title: A system for generalized mutagenesis of Haemophilus ducreyi.
abstract: The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235-9048, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Stevens
authors.forename: L D
authors.initials: LD
authors.lastname: Cope
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
authors.forename: E J
authors.initials: EJ
authors.lastname: Hansen
mesh_terms: D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D018274:Electroporation; D006191:Haemophilus ducreyi / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids
chemical_list: D004251:DNA Transposable Elements; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides