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Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane

Nienke Buddelmeijer1, Martin Krehenbrink, Frédéric Pecorari

  • 1Institut Pasteur, Molecular Genetics Unit and CNRS URA2172, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.

Journal of Bacteriology
|November 4, 2008
PubMed

Insights

The study tracked the Klebsiella oxytoca type II secretion system protein PulD using a fluorescent tag. PulD

Area of Science:

  • Microbiology
  • Molecular Biology
  • Cell Biology

Background:

  • The type II secretion system (T2SS) is crucial for protein secretion in Gram-negative bacteria.
  • PulD is an outer membrane secretin essential for T2SS function in Klebsiella oxytoca.
  • The pilotin PulS is a known chaperone for PulD, but its precise role in localization is not fully understood.

Purpose of the Study:

  • To determine the in vivo cellular localization of the PulD secretin in Escherichia coli.
  • To investigate the influence of the chaperone PulS on PulD localization and stability.
  • To assess the functionality of a PulD-fluorescent protein chimera.

Main Methods:

  • Construction and characterization of a PulD-mCherry fluorescent chimera in E. coli.
  • In vivo fluorescence microscopy to observe protein localization.
  • Subcellular fractionation to confirm membrane localization.
  • Functional assays for pullulanase secretion and phage shock response.

Main Results:

  • PulD-mCherry localized to the outer membrane in the presence of high PulS levels and was functional.
  • At lower PulS levels, PulD-mCherry was less stable and primarily located in the inner membrane.
  • The absence of PulS resulted in polar fluorescent foci, indicating altered localization.
  • PulD-mCherry chimera exhibited properties similar to native PulD, including multimerization.

Conclusions:

  • The pilotin PulS plays a critical role in directing the outer membrane localization and stability of the secretin PulD.
  • PulD localization is dependent on PulS levels, affecting its integration into the outer membrane.
  • The PulD-mCherry chimera serves as a reliable tool for studying secretin localization and T2SS biogenesis.

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