Limits...
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.

Show MeSH

Related in: MedlinePlus

Co-localization assays for MsTAG with MsParA. (A) Schematic representation of construction of co-expression plasmids. MsTAG and MsParA were co-expressed under their respective hsp60 promoters in M. smegmatis (left panel). The GFP fusion expression cassette for expressing GFP-fused MsTAG (left upper panel) and the DsRed2 fusion expression cassette for expressing DsRed2-fused MsParA (left lower panel) were constructed as described in ‘Materials and Methods’. The recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 contained two gene expression cassettes (right panel). (B) MsTAG co-localizes with MsParA. The M. smegmatis double overexpression strain was grown in 7H9 medium to the stage of logarithmic growth. The localization of MsTAG-GFP and MsParA-DsRed2 within single cells (indicated by arrows) was done by fluorescence microscopy. Images of MsTAG-GFP and MsParA-DsRed2 were further subjected to overlay assay. Yellow fluorescence was observed at points where GFP and DsRed2 signals overlapped, indicating co-localization of the two proteins (right panel).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366916&req=5

pone-0038276-g006: Co-localization assays for MsTAG with MsParA. (A) Schematic representation of construction of co-expression plasmids. MsTAG and MsParA were co-expressed under their respective hsp60 promoters in M. smegmatis (left panel). The GFP fusion expression cassette for expressing GFP-fused MsTAG (left upper panel) and the DsRed2 fusion expression cassette for expressing DsRed2-fused MsParA (left lower panel) were constructed as described in ‘Materials and Methods’. The recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 contained two gene expression cassettes (right panel). (B) MsTAG co-localizes with MsParA. The M. smegmatis double overexpression strain was grown in 7H9 medium to the stage of logarithmic growth. The localization of MsTAG-GFP and MsParA-DsRed2 within single cells (indicated by arrows) was done by fluorescence microscopy. Images of MsTAG-GFP and MsParA-DsRed2 were further subjected to overlay assay. Yellow fluorescence was observed at points where GFP and DsRed2 signals overlapped, indicating co-localization of the two proteins (right panel).

Mentions: Since our data indicated physical and functional interactions between MsTAG and MsParA, we predicted that the two proteins would co-localize in vivo in M. smegmatis. To test this hypothesis, we performed co-localization assays using fluorescently labeled proteins. A recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 for expressing GFP-fused MsTAG (Fig. 6A, left upper panel) and DsRed2-fused MsParA under individual hsp60 promoters (Fig. 6A, left lower panel) was designed, constructed and used to make recombinant M. smegmatis strains as described in ‘Materials and Methods’. The fusion proteins were clearly expressed in M. smegmatis at 42°C, and their characteristic green or red fluorescence could be observed by fluorescence microscopy (Fig. 6B).


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

Huang F, He ZG - PLoS ONE (2012)

Co-localization assays for MsTAG with MsParA. (A) Schematic representation of construction of co-expression plasmids. MsTAG and MsParA were co-expressed under their respective hsp60 promoters in M. smegmatis (left panel). The GFP fusion expression cassette for expressing GFP-fused MsTAG (left upper panel) and the DsRed2 fusion expression cassette for expressing DsRed2-fused MsParA (left lower panel) were constructed as described in ‘Materials and Methods’. The recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 contained two gene expression cassettes (right panel). (B) MsTAG co-localizes with MsParA. The M. smegmatis double overexpression strain was grown in 7H9 medium to the stage of logarithmic growth. The localization of MsTAG-GFP and MsParA-DsRed2 within single cells (indicated by arrows) was done by fluorescence microscopy. Images of MsTAG-GFP and MsParA-DsRed2 were further subjected to overlay assay. Yellow fluorescence was observed at points where GFP and DsRed2 signals overlapped, indicating co-localization of the two proteins (right panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038276-g006: Co-localization assays for MsTAG with MsParA. (A) Schematic representation of construction of co-expression plasmids. MsTAG and MsParA were co-expressed under their respective hsp60 promoters in M. smegmatis (left panel). The GFP fusion expression cassette for expressing GFP-fused MsTAG (left upper panel) and the DsRed2 fusion expression cassette for expressing DsRed2-fused MsParA (left lower panel) were constructed as described in ‘Materials and Methods’. The recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 contained two gene expression cassettes (right panel). (B) MsTAG co-localizes with MsParA. The M. smegmatis double overexpression strain was grown in 7H9 medium to the stage of logarithmic growth. The localization of MsTAG-GFP and MsParA-DsRed2 within single cells (indicated by arrows) was done by fluorescence microscopy. Images of MsTAG-GFP and MsParA-DsRed2 were further subjected to overlay assay. Yellow fluorescence was observed at points where GFP and DsRed2 signals overlapped, indicating co-localization of the two proteins (right panel).
Mentions: Since our data indicated physical and functional interactions between MsTAG and MsParA, we predicted that the two proteins would co-localize in vivo in M. smegmatis. To test this hypothesis, we performed co-localization assays using fluorescently labeled proteins. A recombinant plasmid pMV261-MsTAG-GFP/MsParA-DsRed2 for expressing GFP-fused MsTAG (Fig. 6A, left upper panel) and DsRed2-fused MsParA under individual hsp60 promoters (Fig. 6A, left lower panel) was designed, constructed and used to make recombinant M. smegmatis strains as described in ‘Materials and Methods’. The fusion proteins were clearly expressed in M. smegmatis at 42°C, and their characteristic green or red fluorescence could be observed by fluorescence microscopy (Fig. 6B).

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