Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.
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 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 parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.
Kolodrubetz
D
D
Department of Microbiology, University of Texas Health Science Center at Science Center at San Antonio 78284, USA.
Phillips
L H
LH
Ezzo
P J
PJ
Kraig
E
E
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005098:Exotoxins / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D005098:Exotoxins; C018579:leukotoxin