A novel type II restriction and modification (R-M) system, Sth368I, which confers resistance to ST84, was found in Streptococcus thermophilus CNRZ368 but not in the very closely related strain A054. Partial sequencing of the integrative conjugative element ICESt1, carried by S. thermophilus CNRZ368 but not by A054, revealed a divergent cluster of two genes, sth368IR and sth368IM. The protein sequence encoded by sth368IR is related to the type II endonucleases R.LlaKR2I and R.Sau3AI, which recognize and cleave the sequence 5-GATC-3. The protein sequence encoded by sth368IM is very similar to numerous type II 5-methylcytosine methyltransferases, including M.LlaKR2I and M.Sau3AI. Cell extracts of CNRZ368 but not A054 were found to cleave at the GATC site. Furthermore, the C residue of the sequence 5-GATC-3 was found to be methylated in CNRZ368 but not in A054. Cloning and integration of a copy of sth368IR and sth368IM in the A054 chromosome confers on this strain phenotypes similar to those of CNRZ368, i.e., phage resistance, endonuclease activity of cell extracts, and methylation of the sequence 5-GATC-3. Disruption of sth368IR removes resistance and restriction activity. We conclude that ICESt1 encodes an R-M system, Sth368I, which recognizes the sequence 5-GATC-3 and is related to the Sau3AI and LlaKR2I restriction systems.Streptococcus thermophilus is extensively used as a starter in the manufacture of cheese and yogurt with other lactic acid bacteria, like Lactococcus lactis or Lactobacillus delbrueckii subsp. bulgaricus. The proliferation of bacteriophages is one of the main reasons for the failure of these fermentation processes. Since it is difficult to avoid contamination, the strains used as starters should be highly resistant to a large array of phages. In the best known lactic acid bacterium, L. lactis, four types of natural defense mechanisms against bacteriophages have been identified on the basis of their modes of action: blocking of phage adsorption, blocking of phage DNA penetration, abortive infection, and restriction-modification (R-M) systems (11). In this species, the resistance is generally encoded by plasmids, and several different mechanisms can be carried on one plasmid (18). The genes of 10 R-M systems have been cloned from L. lactis strains: eight of the systems are encoded by plasmids, and only two are encoded by the chromosome (11). Some of these plasmids, like pTR2030, are conjugative, allowing easy introduction by conjugative transfer into phage-sensitive strains of commercial importance. The resulting strains have been used successfully by the dairy industry (1, 33).In contrast, very few phage defense mechanism have been described in S. thermophilus. This could be due to the scarcity of plasmids in this species and/or to the more recent progress in its genetics. Most of the strains of S. thermophilus appear to be plasmid free except for a few isolates that contain a single relatively small plasmid (25). None is conjugative. Four type II R-M systems have been well characterized in S. ...
