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 methyltrans-ferases, 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 con-jugative, 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. thermophilus: Sth134I (35) is an isoschizomer of HpaII and MspI, and Sth117I (36), Sth455I (15), and SslI (3) are isoschizomers of BstNI and EcoRII. However, their genes have been neither cloned nor sequenced. A site-specific integrative element, ICESt1,was found to be integrated in the 3 end of fda of S. thermophilus CNRZ368, an open reading frame (ORF) encoding a putative fructose-1,6-bisphosphate aldolase (6). It excises by site-specific recombi-nation. Partial sequencing of the right end of this element reveals ORFs encoding proteins related to those of some con-jugative plasmids and conjugative transposons. Therefore, ICESt1 could be an integrative conjugative element. The results presented in this study show that ICESt1 carries the genes encoding a type II R-M system, Sth368I, which recognizes the sequence 5-GATC-3. These genes were cloned and sequenced. They are related to those encoding LlaKR2I of L. lactis (38) and Sau3AI of Staphylococcus aureus (34), two type II R-M systems which also recognize GATC sequences. MATERIALS AND METHODS Bacterial strains and media. The Escherichia coli, S. thermophilus, and L. lactis strains used in this study are listed in Table 1. E. coli strains were grown at 37°C on Luria-Bertani medium supplemented with 170 g of chloramphenicol/ml [strains containing pBC KS() (Stratagene, La Jolla, Calif.)]-derived plasmids], 50 g of ampicillin/ml [strains containing pBluescript SK() (Stratagene)-derived plasmids], or 150 g of erythromycin/ml (strains containing pGHost9-derived plasmids). The S. thermophilus strains were grown at 42°C in M17 broth containing 5 g of lactose/liter (M17L) supplemented when appropriate with 2 g of erythromycin/ml (strains containing integrated pGHost9-derived plasmids) or at 30°C in M17L broth containing 5 g of erythromycin/ml (strains containing free pGHost9-derived plasmids). L. lactis MG1363 was grown at 30°C in M17 broth containing 0.2 M glucose (M17G) supplemented with 5 g of erythromy-cin/ml (strains containing pGHost9-derived plasmids).