Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation

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 multiply 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 (gamma = 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. Actinobacillus (Haemophilus) pleuropneumoniae is a prominent cause of respiratory infections in swine. Clinical isolates of A pleuropneumoniae have been reported to be resistant to tetracycline, triple sulfonamides, ampicillin, and streptomycin. There has been particular concern over the increasing incidence of resistance to chloramphenicol, which may be related to the extensive use of this antibiotic for treatment of swine pleuropneumonia. In 1980, 95% of the strains of A pleuropneumoniae isolated from the St-Hyacinthe region of Quebec, Canada, were found to be sensitive to chloramphenicol; whereas in 1982, only 57% of surveyed strains were still sensitive to this antibiotic. Resistance to ampicillin, streptomycin, and sulfadiazine in A pl europneumoniae strains has been shown to be plasmid-mediated. The purpose of the study reported here was to use electroporation to analyze plasmids carried by a multiply antibiotic-resistant clinical isolate of A pleuropneumoniae. Electroporation involves the use of brief high-voltage electrical discharges to induce reversible permeability in both prokaryotic and eukaryotic membranes. Using a 5.0-kb A pleuropneumoniae plasmid encoding resistance to chloramphenicol, we have optimized electroporation as a means to transform this species. Conditions permitting an efficiency of over 10(5) transformants (Tfs)/microgram of plasmid DNA are described.