title: Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.
abstract: Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048.
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
authors.forename: J L
authors.initials: JL
authors.lastname: Latimer
authors.forename: S E
authors.initials: SE
authors.lastname: Thomas
authors.forename: M
authors.initials: M
authors.lastname: Helminen
authors.forename: W L
authors.initials: WL
authors.lastname: Albritton
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
mesh_terms: D015139:Blotting, Southern; D015153:Blotting, Western; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006191:Haemophilus ducreyi / Q000235:genetics*; D016296:Mutagenesis; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection
chemical_list: D004269:DNA, Bacterial