title: Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.
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 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.
authors.affiliation: Department of Microbiology, University of Texas Health Science Center at Science Center at San Antonio 78284, USA.
authors.forename: D
authors.initials: D
authors.lastname: Kolodrubetz
authors.forename: L H
authors.initials: LH
authors.lastname: Phillips
authors.forename: P J
authors.initials: PJ
authors.lastname: Ezzo
authors.forename: E
authors.initials: E
authors.lastname: Kraig
mesh_terms: 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
chemical_list: D004269:DNA, Bacterial; D005098:Exotoxins; C018579:leukotoxin