Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site

The transposon Tn916, when introduced into Pasteurella multocida by electroporation on a nonreplicating plasmid, integrates into the bacterial chromosome. Efficiencies of approximately 8x10(4) mutants/microg of plasmid DNA were obtained. Restriction digestion and Southern analysis indicate that the Tn916 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 HhaI; molecular cloning or amplification by polymerase chain reaction was not required. The Tn916 element has a single HhaI 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. Tn916 integration adds a 12 kb segment to the genomic HhaI 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 Tn916 element was used to determine the sequence at the disruption site. In summary, Tn916 can be used to disrupt and to clone genes of P. multocida in a rapid and facile fashion.