Characterization of a Novel Type II Restriction-Modification System, Sth 368I, Encoded by the Integrative Element ICE St1 of Streptococcus thermophilus CNRZ368

Abstract

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. ...

Biology of Three ICE Families: SXT/R391, ICE Bs1 , and ICE St1 /ICE St3

“…fda is also the target for the integration of another closely related ICE named ICESt3, initially identified in S. thermophilus CNRZ385 (102). Despite the challenge to genetically manipulate S. thermophilus, ICESt1 and ICESt3 have been studied over the past decade for their conjugative properties, their regulation, and the adaptive traits that they confer to their host as well as for their evolution by accretionmobilization (91,102,103,104,105,106). Diverse ICEs of streptococci are closely related to ICESt1 and ICESt3 and thus belong to the same family based on the strong similarities of their respective regulation and conjugation modules (91).…”

“…6). The variable region of ICESt1 encodes functions conferring resistance to bacteriophage infection, i.e., a type II restrictionmodification system named Sth368I and a putative Abi resistance system (70,106). The variable region of ICESt3 harbors two ORFs, orf385F and orf385G, encoding two putative methyltransferases that may belong to a type II restriction-modification system but no gene coding for an associated restriction enzyme has been found (102).…”

Discovering lactic acid bacteria by genomics

“…Although a moderate thermophile, it is phylogenetically related to the more mesophilic lactococci and has a comparable low G+C ratio (40%). Genes coding for metabolic pathways involved in sugar catabolism (Poolman et al 1989;Vaughan et al 2001), protein and peptide utilization (Fernandez-Espla et al 2000;Garault et al 2002), polysaccharide production (Almirón-Roig et al 2000), the stress response system (Perrin et al 1999), and phage resistance mechanisms (Solow & Somkuti 2000;Burrus 2001) have been sequenced and characterized. Although most strains do not harbor plasmids, other mobile elements have been reported (Guedon et.…”

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques to Lower Phage Contamination in Dairy Fermentation

“…This can be an obstacle in generating novel phage-resistant strains via conjugational events [153]. Yet, studies performed by Burrus et al (2001) revealed the presence of an integrative conjugative element ICRSt1 in S. thermophilus strain CNRZ368, shown to encode a II-type R/M system that provided resistance to phage φST84 infection [154]. Identification of a phage defense system on an integrative element suggests that also such genetic elements as transposons can be responsible for the spread of phage-resistance mechanisms within bacterial populations.…”