JOURNAL OF BACTERIOLOGY, Feb. 2008, p. 1390–1400 Vol. 190, No. 4 0021-9193/08/$08.000 doi:10.1128/JB.01412-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved. Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus He´le`ne Deveau,1 Rodolphe Barrangou,2 Josiane E. Garneau,1 Jessica Labonte´,1 Christophe Fremaux,3 Patrick Boyaval,3 Dennis A. Romero,2 Philippe Horvath,3 and Sylvain Moineau1* De´partement de Biochimie et de Microbiologie, Faculte´ des Sciences et de Ge´nie, Groupe de Recherche en E´cologie Buccale, Faculte´ de Me´decine Dentaire, Fe´lix d’He´relle Reference Center for Bacterial Viruses, Universite´ Laval, Quebec City, Quebec G1V 0A6, Canada1; Danisco, USA, Inc., 3329 Agriculture Drive, Madison, Wisconsin 537162; and Danisco France SAS, BP10, F-86220 Dange´-Saint-Romain, France3 Received 31 August 2007/Accepted 21 November 2007 Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1. Streptococcus thermophilus is one of the most industrially important lactic acid bacteria since it is widely used for the manufacture of yogurt and a number of cheeses. Several strains of this low GC gram-positive species are used in large-scale milk fermentations because each strain possesses its own distinctive properties that are suitable for the manufacture of an array of fermented products, each with unique organo- leptic properties. The systematic use of the same S. ther- mophilus strains in dairy processes has been impaired by the ubiquitous presence of virulent phages. Consequently, S. thermophilus bacteriophages have been the subject of extensive research in recent years with the aim of preventing their mul- tiplication (8, 12). S. thermophilus phages, like their hosts, are rather homoge- nous since they all belong to one polythetic species containing both temperate and virulent phages (9, 13). S. thermophilus phages are morphologically similar to coliphage lambda and accordingly belong to the Siphoviridae family. S. thermophilus phages are currently classified into two groups based on their general DNA packaging scheme (cos or pac) and the compo- sition of their structural proteome (27). Seven complete ge- nome sequences of S. thermophilus phages are publicly avail- able, including those of the cos-type phages DT1, Sfi19, Sfi21, and 7201 and the pac-type phages O1205, Sfi11, and 2972 (28). Information on phage-host interactions has increased appre- ciably in recent years. It is well known that bacteria have a plethora of mechanisms to fight a diverse phage population (10). Traditionally in lactic acid bacteria, natural phage de- fense systems are divided in four main groups, namely, the inhibition of phage adsorption, the inhibition of DNA ejection, restriction-modification systems, and abortive infection (Abi) systems (10, 24). Globally, these mechanisms have been exten- sively studied in Lactococcus lactis, as well as in Escherichia coli (10). Unfortunately, few of these natural phage resistance mechanisms have been found in S. thermophilus (35). To cope with virulent phages and the lack of known defense mecha- nisms, the dairy industry has developed protocols to rapidly isolate bacteriophage-insensitive S. thermophilus mutants (BIMs) (39). These BIMs are spontaneous, naturally occurring phage-resistant descendants that survive exposure to virulent phages. Up until recently, the mechanism responsible for this resistance was often attributed to mutations in the phage re- ceptors (2, 15). The complete genome sequence of three S. thermophilus host strains is now available (4, 11, 31). Comparative analyses of these closely related S. thermophilus strains have revealed that genetic polymorphisms primarily occur at a few hypervari- able regions, including three CRISPR loci (4, 5, 22, 31). These CRISPR loci have been found in a wide range of bacterial genomes (19, 23, 30). They are composed of 21 to 48-bp direct DNA repeats interspersed with nonrepetitive spacers of similar length. The direct repeats are highly conserved, while the num- ber and sequence of the spacers are diverse, even among strains of a same species. Sequence similarities between spac- * Corresponding author. Mailing address: Groupe de Recherche en E´cologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, Quebec City, Quebec G1V 0A6, Canada. Phone: (418) 656-3712. Fax: (418) 656-2861. E-mail: Sylvain.Moineau@bcm.ulaval.ca. Published ahead of print on 7 December 2007. 