CHARACTERIZATION OF THE PAC251 RESTRICTION-MODIFICATION GENES ISOLATED FROM THE ENDOGENOUS PRA2 PLASMID OF PSEUDOMONAS-ALCALIGENES NCIB-9867

Genes for the class II Pseudomonas alcaligenes NCIB 9867 restriction-m odification (R-M) system, Pac25I, have been cloned from its 33-kb endo genous plasmid, pRA2. The Pac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The dedu ced amino acid sequence of the Pac25I methylase revealed significant s imilarity with the XcyI, XmaI, Cfr9I, and SmaI methylases. High sequen ce similarity was displayed between the Pac25I endonuclease and the Xc yI, XmaI, and Cfr9I endonucleases which cleave between the external cy tosines of the recognition sequence (i.e., 5'-C down arrow CCGGG-3') a nd are thus perfect isoschizomers. However, no sequence similarity was detected between the Pac25I endonuclease and the SmaI endonuclease wh ich cleaves between the internal CpG of the recognition sequence (i.e. , 5'-CCC down arrow GGG-3'). Both the Pac25I methylase and endonucleas e were expressed in Escherichia coli. An open reading frame encoding a protein which shows significant similarity to invertases and resolvas es was located immediately upstream of the Pac25I R-M operon. In addit ion, a transposon designated Tn5563 was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests ho rizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria. (C) 1998 Academic Press.