Tn10 insertional mutagenesis in Pasteurella multocida

The objective of this study was to identify a transposon that will randomly and stably integrate in the genome of Pasteurella multocida. Using the suicide conjugative delivery system, pLOF, containing a kanamycin resistance marked Tn10 insertion element, we determined that Tn10 is effective in producing insertional mutations in this species. Combined conjugation/transposition events occurred once per 10,000 donor cells. Only 1.4% of the isolates resulted from vector integration and the genomic insertions were random and stable as determined by DNA/DNA hybridization after 10 serial subcultures lacking antibiotic selection. Twenty-two percent of the isolates were auxotrophic, 4% were tryptophan auxotrophs. Mutants derived by this method will be useful for studying putative virulence determinants of Pasteurella.