1390 ers and extrachromosomal elements such as phages and plas- mids led to the hypothesis that the CRISPR loci as well as CRISPR-associated genes (cas) play a role in protecting cells from the invasion of foreign DNA (5, 20, 30, 36, 37). In fact, it was recently demonstrated that CRISPR1/cas provides resis- tance against virulent phages in S. thermophilus (2). We recently used S. thermophilus strain DGCC7710 and the virulent pac-type phages 2972 and 858 to show that CRISPR plays a role in the development of BIMs (2). Specifically, we found that in response to challenges with phage 858 and/or 2972, S. thermophilus DGCC7710 integrates new spacers de- rived from the phage genomes, generating a phage-resistant phenotype. The specificity of the resistance was determined by the identity between spacer and phage sequences (2). While the insertion of new spacers provided significant phage resis- tance, a small population of phages was able to infect the BIMs. This suggested that both CRISPR locus and phage genomic regions are subject to rapid evolutionary changes (2). In the present study, we investigated the role of one of these CRISPR loci (CRISPR1) in phage-host interactions in S. ther- mophilus in greater detail. First, we demonstrated that this phage resistance mechanism is unique since it does not corre- spond to any known natural prokaryote antiviral barrier. More- over, we analyzed BIMs derived from another S. thermophilus strain, namely, SMQ-301 (40), and show that its CRISPR1 locus can provide resistance against cos-type S. thermophilus phages. Finally, the homologous spacer region in the phage genome, which we propose to name proto-spacer, was analyzed for phage mutants infecting BIMs. Some of these phages showed a direct response to CRISPR1 by either a single nu- cleotide mutation or a deletion, in the proto-spacer region. Interestingly, in other phage mutants, a single nucleotide mu- tation was found in a short region (AGAA) that is located two nucleotides downstream of the proto-spacer sequence in the phage genome. MATERIALS AND METHODS Bacterial strains, phages, and microbiological assays. S. thermophilus host strains and their BIM derivatives (Tables 1 and 2) were grown in M17 broth supplemented with 0.5% lactose (LM17) at 42°C. Phages were propagated in LM17 supplemented with 10 mM calcium chloride. High-titer phage lysates were obtained as described elsewhere (28). The efficiency of plaquing (EOP) was determined by dividing the phage titer obtained by plating on a BIM by the titer obtained by plating the same phage on a sensitive host strain. Phage adsorption assays were performed as reported previously (16). Cell survival was assayed (3) by using a multiplicity of infection (MOI) of 5. For the efficiency of center of infection (ECOI) experiments, cells in exponential-phase (i.e., an optical density TABLE 1. Phage sensitivity of isolated BIMs derived from S. thermophilus strain DGCC7710 Strain Phage used for challenge Sensitivity to phagea: Source or reference 2972 858 DGCC7710 28 DGCC7710858 S1S2 858 – 2 DGCC7710858 S3 858 – – 2 DGCC77102972 S4 2972 – 2 DGCC77102972 S5 2972 – 2 DGCC77102972 S6 2972 – – 2 DGCC77102972 S7 2972 – – 2 DGCC77102972 S8 2972 – 2 DGCC77108582972 S9S10S11S12 2972 858 – – 2 DGCC77108582972 S13S14 2972 858 – – 2 DGCC77102972 S15 2972 – – This study Derivatives of DGCC7710858 S1S2 DGCC7710858 S1S2 2972 S16S17 2972 – – This study DGCC7710858 S1S2 2972 S18S17 2972 – – This study DGCC7710858 S1S2 2972 S19 2972 – – This study Derivatives of DGCC77102972 S4 DGCC77102972 S4 858 S30 858 – – This study DGCC77102972 S4 858 S32 858 – – This study DGCC77102972 S4 858 S33 858 – – This study Derivatives of DGCC77102972 S6 DGCC77102972 S6 2972.S6B S20 2972.S6B – – This study DGCC77102972 S6 2972.S6B S30 2972.S6B – – This study DGCC77102972 S6 