CHARACTERIZATION OF A PASTEURELLA-MULTOCIDA PLASMID AND ITS USE TO EXPRESS RECOMBINANT PROTEINS IN PASTEURELLA-MULTOCIDA

The complete nucleotide sequence of a naturally occurring 5.36-kb stre ptomycin and sulphonamide resistance plasmid, designated pIG1, isolate d from type D Pasteurella multocida was determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were show n by sequence homologies and functional characterizations to be involv ed in the replication and mobilization of pIG1, respectively. The rema ining sequence carried an unusual arrangement of streptomycin- and sul phonamide-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 sequ ences. A 4.5-kb recombinant plasmid was constructed by replacing the a ntibiotic resistance genes of pIG1 with a kanamycin resistance gene an d seven unique restriction sites. The resulting plasmid, designated pI G112, stably replicates in P. multocida, Pasteurella haemolytica, Acti nobacillus pleuropneumoniae, and Escherichia coli and can be introduce d into these organisms by either transformation or conjugation. This v ector exists at approximately 70 copies per cell in P. multocida and a pproximately 20 copies per cell in E. coli. To demonstrate plasmid-bor ne gene expression in P. multocida, the P. multocida dermonecrotic tox in gene, toxA, and a genetically modified form of this gene were clone d into pIG112 and expressed in high amounts in a nontoxigenic P. multo cida strain. Cell culture assays demonstrated that nontoxigenic P. mul tocida expressing toxA was cytopathic, whereas a strain expressing the modified toxA derivative was not. (C) 1997 Academic Press.