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Related Experiment Videos

Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system.

Jonathan Livny1, David I Friedman

  • 1Program in Cell and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.

Molecular Microbiology
|March 11, 2004
PubMed
Summary

We developed a reporter system to study Shiga toxin (Stx) producing E. coli (STEC) prophage induction. This system reveals that STEC lysogens with Stx prophages are more sensitive to induction, aiding toxin release studies.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Bacteriology

Background:

  • Shiga toxin (Stx) genes in STEC are carried by lambdoid prophages.
  • Prophage induction in STEC lysogens is thought to be crucial for Stx production and release.

Purpose of the Study:

  • To develop a novel reporter system, Selectable In Vivo Expression Technology (SIVET), to study STEC prophage induction.
  • To investigate the frequency and mechanisms of spontaneous prophage induction in STEC.

Main Methods:

  • Developed SIVET, a reporter system based on a defective H-19B prophage with a TnpR resolvase and a cat-tet gene cassette.
  • Utilized SIVET to quantify spontaneous prophage induction rates during bacterial growth.
  • Analyzed factors influencing induction, including cellular physiology and prophage type.

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Main Results:

  • Approximately 0.005% of H-19B lysogens spontaneously induced per generation in LB media.
  • Cellular physiology, like RecA protein levels, influences spontaneous induction more than repressor levels.
  • Lysogens with Stx-encoding prophages showed higher induction rates than those with non-Stx prophages.
  • Co-infection with two different prophages (933W and H-19B) typically resulted in the induction of both.

Conclusions:

  • SIVET is an effective tool for studying STEC prophage induction dynamics.
  • Stx-encoding prophages may possess enhanced sensitivity to induction signals.
  • Spontaneous prophage induction is a complex process influenced by both bacterial physiology and prophage genetics.