A system for generalized mutagenesis of Haemophilus ducreyi

M. K. Stevens, L. D. Cope, J. D. Radolf, E. J. Hansen

Research output: Contribution to journalArticle

11 Citations (Scopus)

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- Δ3, 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 μg of plasmid pMS1 carrying Tn1545-Δ3 resulted in the production of 104 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-Δ3 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-Δ3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.

Original languageEnglish (US)
Pages (from-to)2976-2982
Number of pages7
JournalInfection and Immunity
Volume63
Issue number8
StatePublished - 1995

Fingerprint

Haemophilus ducreyi
Mutagenesis
Kanamycin
Electroporation
Southern Blotting
Chromosomes
Kanamycin Resistance
Phenotype
DNA Transposable Elements
Insertional Mutagenesis
Virulence Factors
Plasmids
lipid-linked oligosaccharides
DNA

ASJC Scopus subject areas

  • Immunology

Cite this

Stevens, M. K., Cope, L. D., Radolf, J. D., & Hansen, E. J. (1995). A system for generalized mutagenesis of Haemophilus ducreyi. Infection and Immunity, 63(8), 2976-2982.

A system for generalized mutagenesis of Haemophilus ducreyi. / Stevens, M. K.; Cope, L. D.; Radolf, J. D.; Hansen, E. J.

In: Infection and Immunity, Vol. 63, No. 8, 1995, p. 2976-2982.

Research output: Contribution to journalArticle

Stevens, MK, Cope, LD, Radolf, JD & Hansen, EJ 1995, 'A system for generalized mutagenesis of Haemophilus ducreyi', Infection and Immunity, vol. 63, no. 8, pp. 2976-2982.
Stevens MK, Cope LD, Radolf JD, Hansen EJ. A system for generalized mutagenesis of Haemophilus ducreyi. Infection and Immunity. 1995;63(8):2976-2982.
Stevens, M. K. ; Cope, L. D. ; Radolf, J. D. ; Hansen, E. J. / A system for generalized mutagenesis of Haemophilus ducreyi. In: Infection and Immunity. 1995 ; Vol. 63, No. 8. pp. 2976-2982.
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