Cloned restriction/modification system from Pseudomonas aeruginosa

Abstract

DNA fragments from Pseudomonas aeruginosa carrying the PaeR7 restriction/modification genes have been cloned in the plasmid vector pBR322 and propagated in Escherichia coli. A subclone (pPAORM3.8) has been constructed that contains the complete restriction/modification system on a 3.8-kilobase DNA fragment. Digestion of the pPAORM3.8 plasmid with nuclease BAL-31 has yielded two types of clones. One type contains an active methylase gene but no active endonuclease gene; such clones will modify the DNA but not restrict the growth of incoming phage in vivo. The second type contains an active endonuclease gene but no active methylase gene, as judged both by in vivo tests and by the activity of the cell extracts in vitro. Although extracts of cells containing these plasmids display restriction endonuclease activity, these bacteria are unable to restrict the growth of incoming phage. Furthermore, chromosomal and phage DNA isolated from these host cells are not protected against cleavage by PaeR7 in vitro. The properties of PaeR7 endonuclease and methylase enzymes have also been examined. The PaeR7 restriction endonuclease recognizes and cleaves the sequence C decreased T-C-G-A-G, as does Xho I. However, there exists a canonical Xho I site at 26.5% on the adenovirus 2 genome which is totally refractory to PaeR7 cleavage but is cut by Xho I. Under conditions of low salt, high glycerol, and high enzyme concentrations, a "PaeR7" activity is found that is similar to that observed for EcoRI. Finally, evidence is presented that the PaeR7 methylase modifies the adenine residue within the recognition sequence.