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.
Antonenko
V
V
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336, München, Germany
Pawlow
V
V
Heesemann
J
J
Rakin
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D008745:Methylation; D008957:Models, Genetic; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics; D015008:Yersinia enterocolitica / Q000201:enzymology* / Q000235:genetics
D015280:DNA Restriction-Modification Enzymes; C059681:DNA modification methylase PstI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific