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In vitro characterization of the phage lysis protein MS2-L

Julija Mezhyrova1, Janosch Martin2, Clara Börnsen3

  • 1Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt am Main 60438, Germany.

Microbiome Research Reports
|December 4, 2023
PubMed

Insights

Bacterial lysis peptide MS2-L forms large oligomeric complexes within cell membranes, leading to cell envelope lesions. The chaperone DnaJ interacts with MS2-L but does not affect its oligomerization or lysis mechanism.

Area of Science:

  • Microbiology
  • Structural Biology
  • Biochemistry

Background:

  • MS2-L is a phage toxin from Leviviridae family, mediating bacterial cell lysis via cell envelope lesions.
  • The precise mechanism of MS2-L-induced lysis and its interaction with cellular factors like chaperone DnaJ remain unclear.
  • Understanding MS2-L is crucial for bioengineering applications.

Purpose of the Study:

  • To elucidate the molecular mechanism of MS2-L action and its interaction with DnaJ.
  • To investigate the oligomerization process of MS2-L upon membrane insertion.
  • To provide insights into MS2-L-mediated bacterial cell lysis.

Main Methods:

  • Combined in vitro and in vivo overexpression studies.
  • Cell-free synthesis of MS2-L and derivatives, insertion into nanodiscs or detergent micelles.
  • Native liquid bead ion desorption mass spectrometry and cryo-electron microscopy.
  • Fluorescent protein fusions for in vivo monitoring in E. coli.

Main Results:

  • MS2-L self-assembles into high oligomeric states after membrane insertion, primarily directed by its transmembrane domain.
  • Oligomerization is hindered in detergent environments but not by DnaJ interaction.
  • Cryo-EM reveals lysis initiation in the outer membrane, followed by peptidoglycan disruption and inner membrane disintegration.
  • MS2-L forms complexes similar to ΦX174-E, with oligomeric interfaces in transmembrane domains.

Conclusions:

  • MS2-L oligomerization is essential for its membrane-disrupting and cell-lysing activity.
  • The transmembrane domain is critical for MS2-L oligomerization and complex formation.
  • Higher-order assembly of phage toxins may be a conserved mechanism for membrane disintegration and bacterial lysis.
Keywords:
Phage toxinscell-free expressionmolecular assembliesnanodiscsnative mass spectrometry

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