Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system.
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.
Program in Cell and Molecular Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Jonathan
J
Livny
David I
DI
Friedman
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics* / Q000502:physiology; D003090:Coliphages / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D015967:Gene Expression Regulation, Viral; D008242:Lysogeny; D022603:Shiga Toxins / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
D022603:Shiga Toxins; D014764:Viral Proteins