title: Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation.
abstract: An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.
authors.affiliation: Department of Medical Microbiology, Stanford University School of Medicine, CA 94305.
authors.forename: G
authors.initials: G
authors.lastname: Lalonde
authors.forename: J F
authors.initials: JF
authors.lastname: Miller
authors.forename: L S
authors.initials: LS
authors.lastname: Tompkins
authors.forename: P
authors.initials: P
authors.lastname: O'Hanley
mesh_terms: D000188:Actinobacillus / Q000235:genetics*; D000668:Ampicillin Resistance / Q000235:genetics; D000818:Animals; D002702:Chloramphenicol Resistance / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D011815:R Factors; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial