Stepwise cloning and molecular characterization of the HgiDI restriction-modification system from Herpetosiphon...

The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E. coli in a two-step procedure. Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC). Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material. The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons. In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far. Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.