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

Mol Microbiol. 2004 Mar;51(6):1691-704. doi: 10.1111/j.1365-2958.2003.03934.x.

Abstract

Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the lambda family, such as H-19B. The subpopulation of STEC lysogens with induced prophages has been postulated to contribute significantly to Stx production and release. To study induced STEC, we developed a selectable in vivo expression technology, SIVET, a reporter system adapted from the RIVET system. The SIVET lysogen has a defective H-19B prophage encoding the TnpR resolvase gene downstream of the phage PR promoter and a cat gene with an inserted tet gene flanked by targets for the TnpR resolvase. Expression of resolvase results in excision of tet, restoring a functional cat gene; induced lysogens survive and are chloramphenicol resistant. Using SIVET we show that: (i) approximately 0.005% of the H-19B lysogens are spontaneously induced per generation during growth in LB. (ii) Variations in cellular physiology (e.g. RecA protein) rather than in levels of expressed repressor explain why members of a lysogen population are spontaneously induced. (iii) A greater fraction of lysogens with stx encoding prophages are induced compared to lysogens with non-Stx encoding prophages, suggesting increased sensitivity to inducing signal(s) has been selected in Stx encoding prophages. (iv) Only a small fraction of the lysogens in a culture spontaneously induce and when the lysogen carries two lambdoid prophages with different repressor/operators, 933W and H-19B, usually both prophages in the same cell are induced.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacteriophage lambda / genetics*
  • Bacteriophage lambda / physiology
  • Coliphages / genetics*
  • Coliphages / physiology
  • Coliphages / ultrastructure
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli / virology
  • Gene Expression Regulation, Viral
  • Lysogeny*
  • Shiga Toxins / biosynthesis
  • Shiga Toxins / genetics*
  • Transcription, Genetic
  • Viral Proteins / genetics

Substances

  • Shiga Toxins
  • Viral Proteins