DIRECTED GENOMIC INTEGRATION IN ACTINOBACILLUS-ACTINOMYCETEMCOMITANS - GENERATION OF DEFINED LEUKOTOXIN-NEGATIVE MUTANTS

To develop targeted gene integration in the periodontal pathogen Actin obacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistan t plasmid containing a segment of the leukotoxin gene was electroporat ed into strain JP2. Ln all of the stable spectinomycin-resistant trans formants that arose, the plasmid had recombined into the genomic leuko toxin locus since ColE1-based vectors cannot replicate extrachromosoma lly in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the le ukotoxin-producing strain JP2 with a ColE1-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 p arental strain. These results demonstrate that integration-based appro aches can be used for generating isogenic mutants in specific virulenc e genes in A. actinomycetemcomitans.