Mini-Mu technology in Salmonella typhi: isolation of stable MudJ operon fusions by cis complementation.
This paper describes a method to achieve stable MudJ insertions in the Salmonella typhi Ty2 chromosome. The method is a modification of the genetic complementation system described previously for Salmonella typhimurium, which consists in placing the defective transposon (MudJ) near the transposase genes of a helper Mu phage on a single DNA fragment. This fragment is then introduced into a new bacterial host by means of P22 transduction. We constructed a S. typhi strain which carries MudJ and the Mu helper phage in the chromosome. This strain was induced to lytic growth and the lysate was used to infect S. typhi Ty2. The frequency of mutation was 2.0 x 10(-6) mutants per recipient bacterium. Superinfection with the Mu helper phage was about 1%. To determine the number of MudJ insertions, several mutants were subjected to Southern blot analysis. From a total of 25 mutants analyzed, only 4 contained more than one insertion. Our procedure compares well with the method described previously for the S. typhimurium-P22 system and can be applied to other Mu sensitive bacteria.
Contreras
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Departamento de Bioquímica y Biología Molecular, Universidad de Chile, Santiago.
Obreque
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D010583:Bacteriophage mu / Q000302:isolation & purification; D009876:Operon; D012485:Salmonella typhi / Q000235:genetics*; D014161:Transduction, Genetic