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Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

Baronian G, Ginda K, Berry L, Cohen-Gonsaud M, Zakrzewska-Czerwińska J, Jakimowicz D, Molle V - PLoS ONE (2015)

Bottom Line: The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation.Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type.In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, Centre National de la Recherche Scientifique, UMR 5235, Montpellier, France.

ABSTRACT
Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus

Localization of ParB and derivatives.(A) Subcellular localization of ParB-GFP in M. smegmatis mc2155ΔparB strain. Are shown Differential Intereference Contrast image (DIC), GFP fluorescence (GFP) and a merged image of DIC and GFP fluorescence (Merge). Localization of ParB isoforms are shown in three different panels: the upper panel shows wild-type ParB (ParB), the middle panel shows phosphoablative ParB (ParB_Ala) and the lower panel shows phosphomimetic ParB (ParB_Asp). Scalebar 2μm (B) Immunoblotting of ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains. Crude extracts of M. smegmatis mc2155ΔparB complemented with pVV16_egfp_parB, pVV16_egfp_parB_Ala or pVV16_egfp_parB_Asp were electrophoresed on SDS-PAGE gel, ParB-GFP derivatives were then detected by immoblotting using anti-GFP antibody according to the manufacturer’s instructions (Santa Cruz) and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR).
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pone.0119907.g005: Localization of ParB and derivatives.(A) Subcellular localization of ParB-GFP in M. smegmatis mc2155ΔparB strain. Are shown Differential Intereference Contrast image (DIC), GFP fluorescence (GFP) and a merged image of DIC and GFP fluorescence (Merge). Localization of ParB isoforms are shown in three different panels: the upper panel shows wild-type ParB (ParB), the middle panel shows phosphoablative ParB (ParB_Ala) and the lower panel shows phosphomimetic ParB (ParB_Asp). Scalebar 2μm (B) Immunoblotting of ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains. Crude extracts of M. smegmatis mc2155ΔparB complemented with pVV16_egfp_parB, pVV16_egfp_parB_Ala or pVV16_egfp_parB_Asp were electrophoresed on SDS-PAGE gel, ParB-GFP derivatives were then detected by immoblotting using anti-GFP antibody according to the manufacturer’s instructions (Santa Cruz) and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR).

Mentions: Phosphorylation appears to prevent the interaction between ParB and ParA. Since ParB location is tightly dependent on the interaction with ParA we decided to investigate if phosphorylation could affect ParB complexes occurrence, number and/or localization in vivo. Wild-type ParB, phosphoablative and phosphomimetic ParB mutants were fused to EGFP using pVV16_egfp vector under hsp60 promoter and the resulting constructs were introduced into M. smegmatis mc2155ΔparB strain [56]. M. tuberculosis ParB was fully functional in M. smegmatis cells, as demonstrated by the complementation of the parB deletion phenotype (2.52% anucleate cells, S3 Table). The localization of M. tuberculosis ParB resembled the native protein localization with two complexes in proximity of the cell poles (about 20 and 80% of cell length) (Fig. 5A). Such a positioning was observed in most analysed cells, however a fraction of cells with a diffuse fluorescence could also be observed. Both phosphomimetic and phosphoablative ParB mutant did not form clear foci in most of the cells but were mostly dispersed throughout the cell, either as a diffuse fluorescence (ParB_Asp) or irregularly spaced, faint foci (ParB_Ala) (Fig. 5A). This observation is in agreement with the lack of ParB_Ala and ParB_Asp complementation of segregation phenotype of M. smegmatis parB deletion mutant (S3 Table). While mislocalisation was expected in case of phosphomimetic mutant which does not bind DNA, it seems surprising for ParB_Ala. This suggests that although ParB_Ala is able to interact with DNA and ParA, the lack of phosphoregulation severely affects its localization and function. That could result from influence of phosphorylation on the dynamics of ParA-ParB-parS interactions. For example altered phosphorylation state of ParB could affect either stability of the nucleoprotein complex or ParB stimulated ParA ATPase activity changing its dynamics and localisation. Moreover, western blot analysis from ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains revealed proper expression of the GFP-recombinant ParB proteins in each strain (Fig. 5B).


Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.

