CHARACTERIZATION OF A RESTRICTION-ENDONUCLEASE, PHAI, FROM PASTEURELLA-HAEMOLYTICA SEROTYPE A1 AND PROTECTION OF HETEROLOGOUS DNA BY A CLONED PHAI METHYLTRANSFERASE GENE

Pasteurella haemolytica is the leading cause of economic loss to the b eef cattle industry in the United States and an important etiologic ag ent worldwide. Study of P. haemolytica is hindered by researchers' ina bility to genetically manipulate the organism. A new restriction endon uclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymo l. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strai n NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5 -base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage o ccurs 5 bases 3' from the former recognition site and 9 bases 5' from the latter recognition site. A gene encoding a methyltransferase which protects against PhaI cleavage was cloned from P. haemolytica NADC-D6 0 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemo lytica when introduced by electroporation, the same plasmid DNA obtain ed from E. coli which contained a cloned PhaI methyltransferase gene c ould do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.