title: Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.
abstract: Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.
authors.affiliation: Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6076.
authors.forename: M A
authors.initials: MA
authors.lastname: Mitchell
authors.forename: K
authors.initials: K
authors.lastname: Skowronek
authors.forename: L
authors.initials: L
authors.lastname: Kauc
authors.forename: S H
authors.initials: SH
authors.lastname: Goodgal
mesh_terms: D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004558:Electric Stimulation; D005819:Genetic Markers; D006193:Haemophilus influenzae / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D003850:Deoxyribonuclease I