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  • Title: Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system.
    Author: Livny J, Friedman DI.
    Journal: Mol Microbiol; 2004 Mar; 51(6):1691-704. PubMed ID: 15009895.
    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.
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