A SYSTEM FOR GENERALIZED MUTAGENESIS OF HAEMOPHILUS-DUCREYI

The lack of a generalized mutagenesis system for Haemophilus ducreyi h as hampered efforts to identify virulence factors expressed by this se xually transmitted pathogen. To address this issue, the transposable e lement Tn1545-Delta 3, which encodes resistance to kanamycin, was eval uated for its ability to insert randomly into the H. ducreyi chromosom e and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 mu g of plasmid pMS1 carrying Tn1545-Delta 3 resulted in the pr oduction of 10(4) kanamycin-resistant transformants; Southern blot ana lysis of a number of these transformants indicated that insertion of t he transposon into the chromosome occurred at a number of different si tes. 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 kanamy cin did not affect the stability of the transposon insertion. To confi rm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-Delta 3 was clone d from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resi stant transformants, the majority of which had the same altered LOS ph enotype as the original mutant. Southern blot analysis confirmed the o ccurrence of proper allelic exchange in the LOS-deficient transformant s obtained in this backcross experiment. The ability of Tn1545-Delta 3 to produce insertion mutations in H. ducreyi should facilitate geneti c analysis of this pathogen.