2972.S6B S31 2972.S6B – – This study Derivatives of DGCC77102972 S6 2972.S6B S20 DGCC77102972 S6 2972.S6B S20 2972.S20A S21S22 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S23 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S24 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S25S26 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S27 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S28 2972.S20A – – This study DGCC77102972 S6 2972.S6B S20 2972.S20A S29 2972.S20A – – This study a , Sensitive to phage; –, insensitive to phage (EOP 104). VOL. 190, 2008 CRISPR-ENCODED RESISTANCE IN S. THERMOPHILUS 1391 at 600 nm of 0.6) were infected by using an MOI of 5. Phages were first allowed to adsorb for 15 min, and then the unbound phages were removed by a quick centrifugation. The pellet of infected cells was washed twice with fresh LM17 broth. ECOI formation was calculated by dividing the phage titer obtained by plating resistant infected cells by the titer obtained with the sensitive infected strain. One-step growth curves (32) were performed by using an MOI of 0.2. The burst size was determined by dividing the average titer after the rise period by the average titer before the bacteria began to release virions. For each microbiolog- ical test, the mean value and the standard deviation were calculated from three independent experiments. Isolation of BIMs. BIMs were obtained by challenging sensitive S. thermophi- lus strains with virulent phages 2972 (28), 858 (28), or DT1 (40) or with mutant phages (see below). BIMs were also obtained by challenging S. thermophilus strains with a mixture of phages 2972 and 858 at a ratio of 1:1. Briefly, 100 l of an overnight culture of S. thermophilus was used to inoculate 10 ml of LM17, which was incubated at 42°C until the optical density at 600 nm reached 0.3. Phages and calcium chloride were then added at final concentrations of 107 PFU/ml and 10 mM, respectively. The phage-containing culture was incubated at 42°C for 24 h and monitored for lysis. A total of 100 l of the lysed culture was then used to inoculate 10 ml of fresh LM17. The remaining lysate was centri- fuged, and the pellet was transferred into another tube containing 10 ml of LM17 broth. These two cultures were incubated at 42°C for 16 h, serially diluted (1/10), and plated on LM17. The phage sensitivity of the isolated BIMs was first esti- mated by a spot test (33). Isolation and characterization of mutant phages. All phage mutants were single-plaque purified three times and propagated as previously described (34). Twenty well-defined plaques from four BIMs were isolated and analyzed. The individual phages were propagated on the BIM used to isolate them and were designated by the name of the parental wild-type phage followed by the new spacer number in the host strain. A different letter was added to identify each distinct phage mutant. Phage DNA was isolated as described previously from 1 ml of phage lysate (34). Restriction endonucleases (Roche Diagnostics) were used as recommended by the manufacturer. When necessary, restricted phage DNA samples were heated for 10 min at 70°C to prevent cohesive end ligation. The DNA fragments were separated on 0.8% agarose gels in 1 TAE buffer and UV visualized after staining with ethidium bromide. DNA sequencing of the 858 phage genome. Genomic DNA of virulent phage 858 was isolated by using Qiagen Lambda Maxi kit with previously described modifications (14). The primers used to sequence the genome of virulent phage 2972 (28) were used to begin sequencing the genome of the closely related phage 858, using isolated phage DNA as a template. New primers were designed from the nucleotide sequence, and primer walking on the two DNA strands was used to complete the sequencing of the genome. An ABI Prism 3700 at the genomic platform of the Centre Hospitalier de l’Universite´ Laval was used for the sequencing. ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and GeneMark.hmm (http://opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi) were used for DNA sequence searches (29). PSI-BLAST and Advanced BLAST Search 2.1 were also used for sequence comparisons (http://www.ncbi.nlm.nih .gov/BLAST) (1). Only the best match is presented. Sequencing the CRISPR1 locus in S. thermophilus strains and the proto- spacer region in the phage genomes. The CRISPR1 locus of the BIMs (including repeats and spacers) and the proto-spacer region in the phage genomes were sequenced from PCR products. Total bacterial DNA was prepared as described previously (21). Computer-assisted DNA analyses of the CRISPR loci were performed by using the Staden Package (38) (http://staden.sourceforge.net/), CLUSTALW (http://www.ebi.ac.uk/clustalw/), and DNA Display (http://www .mrgtech.ca/DNA). Nucleotide sequence accession numbers. The complete genomic sequences of the wild-type phages analyzed here are available under the indicated GenBank accession numbers: DT1 (AF085222), 2972 (AY699705), and 858 (EF529515). RESULTS Characterization of the phage resistance system. Phage re- sistance mechanisms are typically characterized by using a series of microbiological assays to determine their general mode of action. Wild-type virulent phage 2972, phage-sen- sitive strain S. thermophilus DGCC7710, and previously isolated S. thermophilus BIMs DGCC7710858 S3, DGCC 77102972 S4, and DGCC77102972 S4858S32 (2) were se- lected for these assays (Table 1 and Fig. 1). Strains DGCC7710858 S3 and DGCC77102972 S4 are BIM deriva- tives of DGCC7710 that have acquired a single new spacer in their respective CRISPR1 locus after a phage challenge, while DGCC77102972 S4858S32 is a second-generation BIM de- rived from the first-generation BIM DGCC77102972 S4, and thus it has acquired a second spacer after a second phage chal- lenge. Phage adsorption assays showed that phage 2972 adsorbed at the same level (90%) to phage-sensitive and phage-resistant strains (Table 3), indicating that the addition of a new spacer in CRISPR1 did not prevent phage adsorption. The isolation of second generation of CRISPR BIMs through the addition of new spacers (originating from the phage genome) precludes that this defense mechanism prevents the ejection of the phage DNA into the cell. Ruling out restriction-modification systems was the isolation of two mutants of phage 2972 from DGCC7710858 S3 or DGCC77102972 S4 (at a frequency of 105) that propagate on their respective BIM hosts and wild- type DGCC7710 with equal efficiency (EOP 1.0), even after passage through DGCC7710 (data not shown). One character- istic feature of Abi mechanisms is the high cell mortality fol- lowing the abortion of phage infection (10). Cell survival assays showed that between 64 to 73% of the phage-resistant S. ther- mophilus strains survive the phage infection (Table 3), indicat- ing that CRISPR1 is not an Abi mechanism (10). Interestingly, some infected cells still released virions (Table 3). The burst size, however, was significantly reduced, decreasing from 190 new phages per sensitive host cell (DGCC7710) to between 6 and 28 new virions, depending on the infected BIM strain (Table 3). Of note, the phage latency period was similar in the sensitive and first-generation BIMs (between 39 to 44 min at 42°C) but longer in the second-generation BIM (Table 3). Collectively, these results indicate that CRISPR1 is indeed a novel phage resistance system. Iterative addition of spacers. It has been shown that the addition of a new spacer in the CRISPR1 locus can increase the phage resistance of a particular strain (2). However, the replication of a small number of phages still occurs (Table 3). It has also been reported that the random acquisition of mul- tiple spacers (up to 4) after only one phage challenge can lead to increased phage resistance (2). We wanted to determine whether successive phage challenges and the subsequent iter- ative addition of spacers could lead to even greater phage protection. For these experiments, we used S. thermophilus DGCC7710 derivatives that are still sensitive to virulent phage 2972 TABLE 2. Phage sensitivity of isolated BIMs derived from S. thermophilus strain SMQ-301 Strain Phage used for challenge Sensitivity to phage DT1a Source or reference SMQ-301 40 SMQ-301DT1 S34S35 DT1 – This study SMQ-301DT1 S36S37 DT1 – This study SMQ-301DT1 S38S36S37 DT1 – This study SMQ-301DT1 S39 DT1 – This study a , Sensitive to phage; –, insensitive to phage (EOP 104). 