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Genetics, Vol. 167, 1069-1077, July 2004, Copyright © 2004
doi:10.1534/genetics.104.026682
Insertions of Mini-Tn10 Transposon T-POP in Salmonella enterica sv. typhi
Alejandro A. Hidalgo*,
A. Nicole Trombert*,
J. C. Castro-Alonso*,
Carlos A. Santiviago*,
Bruno R. Tesser*,
Philip Youderian
and
Guido C. Mora*,1
* Unidad de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340 Santiago, Chile
Department of Biology, Texas A&M University, College Station, Texas 83843-3052
1 Corresponding author: Unidad de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.
E-mail: gmora{at}genes.bio.puc.cl
We have mutagenized a clinical strain of Salmonella enterica sv. typhi with mini-transposon Tn10dTet (T-POP) to obtain conditional lethal (tetracycline-dependent) mutants with T-POP insertions upstream of essential genes. Generalized transducing phage P22 was used to introduce T-POP from a S. typhimurium donor into a S. typhi recipient. Chromosomal DNA was purified from the mutagenized donor strains, fragmented, and then electroporated into S. typhi to backcross the original T-POP insertions. Four tetracycline-dependent mutants with two distinct terminal phenotypes were found among 1700 mutants with T-POP insertions. When grown in the absence of tetracycline, two of the four tetracycline-dependent mutants arrest at a late stage in the cell cycle, can be rescued by outgrowth in media with tetracycline, and define a reversible checkpoint late in the cell cycle. One of these insertions creates an operon fusion with a gene, yqgF, that is conserved among gram-negative bacteria and likely encodes an essential Holliday junction resolvase. T-POP insertions can be used not only to identify essential S. typhi genes but also to reveal novel phenotypes resulting from the depletion of their products.
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