Baronian G, Ginda K, Berry L, Cohen-Gonsaud M, Zakrzewska-Czerwińska J, Jakimowicz D, Molle V - PLoS ONE (2015)

Localization of ParB and derivatives.(A) Subcellular localization of ParB-GFP in M. smegmatis mc2155ΔparB strain. Are shown Differential Intereference Contrast image (DIC), GFP fluorescence (GFP) and a merged image of DIC and GFP fluorescence (Merge). Localization of ParB isoforms are shown in three different panels: the upper panel shows wild-type ParB (ParB), the middle panel shows phosphoablative ParB (ParB_Ala) and the lower panel shows phosphomimetic ParB (ParB_Asp). Scalebar 2μm (B) Immunoblotting of ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains. Crude extracts of M. smegmatis mc2155ΔparB complemented with pVV16_egfp_parB, pVV16_egfp_parB_Ala or pVV16_egfp_parB_Asp were electrophoresed on SDS-PAGE gel, ParB-GFP derivatives were then detected by immoblotting using anti-GFP antibody according to the manufacturer’s instructions (Santa Cruz) and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4373775&req=5

pone.0119907.g005: Localization of ParB and derivatives.(A) Subcellular localization of ParB-GFP in M. smegmatis mc2155ΔparB strain. Are shown Differential Intereference Contrast image (DIC), GFP fluorescence (GFP) and a merged image of DIC and GFP fluorescence (Merge). Localization of ParB isoforms are shown in three different panels: the upper panel shows wild-type ParB (ParB), the middle panel shows phosphoablative ParB (ParB_Ala) and the lower panel shows phosphomimetic ParB (ParB_Asp). Scalebar 2μm (B) Immunoblotting of ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains. Crude extracts of M. smegmatis mc2155ΔparB complemented with pVV16_egfp_parB, pVV16_egfp_parB_Ala or pVV16_egfp_parB_Asp were electrophoresed on SDS-PAGE gel, ParB-GFP derivatives were then detected by immoblotting using anti-GFP antibody according to the manufacturer’s instructions (Santa Cruz) and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR).
Mentions: Phosphorylation appears to prevent the interaction between ParB and ParA. Since ParB location is tightly dependent on the interaction with ParA we decided to investigate if phosphorylation could affect ParB complexes occurrence, number and/or localization in vivo. Wild-type ParB, phosphoablative and phosphomimetic ParB mutants were fused to EGFP using pVV16_egfp vector under hsp60 promoter and the resulting constructs were introduced into M. smegmatis mc2155ΔparB strain [56]. M. tuberculosis ParB was fully functional in M. smegmatis cells, as demonstrated by the complementation of the parB deletion phenotype (2.52% anucleate cells, S3 Table). The localization of M. tuberculosis ParB resembled the native protein localization with two complexes in proximity of the cell poles (about 20 and 80% of cell length) (Fig. 5A). Such a positioning was observed in most analysed cells, however a fraction of cells with a diffuse fluorescence could also be observed. Both phosphomimetic and phosphoablative ParB mutant did not form clear foci in most of the cells but were mostly dispersed throughout the cell, either as a diffuse fluorescence (ParB_Asp) or irregularly spaced, faint foci (ParB_Ala) (Fig. 5A). This observation is in agreement with the lack of ParB_Ala and ParB_Asp complementation of segregation phenotype of M. smegmatis parB deletion mutant (S3 Table). While mislocalisation was expected in case of phosphomimetic mutant which does not bind DNA, it seems surprising for ParB_Ala. This suggests that although ParB_Ala is able to interact with DNA and ParA, the lack of phosphoregulation severely affects its localization and function. That could result from influence of phosphorylation on the dynamics of ParA-ParB-parS interactions. For example altered phosphorylation state of ParB could affect either stability of the nucleoprotein complex or ParB stimulated ParA ATPase activity changing its dynamics and localisation. Moreover, western blot analysis from ParB-GFP derivatives in M.smegmatis mc2155ΔparB complemented strains revealed proper expression of the GFP-recombinant ParB proteins in each strain (Fig. 5B).

Bottom Line: The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation.Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type.In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, Centre National de la Recherche Scientifique, UMR 5235, Montpellier, France.

ABSTRACT
Here, we present for the first time that Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus