Directed genomic integration in Actinobacillus actinomycetemcomitans: Generation of defined leukotoxin-negative mutants

D. Kolodrubetz, L. H. Phillips, P. J. Ezzo, E. Kraig

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin- producing strain JP2 with a CnlE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had <0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

Original languageEnglish (US)
Pages (from-to)2780-2784
Number of pages5
JournalInfection and Immunity
Volume63
Issue number7
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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