Characterization of a restriction endonuclease, PhaI, from Pasteurella haemolytica serotype A1 and protection of heterologous DNA by a cloned PhaI methyltransferase gene

Abstract

Pasteurella haemolytica is the leading cause of economic loss to the beef cattle industry in the United States and an important etiologic agent worldwide. Study of P. haemolytica is hindered by researchers' inability to genetically manipulate the organism. A new restriction endonuclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymol. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5-base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage occurs 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-D60 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemolytica when introduced by electroporation, the same plasmid DNA obtained from E. coli which contained a cloned PhaI methyltransferase gene could do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.