Insertions of mini-Tn10 transposon T-POP in Salmonella enterica sv. typhi.
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.
Unidad de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340 Santiago, Chile.
Alejandro A
AA
Hidalgo
A Nicole
AN
Trombert
J C
JC
Castro-Alonso
Carlos A
CA
Santiviago
Bruno R
BR
Tesser
Philip
P
Youderian
Guido C
GC
Mora
D017100:Bacteriophage P22; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D045565:Holliday Junction Resolvases / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012485:Salmonella typhi / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013752:Tetracycline
D017931:DNA Primers; D004251:DNA Transposable Elements; D045565:Holliday Junction Resolvases; D013752:Tetracycline