Characterization of a novel type II restriction-modification system, Sth368I, encoded by the integrative element ICESt1 of Streptococcus thermophilusCNRZ368

A novel type II restriction and modification (R-M) system, Sth368I, which c onfers resistance to phi ST84, was found in Streytococcus thermophilus CNRZ 368 but not in the very closely related strain A054., Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNR Z368 but not by A054, revealed a divergent cluster of two genes, sth368IR a nd sth368IM, The protein sequence encoded by sth368IR is related to the typ e LI endonucleases R.LlaKR2I and R,Sau3AI, which recognize and cleave the s equence 5'-GATC-3', The protein sequence encoded by sth368IM is very simila r to numerous type II 5-methylcytosine methyltransferases, including M.LlaK R2I and M.Sau3AI, Cell extracts of CNRZ368 but not A054 were found to cleav e at the GATC site. Furthermore, the C residue of the sequence 5'-GATC-3' w as found to be methylated in CNRZ368 but not in A054, Cloning and integrati on of a copy of sth368IR and sth3681M in the A054 chromosome confers on thi s strain phenotypes similar to those of CNRZ368, i,e,, phage resistance, en donuclease activity of cell extracts, and methylation of the sequence 5'-GA TC-3', Disruption of sth368IR removes resistance and restriction activity, We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes th e sequence 5'-GATC-3' and is related to the Sau3AI and LlaKR2I restriction systems.