Cloning and expression of the Pst I restriction-modification system in Escherichia coli.

Abstract

The Pst I restriction-modification system of Providencia stuartii 164 (Proteus inconstans) was cloned and characterized. Transformants of E. coli carrying the Pst I gene system inserted into the cloning vector pBR322 were selected on the basis of acquired resistance to bacteriophage .lambda. infection. Pst I endonuclease was detected in osmotic shock fluid from each of the resistant clones. Plasmid and chromosomal DNA from these clones could not be digested by Pst I, indicating that the gene for the corresponding modification enzyme had also been cloned and was being expressed. The smallest recombinant plasmid encoding both activities, pPst201, contains an insert of .apprx. 4000 base pairs. In vitro transcription studies indicate that this DNA fragment also contains the endogenous promoter(s) of the system. When pPst201 was introduced into a minicell-producing strain of E. coli, 2 new proteins, 32,000 and 35,000 daltons, were synthesized. These were probably assigned the Pst I modification (methylase) and restriction enzymes, respectively. The active form of the restriction enzyme is a dimer, as determined by gel filtration. Constructed transformants of P. stuartii 164 that carry the Pst I system inserted into pBR322 produce .apprx. 10 times more Pst I endonuclease activity than does the native strain.