title: Phosphorylation of Mycobacterium tuberculosis ParB participates in regulating the ParABS chromosome segregation system.
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.
authors: 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.
authors: Grégory
authors: G
authors: Baronian
authors: University of Wroclaw, Faculty of Biotechnology, Department of Molecular Microbiology, Poland.
authors: Katarzyna
authors: K
authors: Ginda
authors: 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.
authors: Laurence
authors: L
authors: Berry
authors: Centre de Biochimie Structurale, Centre National de la Recherche Scientifique, UMR 5048, Universités Montpellier I et II, Montpellier, France.
authors: Martin
authors: M
authors: Cohen-Gonsaud
authors: University of Wroclaw, Faculty of Biotechnology, Department of Molecular Microbiology, Poland.
authors: Jolanta
authors: J
authors: Zakrzewska-Czerwińska
authors: University of Wroclaw, Faculty of Biotechnology, Department of Molecular Microbiology, Poland.
authors: Dagmara
authors: D
authors: Jakimowicz
authors: 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.
authors: Virginie
authors: V
authors: Molle
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D020090:Chromosome Segregation; D002876:Chromosomes, Bacterial / Q000378:metabolism*; D004269:DNA, Bacterial; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010766:Phosphorylation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial