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Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system
Author(s) -
Livny Jonathan,
Friedman David I.
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2003.03934.x
Subject(s) - biology , selectable marker , encoding (memory) , computational biology , genetics , plasmid , gene , neuroscience
Summary Shiga toxin (Stx) genes in Stx producing Escherichia coli (STEC) are encoded in prophages of the λ 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 s electable in vivo e xpression t echnology, 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 P R 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.