Characterization of a novel unique restriction-modification system from Yersinia enterocolitica O:8 1B

Genetic manipulations with enteropathogenic Yersinia enterocolitica O:8 are complicated by the presence of an efficient PstI-like YenI restriction-modification (R-M) system. We have characterized the YenI R-M system in Y. enterocolitica O:8, biotype 1B. A 5039 bp DNA fragment of the pSAK2 recombinant plasmid carrying the yenI locus was used to determine the nucleotide sequence. DNA sequence analysis identified a single 2481 bp open reading frame (ORF) that encodes an 826 amino acid large polypeptide having an apparent molecular mass of 93 kDa. The N-terminal part of the YenI ORF has 45 and 40% identity to PstI and BsuI methyltransferases (MTases), respectively; while the C-terminal part depicts 55 and 45% identity to endonucleases (ENases) of both isoschyzomeric enzymes. The yenI gene was cloned into pT7-5 plasmid and has been shown to encode a single polypeptide of expected molecular mass. A specific recognition sequence, typical to the type II R-M systems and single peptide organization, typical to type IV R-M systems, make YenI unique among known restriction-modification systems. We have constructed a truncated recombinant variant of YenI enzyme, which conserved only MTase activity, and that can be applied to YenI methylation of the DNA to be transformed into Y. enterocolitica O:8 biotype 1B strains.