Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4

Claus Sternberg, Leo Eberl, Juan M. Sanchezromero, Michael Christian Givskov, Søren Molin, Victor de Delorenzo

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    Abstract

    The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene, The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene, The high frequencies of marker excision obtained, with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event, Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida, This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.
    Original languageEnglish
    JournalJournal of Bacteriology
    Volume177
    Issue number1
    Pages (from-to)52-58
    ISSN0021-9193
    Publication statusPublished - 1995

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