Transposon Tn916insertional mutagenesis ofPasteurella multocidaand direct sequencing of disruption site

The transposon Tn 916 , when introduced into Pasteurella multocida by electroporation on a nonreplicating plasmid, integrates into the bacterial chromosome. Efficiencies of approximately 8×10 4 mutants/μg of plasmid DNA were obtained. Restriction digestion and Southern analysis indicate that the Tn 916 element integrates in a quasi-random fashion throughout the genome. Most transformants had a single copy of the transposon but approximately 5% had two copies. Furthermore, the nucleotide sequence at the disruption site of any desired mutant was obtained by capitalizing on the differential sensitivity of the transposon and the genome to the restriction enzyme Hha I; molecular cloning or amplification by polymerase chain reaction was not required. The Tn 916 element has a single Hha I site. On the other hand, this restriction enzyme frequently cleaves the P. multocida chromosome with the vast majority of the resulting genomic fragments being less than 7 kb in length. Tn 916 integration adds a 12 kb segment to the genomic Hha I fragment at the site of disruption. The resulting chimeric DNA fragment was isolated on the basis of size from digests of mutant genomic DNA separated on agarose gels. DNA sequencing with primers corresponding to the terminus of the Tn 916 element was used to determine the sequence at the disruption site. In summary, Tn 916 can be used to disrupt and to clone genes of P. multocida in a rapid and facile fashion.