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Related Experiment Videos

Replacement recombination in Lactococcus lactis.

K J Leenhouts1, J Kok, G Venema

  • 1Institute of Genetics, University of Groningen, The Netherlands.

Journal of Bacteriology
|August 1, 1991
PubMed
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This study demonstrates that integrating a plasmid into Lactococcus lactis can inactivate the X-prolyl dipeptidyl aminopeptidase (pepXP) gene through replacement recombination. Low-frequency recombination events also led to similar gene inactivation outcomes.

Area of Science:

  • * Molecular Biology
  • * Microbial Genetics

Background:

  • * The X-prolyl dipeptidyl aminopeptidase gene (pepXP) is present in a 5.3-kb fragment on the pUC18-derived integration plasmid pML336.
  • * This gene was targeted for inactivation using an erythromycin resistance marker insertion within its coding sequence.

Purpose of the Study:

  • * To investigate the integration of plasmid pML336 into the Lactococcus lactis chromosome.
  • * To analyze the mechanisms of gene inactivation and plasmid integration in L. lactis.
  • * To determine the frequency of recombination events leading to pepXP gene inactivation.

Main Methods:

  • * Electrotransformation of Lactococcus lactis with covalently closed circular DNA of plasmid pML336.
  • * Selection of erythromycin-resistant transformants to identify successful integration and/or inactivation events.

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  • * Analysis of recombination events, including double-crossover (replacement) and Campbell-type integration.

    • Main Results:

    • * In 2% of transformants, the pepXP gene was inactivated via replacement recombination between the plasmid and the L. lactis chromosome.
    • * Other transformants contained a Campbell-type integrated plasmid copy without pepXP gene inactivation.
    • * Loss of plasmid DNA from integrated copies occurred at low frequencies (2.8-8.5 x 10^-6) via recombination between 1.6-kb repeats, mimicking replacement recombination outcomes.

    Conclusions:

    • * Plasmid integration into L. lactis can lead to targeted gene inactivation through specific recombination mechanisms.
    • * Both replacement and Campbell-type integration pathways are significant in L. lactis genetic manipulation.
    • * Spontaneous recombination events can result in chromosomal structures similar to those achieved through targeted gene replacement.