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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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Construction of the MsParA (Ms6939) knockout strain of M. smegmatis and Southern blot assays.(A) Schematic representation of the recombination strategy for the removal of MsParA from the genome of M. smegmatis. (B) A map of the recombinant vector pMindMsParA containing upstream and downstream sequences of MsParA, and the gene that confers resistance against hygromycin. (C) Schematic representation of the size of a BstE II-digested DNA fragment from the genomic DNA of Msm/WT strain (upper panel) and MsParA knockout strain (lower panel). The probe is indicated with a black bar. (D) Southern blot assays. A 300 bp probe corresponding to the sequences of the MsParA upstream genomic fragment of M. smegmatis was obtained by PCR and labeled with digoxigenin dUTP (Boehringer Mannheim, Inc., Germany). The probe was used to detect the size change of the BstE II-digested genomic fragment of M. smegmatis before and after recombination.
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pone-0038276-g001: Construction of the MsParA (Ms6939) knockout strain of M. smegmatis and Southern blot assays.(A) Schematic representation of the recombination strategy for the removal of MsParA from the genome of M. smegmatis. (B) A map of the recombinant vector pMindMsParA containing upstream and downstream sequences of MsParA, and the gene that confers resistance against hygromycin. (C) Schematic representation of the size of a BstE II-digested DNA fragment from the genomic DNA of Msm/WT strain (upper panel) and MsParA knockout strain (lower panel). The probe is indicated with a black bar. (D) Southern blot assays. A 300 bp probe corresponding to the sequences of the MsParA upstream genomic fragment of M. smegmatis was obtained by PCR and labeled with digoxigenin dUTP (Boehringer Mannheim, Inc., Germany). The probe was used to detect the size change of the BstE II-digested genomic fragment of M. smegmatis before and after recombination.

Mentions: Previous studies have suggested that either increasing or decreasing ParA expression level in M. smegmatis affects bacterial growth [21], [22]. In this study, we first constructed a parA-deleted mutant M. smegmatis strain to further analyze the effects of ParA on mycobacterial growth and cell morphology. As shown in Figure 1A, an MsParA-deleted mutant M. smegmatis strain was generated using gene replacement strategy (Fig. 1). A knockout plasmid pMindMsParA containing the Up and Down regions of the MsParA gene was constructed (Fig. 1B). Deletion of MsParA in the mutant strain was further confirmed by a Southern blot assay as shown in Figure 1D. Signal bands of about 1.0 kb and 470 bp were detected in the BstE II-digested genomic DNA of the mutant and wildtype strains (Fig. 1D), respectively, which is consistent with the deletion of MsParA from the chromosomal DNA of M. smegmatis in the mutant strain (Fig. 1C).


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

Huang F, He ZG - PLoS ONE (2012)

Construction of the MsParA (Ms6939) knockout strain of M. smegmatis and Southern blot assays.(A) Schematic representation of the recombination strategy for the removal of MsParA from the genome of M. smegmatis. (B) A map of the recombinant vector pMindMsParA containing upstream and downstream sequences of MsParA, and the gene that confers resistance against hygromycin. (C) Schematic representation of the size of a BstE II-digested DNA fragment from the genomic DNA of Msm/WT strain (upper panel) and MsParA knockout strain (lower panel). The probe is indicated with a black bar. (D) Southern blot assays. A 300 bp probe corresponding to the sequences of the MsParA upstream genomic fragment of M. smegmatis was obtained by PCR and labeled with digoxigenin dUTP (Boehringer Mannheim, Inc., Germany). The probe was used to detect the size change of the BstE II-digested genomic fragment of M. smegmatis before and after recombination.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038276-g001: Construction of the MsParA (Ms6939) knockout strain of M. smegmatis and Southern blot assays.(A) Schematic representation of the recombination strategy for the removal of MsParA from the genome of M. smegmatis. (B) A map of the recombinant vector pMindMsParA containing upstream and downstream sequences of MsParA, and the gene that confers resistance against hygromycin. (C) Schematic representation of the size of a BstE II-digested DNA fragment from the genomic DNA of Msm/WT strain (upper panel) and MsParA knockout strain (lower panel). The probe is indicated with a black bar. (D) Southern blot assays. A 300 bp probe corresponding to the sequences of the MsParA upstream genomic fragment of M. smegmatis was obtained by PCR and labeled with digoxigenin dUTP (Boehringer Mannheim, Inc., Germany). The probe was used to detect the size change of the BstE II-digested genomic fragment of M. smegmatis before and after recombination.
Mentions: Previous studies have suggested that either increasing or decreasing ParA expression level in M. smegmatis affects bacterial growth [21], [22]. In this study, we first constructed a parA-deleted mutant M. smegmatis strain to further analyze the effects of ParA on mycobacterial growth and cell morphology. As shown in Figure 1A, an MsParA-deleted mutant M. smegmatis strain was generated using gene replacement strategy (Fig. 1). A knockout plasmid pMindMsParA containing the Up and Down regions of the MsParA gene was constructed (Fig. 1B). Deletion of MsParA in the mutant strain was further confirmed by a Southern blot assay as shown in Figure 1D. Signal bands of about 1.0 kb and 470 bp were detected in the BstE II-digested genomic DNA of the mutant and wildtype strains (Fig. 1D), respectively, which is consistent with the deletion of MsParA from the chromosomal DNA of M. smegmatis in the mutant strain (Fig. 1C).

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