1392 DEVEAU ET AL. J. BACTERIOL. or virulent phage 858. We selected S. thermophilus DGCC77102972 S4 obtained after a challenge with phage 2972 (but still sensitive to 858) and S. thermophilus DGCC7710858 S1S2 obtained after a challenge with phage 858 (but still sensitive to 2972) (2). Both BIMs were infected with the appropriate virulent phage (DGCC77102972 S4 with 858 and DGCC7710858 S1S2 with 2972) using the BIM isola- tion procedure previously described. For each challenge, sev- eral new BIMs were obtained, and three were selected for further characterization. In all cases, one or two new spacers were acquired at the leader end of the CRISPR1 locus (Table 1), which provided resistance to the second phage (Table 4). Interestingly, the EOP of the phage 2972 was decreased with the addition of a second spacer identical to the phage 2972 in the strain DGCC77102972 S4858S32, indicating that accu- mulating spacers can increase the overall phage resistance of the host (Table 4). Moreover, S. thermophilus DGCC77102972 S6, a BIM re- sistant to both wild-type phages 2972 and 858 (Table 1), was challenged with a mutant phage (2972.S6B, Fig. 2) that is capable of bypassing the CRISPR1-mediated resistance due to a single nucleotide mutation in the S6 targeted region of its FIG. 1. S. thermophilus CRISPR1 locus overview and newly acquired spacers in various phage-resistant mutants. (A) Repeat/spacer region of strain DGCC7710 and the selected BIM named DGCC77102972 S15. Repeats are shown as black diamonds, spacers are numbered in gray boxes, and the leader (L) is shown as a white box. The terminal repeat of CRISPR1 locus is represented with a letter “T” inside the black diamond. (B) The spacer content at the 5 end of the locus in various phage-resistant mutants is represented. The newly added spacers are indicated in white boxes with a designation containing the prefix S, followed by a number. The 3 end of CRISPR1 in BIMs is identical to that of the wild-type strain. (C) Repeat/spacer region of S. thermophilus strain SMQ-301 and its derivatives. TABLE 3. Microbiological effects of the new spacer presence on the bacteriophage 2972 Strain Mean SDa EOP Adsorption (%) ECOI (%) Cell survival (%) Burst size Latent period (min) DGCC7710 1 89.3 2.6 100
1 190 33 40 3 DGCC7710858 S3 (1.7 0.9) 105 94.3 6.0 18.6 1.9 72.5 3.5 7 2 39 2 DGCC77102972 S4 (1.8 2.0) 105 90.3 5.3 19.7 3.7 64.7 5.2 28 2 44 4 DGCC77102972 S4 858 S32
107 91.9 6.4 10.8 8.1 66.1 10.5 6 4 55 5 a Results are the means of three independent experiments. VOL. 190, 2008 CRISPR-ENCODED RESISTANCE IN S. THERMOPHILUS 1393 genome (see below). Three new BIMs that were resistant to phage 2972.S6B were obtained and their CRISPR1 loci an- alyzed. These second-generation BIMs had also acquired a new and unique spacer at the leader end of their CRISPR1 (Table 1). Subsequently, a second-generation BIM (DGCC 77102972 S62972.S6BS20) was challenged with another phage mutant (2972.S20A), resulting in a set of third-generation BIMs. Seven distinct third-generation BIMs were obtained, each of them had acquired one or two new spacers at the leader end of CRISPR1 (Table 1 and Fig. 1B). Taken alto- gether, these data clearly indicate that iterative addition of spacers is possible, resulting in increased phage resistance of these isogenic S. thermophilus strains. It also confirms that CRISPR loci, as well as phages, rapidly change in response to each other. CRISPR1 analysis of another phage-host system in S. ther- mophilus. Until now (see above and reference 2), the study of S. thermophilus CRISPR1 was performed with a single host and two phages, i.e., S. thermophilus DGCC7710 and virulent pac-type phages 2972 and 858 (and their phage mutants). To determine whether the main conclusions described above ap- ply to another S. thermophilus phage-host system, we isolated BIMs from S. thermophilus SMQ-301 