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

Grégory Baronian1, Katarzyna Ginda2, Laurence Berry1

  • 1Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, Centre National de la Recherche Scientifique, UMR 5235, Montpellier, France.

Plos One
|March 26, 2015
PubMed

Insights

Mycobacterium tuberculosis ParB protein phosphorylation by kinases regulates its DNA binding and interaction with ParA. This phosphorylation acts as a molecular switch, controlling the localization and function of the chromosome partitioning complex.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • ParB and ParA are essential for bacterial chromosome segregation in Mycobacterium tuberculosis.
  • ParB binds to parS DNA sequences and interacts with ParA, which is crucial for proper localization.
  • Understanding ParB regulation is key to deciphering chromosome segregation mechanisms.

Purpose of the Study:

  • To investigate the phosphorylation of Mycobacterium tuberculosis ParB.
  • To determine the role of ParB phosphorylation in its DNA-binding activity, ParA interaction, and cellular localization.
  • To identify the specific phosphorylation sites on ParB.

Main Methods:

  • In vitro phosphorylation assays using mycobacterial Ser/Thr protein kinases.
  • Mass spectrometry to identify phosphorylation sites on ParB.
  • Site-directed mutagenesis to create alanine (non-phosphorylatable) and aspartate (phosphomimetic) mutants.
  • Electrophoretic mobility shift assays (EMSAs) to assess DNA-binding.
  • Bacterial two-hybrid assays to study ParA-ParB interactions.
  • Fluorescence microscopy in Mycobacterium smegmatis to observe ParB localization.

Main Results:

  • Mycobacterium tuberculosis ParB is phosphorylated by several Ser/Thr protein kinases in vitro.
  • Mass spectrometry identified phosphorylation on eight threonine and two serine residues.
  • Phosphomimetic mutations in ParB significantly inhibited DNA binding and abolished ParA interaction.
  • Phosphomimetic ParB exhibited delocalized distribution in Mycobacterium smegmatis, unlike wild-type ParB.

Conclusions:

  • Phosphorylation of Mycobacterium tuberculosis ParB by Ser/Thr kinases is a critical regulatory mechanism.
  • Phosphorylation acts as a molecular switch, modulating ParB's DNA-binding, ParA interaction, and subcellular localization.
  • These findings reveal a novel regulatory pathway impacting chromosome partitioning in Mycobacterium tuberculosis.

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