone-0038276-g003: Physical interaction of MsTAG (Ms5082) with MsParA and its effect on mycobacterial growth in response to DNA damage induction.(A) Bacterial two-hybrid assays for the interaction of MsTAG with MsParA performed as described in ‘Materials and Methods’. (B) Co-IP assays. Exponentially growing cells of recombinant M. smegmatis containing MsTAG-expression plasmid were harvested, resuspended and lysed. Co-IP assays were performed as described under ‘Materials and Methods’. Right panel shows a negative control using an unrelated anti-Ms3759 anti-serum. (C) MMS sensitivity assays for the MsTAG-overexpressing M. smegmatis strains. Growth of the recombinant mycobacterial strains were examined in the presence or absence of 0.012% MMS. Aliquots were taken at the indicated times and the CFU was measured. Each analysis was performed in triplicate. Representative growth curves are shown. The recombinant mycobacterial strains are indicated above the panels. (D) Scanning electron microscopy assay of cell morphology. The recombinant mycobacterial strains were grown in the presence of 0.012% MMS and SEM observation was carried out as described in ‘Materials and Methods’. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.

Mentions: In a previous global protein-protein interaction analysis [38], the M. tuberculosis MtParA, encoded by Rv3918c, was linked to MtTAG, encoded by Rv1210. We assayed the potential physical interaction between their two corresponding M. smegmatis homologs–MsParA and MsTAG–to further examine the regulation of ParA. As shown in Figure 3A, in our bacterial two-hybrid assays, the co-transformants containing MsParA and MsTAG grew well on the screening medium. Positive co-transformants (CK+) grew on the medium, whereas negative co-transformants (CK−) were incapable of growth on the same screening medium. No growth was observed for their self-activated controls, or for the co-transformants of MsParA and a non-specific gene (Ms1746). Consistent with previous results [33], a clear interaction between MtParA and MtTAG was detected (Fig. 3A). These results indicated that MsParA physically interacts with MsTAG in M. smegmatis. A further in vitro pull-down assay using purified forms of these proteins also confirmed the specific interaction between them (Suppl Fig. S1).


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

Huang F, He ZG - PLoS ONE (2012)

Physical interaction of MsTAG (Ms5082) with MsParA and its effect on mycobacterial growth in response to DNA damage induction.(A) Bacterial two-hybrid assays for the interaction of MsTAG with MsParA performed as described in ‘Materials and Methods’. (B) Co-IP assays. Exponentially growing cells of recombinant M. smegmatis containing MsTAG-expression plasmid were harvested, resuspended and lysed. Co-IP assays were performed as described under ‘Materials and Methods’. Right panel shows a negative control using an unrelated anti-Ms3759 anti-serum. (C) MMS sensitivity assays for the MsTAG-overexpressing M. smegmatis strains. Growth of the recombinant mycobacterial strains were examined in the presence or absence of 0.012% MMS. Aliquots were taken at the indicated times and the CFU was measured. Each analysis was performed in triplicate. Representative growth curves are shown. The recombinant mycobacterial strains are indicated above the panels. (D) Scanning electron microscopy assay of cell morphology. The recombinant mycobacterial strains were grown in the presence of 0.012% MMS and SEM observation was carried out as described in ‘Materials and Methods’. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038276-g003: Physical interaction of MsTAG (Ms5082) with MsParA and its effect on mycobacterial growth in response to DNA damage induction.(A) Bacterial two-hybrid assays for the interaction of MsTAG with MsParA performed as described in ‘Materials and Methods’. (B) Co-IP assays. Exponentially growing cells of recombinant M. smegmatis containing MsTAG-expression plasmid were harvested, resuspended and lysed. Co-IP assays were performed as described under ‘Materials and Methods’. Right panel shows a negative control using an unrelated anti-Ms3759 anti-serum. (C) MMS sensitivity assays for the MsTAG-overexpressing M. smegmatis strains. Growth of the recombinant mycobacterial strains were examined in the presence or absence of 0.012% MMS. Aliquots were taken at the indicated times and the CFU was measured. Each analysis was performed in triplicate. Representative growth curves are shown. The recombinant mycobacterial strains are indicated above the panels. (D) Scanning electron microscopy assay of cell morphology. The recombinant mycobacterial strains were grown in the presence of 0.012% MMS and SEM observation was carried out as described in ‘Materials and Methods’. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.
Mentions: In a previous global protein-protein interaction analysis [38], the M. tuberculosis MtParA, encoded by Rv3918c, was linked to MtTAG, encoded by Rv1210. We assayed the potential physical interaction between their two corresponding M. smegmatis homologs–MsParA and MsTAG–to further examine the regulation of ParA. As shown in Figure 3A, in our bacterial two-hybrid assays, the co-transformants containing MsParA and MsTAG grew well on the screening medium. Positive co-transformants (CK+) grew on the medium, whereas negative co-transformants (CK−) were incapable of growth on the same screening medium. No growth was observed for their self-activated controls, or for the co-transformants of MsParA and a non-specific gene (Ms1746). Consistent with previous results [33], a clear interaction between MtParA and MtTAG was detected (Fig. 3A). These results indicated that MsParA physically interacts with MsTAG in M. smegmatis. A further in vitro pull-down assay using purified forms of these proteins also confirmed the specific interaction between them (Suppl Fig. S1).

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