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.
Hidalgo
Alejandro A
AA
Unidad de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340 Santiago, Chile.
Trombert
A Nicole
AN
Castro-Alonso
J C
JC
Santiviago
Carlos A
CA
Tesser
Bruno R
BR
Youderian
Philip
P
Mora
Guido C
GC
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