Characterization of aPasteurella multocidaPlasmid and Its Use to Express Recombinant Proteins inP. multocida

Abstract

The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type DPasteurella multocidawas determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively. The remaining sequence carried an unusual arrangement of streptomycin- and sulphonamide-resistant genes when compared to various other plasmids. It appears that the antibiotic resistance region of pIG1 may have evolved by recombination between three different short direct repeat DNA sequences. A 4.5-kb recombinant plasmid was constructed by replacing the antibiotic resistance genes of pIG1 with a kanamycin resistance gene and seven unique restriction sites. The resulting plasmid, designated pIG112, stably replicates inP. multocida, Pasteurella haemolytica, Actinobacillus pleuropneumoniae,andEscherichia coliand can be introduced into these organisms by either transformation or conjugation. This vector exists at approximately 70 copies per cell inP. multocidaand approximately 20 copies per cell inE. coli.To demonstrate plasmid-borne gene expression inP. multocida,theP. multocidadermonecrotic toxin gene,toxA,and a genetically modified form of this gene were cloned into pIG112 and expressed in high amounts in a nontoxigenicP. multocidastrain. Cell culture assays demonstrated that nontoxigenicP. multocidaexpressingtoxAwas cytopathic, whereas a strain expressing the modifiedtoxAderivative was not.