CONSTRUCTION OF CONJUGATIVE SHUTTLE AND SUICIDE VECTORS FOR PASTEURELLA-HAEMOLYTICA AND P. MULTOCIDA

A shuttle cloning vector, pAKA16, and suicide derivatives pAKA19 and p AKA22 have been developed for gene transfer to Pasteurella haemolytica and P. multocida. pAKA16 was constructed by insertion of the lacZ alp ha-peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from P. haemolytica serotype A1. The vec tor encodes ampicillin resistance, and contains at least 14 unique res triction sites and the property of phenotypic identification of recomb inant clones in Escherichia coli by insertional inactivation of beta-g alactosidase activity. It can be transferred by conjugation to P. haem olytica or P. multocida and is stably maintained in both species. The type-II chloramphenicol acetyltransferase-encoding gene (cat), cloned into pAKA16, was stably expressed in both P. haemolytica and P. multoc ida. Plasmids pAKA19 and pAKA22 were constructed by replacement of the origin of DNA replication (ori) of pAKA16 with a Co1E1-type ori from pBR322 or an ori of plasmid R6K (oriR6K) from pJM703.1, respectively. These derivatives replicate in E. coli, but not in either P. haemolyti ca or P. multocida, and are suitable for use as suicide vectors for th ese Pasteurella species.