Abstract
The fate of lysogens following prophage induction has assumed added significance with the finding that in many pathogens virulence genes are carried on prophages and, in some, the production and/or release of the virulence factor is under control of the phage lytic regulatory program. We outline a method for identifying and characterizing from a total lysogen population, the subpopulation in which the prophage is induced. The prophage is genetically altered so that on induction it does not go through the lytic pathway, but does express a resolvase that acts at a reporter cassette located at another site on the bacterial chromosome to irreversibly change the resistance of the bacterium from tetracycline to chloramphenicol. Thus, induced derivatives survive and are easily identified even if they make up a small fraction of the population.
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References
Waldor, M. K., Friedman, D. I. & Adhya, S. L. (2005) Phages; Their role in bacterial pathogenesis and biotechnology (ASM Press, Washington, D.C.).
Tyler, J. S., Livny, J. & Friedman, D. I. (2005) in Phage: Role in Pathogenesis and Biotechnology, eds. K. W. M., Friedman, D. I. & Adhya, S. (ASM Press, Washington, D.C.).
Camilli, A. & Mekalanos, J. J. (1995) Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 18, 671–683.
Livny, J. & Friedman, D. I. (2004) Characterizing spontaneous induction of Stx encoding phages using a selectable reporter system. Mol. Microbiol. 51, 1691–1704.
Eisen, H. A., Fuerst, C. R., Siminovitch, L., Thomas, R., Lambert, L., Pereira da Silva, L. & Jacob, F. (1966) Genetics and physiology of defective lysogeny in K12 (lambda): studies of early mutants. Virology 30, 224–241.
Quandt, J. & Hynes, M. F. (1993) Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127, 15–21.
Powell, B. S., Rivas, M. P., Court, D. L., Nakamura, Y. & Turnbough, C. L., Jr. (1994) Rapid confirmation of single copy lambda prophage integration by PCR. Nucleic Acids Res 22, 5765–5766.
Grindley, N. D. (1983) Transposition of Tn3 and related transposons. Cell 32, 3–5.
Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. & Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U S A 97, 5978–5983.
Ellis, H. M., Yu, D., DiTizio, T. & Court, D. L. (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. U S A 98, 6742–6746.
Lee, S. H., Hava, D. L., Waldor, M. K. & Camilli, A. (1999) Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99, 625–634.
Weisberg, R. A. & Landy, A. (1983) in Lambda II, eds. Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 211–250.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Livny, J., LaRock, C.N., Friedman, D.I. (2009). Identification and Isolation of Lysogens with Induced Prophage. In: Clokie, M.R., Kropinski, A.M. (eds) Bacteriophages. Methods in Molecular Biology™, vol 501. Humana Press. https://doi.org/10.1007/978-1-60327-164-6_22
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DOI: https://doi.org/10.1007/978-1-60327-164-6_22
Publisher Name: Humana Press
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