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Antibacterial toxin colicin N and phage protein G3p compete with TolB for a binding site on TolA

Helen Ridley1, Jeremy H Lakey2

  • 1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

Microbiology (Reading, England)
|December 25, 2014
PubMed

Insights

Colicin N (ColN) uses a unique binding site on TolA, distinct from other colicins, revealing evolutionary diversification in bacterial toxin mechanisms. This interaction is crucial for outer membrane stability and colicin activity.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Structural Biology

Background:

  • Colicins are bacteriocins produced by Escherichia coli that kill related strains.
  • Most colicins utilize pore formation or nuclease activity, involving complex interactions with the bacterial cell envelope.
  • Group A colicins typically engage the TolQRAB complex for translocation into the cell.

Purpose of the Study:

  • To investigate the detailed molecular interactions of Colicin N (ColN) with the TolA protein.
  • To elucidate the unique translocation pathway of ColN compared to other colicins.
  • To understand the role of the TolA-TolB interaction in outer membrane stability and colicin activity.

Main Methods:

  • Biochemical assays to determine binding affinities (KD) between ColN, TolA, and TolB.
  • Site-directed mutagenesis of TolA to introduce proline point mutations.
  • Phenotypic analysis of mutant strains to assess outer membrane stability, colicin activity, and phage protein binding.

Main Results:

  • ColN binds TolA with high affinity (1 µM) via β-strand addition, utilizing the TolB binding site.
  • Single proline mutations in TolA abolish both ColN-TolA and TolA-TolB binding, conferring a complete tol phenotype.
  • The identified TolA mutations destabilize the outer membrane, inhibit ColA and ColE9 activity, and block phage protein binding.

Conclusions:

  • The TolA-TolB β-strand addition is essential for Tol pathway function and outer membrane integrity.
  • ColN employs a distinct translocation route, forming a binary complex with TolA, which may explain its unique toxicity.
  • Evolutionary diversification in colicin mechanisms is evident in their distinct molecular interactions and translocation strategies.

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