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Hetero-oligomeric MspA pores in Mycobacterium smegmatis

Mikhail Pavlenok1, Michael Niederweis2

  • 1Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

FEMS Microbiology Letters
|February 26, 2016
PubMed

Insights

Mycobacterium smegmatis MspA porin heterogeneity was reduced by creating an msp quadruple mutant. This improves MspA nanopore homogeneity for DNA sequencing applications.

Area of Science:

  • Biophysics
  • Molecular Biology
  • Nanotechnology

Background:

  • The MspA porin from Mycobacterium smegmatis is a stable biological nanopore utilized in DNA sequencing.
  • MspA's octameric structure can exhibit heterogeneity due to varying Msp subunit compositions.
  • Hetero-oligomeric MspA pores increase variability, potentially impacting DNA sequencing accuracy.

Purpose of the Study:

  • To develop a method for producing more homogeneous MspA pores for DNA sequencing.
  • To investigate the impact of deleting native msp genes on MspA protein production and pore characteristics.
  • To assess the functional properties of MspA pores derived from a genetically modified M. smegmatis strain.

Main Methods:

  • Genetic engineering of Mycobacterium smegmatis to create an msp quadruple mutant (ML712).
  • Insertion of an inducible porin gene from Mycobacterium tuberculosis into the mutant strain.
  • Isolation and purification of MspA porins from both wild-type and mutant M. smegmatis strains.
  • Lipid bilayer experiments to analyze the functional properties and conductance of MspA pores.

Main Results:

  • The msp quadruple mutant (ML712) successfully eliminated endogenous Msp porin expression.
  • Mutant MspA proteins were produced at levels comparable to wild-type.
  • MspA pores isolated from ML712 exhibited a narrower conductance distribution, indicating increased homogeneity.
  • Functional MspA channels were formed from pores purified from the mutant strain.

Conclusions:

  • The M. smegmatis msp quadruple mutant significantly enhances the homogeneity of MspA pores.
  • This improved homogeneity is beneficial for DNA sequencing applications requiring precise nanopore characteristics.
  • The developed mutant strain may also aid in characterizing other mycobacterial pore proteins.
Keywords:
DNA sequencingassemblynanoporeouter membraneporinprotein purification

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