MOLECULAR GENE CLONING AND NUCLEOTIDE SEQUENCING AND CONSTRUCTION OF AN AROA MUTANT OF PASTEURELLA-HAEMOLYTICA SEROTYPE A1

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by com plementation of the aroA mutation in Escherichia coli K-12 strain AB28 29. The nucleotide sequence of a 2.2-kb fragment encoding aroA predict ed an open reading frame product 434 amino acids long that shows homol ogy to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by cons tructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replace ment plasmid with a 4.2-kb P. haemolytica plasmid which encodes strept omycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a pla smidless strain of serotype IA. Allelic exchange between the replaceme nt plasmid and the chromosome of P. haemolytica gave rise to an ampici llin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on th e same medium lacking tryptophan. Southern blot analysis confirmed tha t aroA of the mutant was inactivated and that the mutant was without a plasmid.