challenged with the vir- ulent cos-type phage DT1 (40), one of the best-characterized cos-type phages in S. thermophilus (15, 16, 17, 26). The analysis of the CRISPR1 locus of wild-type phage-sensitive strain S. thermophilus SMQ-301 revealed the presence of 16 unique spacers, half the number found in DGCC7710. It should be noted that the spacers of SMQ-301 are distinct from those in DGCC7710 (22). It was previously reported that BIMs from S. TABLE 4. EOPs of the wild-type phages on various S. thermophilus strains S. thermophilus strain No. of spacers identical to phage: Mean EOP SDa for phage: 2972 858 2972 858 DGCC7710 0 0 1.0 1.0 DGCC77102972 S4 1 0 (1.8 2.0) 105 0.87 0.04 DGCC77102972 S4 858 S32 2 1
107 (2.4 1.3) 105 DGCC7710858 S1S2 0 1 0.67 0.45 (9.3 9.3) 106 DGCC7710858 S1S2 2972 S19 1 1 (3.1 1.7) 104 (1.6 1.3) 106 a Results are the means of three independent experiments. FIG. 2. Nucleotide sequences in wild-type and mutant phages that correspond to the newly acquired spacers by the S. thermophilus strains. The AGAA motif is highlighted in gray. Each mutation is in boldface and underlined. *, Deletion. 1394 DEVEAU ET AL. J. BACTERIOL. thermophilus SMQ-301 are difficult to obtain (15); however, using the improved protocol described here, four BIMs resis- tant to DT1 were obtained and analyzed (Table 2 and Fig. 1C). In all cases, one to three additional spacers derived from the genome of DT1 were inserted into the CRISPR1 locus (Table 5 and Fig. 3). These findings confirm that CRISPR-mediated phage resistance can protect S. thermophilus against represen- tatives of the two main groups of phages and that it operates through a general mechanism of action. Analysis of the newly acquired spacers in the CRISPR1 locus of S. thermophilus BIMs. The spacer content of 26 BIMs derived from strain S. thermophilus DGCC7710 (generated in the present study and previously described [2]), as well as of the 4 BIMs from S. thermophilus SMQ-301, was analyzed (Ta- ble 1 and Fig. 1). Of the 30 analyzed BIMs, 21 had acquired a single new spacer, seven had acquired two new spacers, one had acquired three new spacers, and one had acquired four new spacers. Thus, the addition of a single new spacer in CRISPR1 appeared to be a common outcome of a phage challenge. The original 32 spacers in DGCC7710 were conserved in all but one first-generation BIM, namely, S. thermophilus DGCC77102972 S15. Interestingly, this BIM had acquired a new spacer at the leader end but lost the first 17 spacers present in wild-type strain S. thermophilus DGCC7710. More- over, two of the four BIMs of SMQ-301 had also lost seven of the original spacers (spacers 4 through 10), suggesting that spacer deletion may occur concomitantly with the addition of new spacers (Table 2 and Fig. 1C). All 30 analyzed BIMs acquired at least one new spacer at the leader end of CRISPR1. Surprisingly, in two BIMs of SMQ- 301, a second new spacer was also added after the third orig- inal spacer. Of note, these two BIMs were also the ones that had lost seven of the original spacers. Thus, the addition of new spacers is clearly polarized toward one end of the CRISPR1 locus, and the acquisition of new spacers within CRISPR1 is also possible, albeit rare. A total of 33 new spacers were acquired by the 26 BIMs derived from S. thermophilus DGCC7710 challenged with vir- ulent phage 2972 or 858. In addition, six distinct spacers were acquired by the four BIMs of S. thermophilus SMQ-301 that was challenged with phage DT1. Analysis of these 39 new spacers showed that 32 of them were 30 nucleotides long (Ta- ble 5). Five spacers were 29 nucleotides long, while the remain- ing two spacers were 31 nucleotides long. Evidently, the addi- tion of a 30-nucleotide-long spacer is the most frequent event, in agreement with the observation that the vast majority of CRISPR1 spacers have a 30-bp length (22). Comparison of the 39 new spacers with three S. thermophilus phage genomes. To compare the newly acquired spacers with the genomic regions of the corresponding phages used in the challenge experiments, the complete genome of virulent phage 858 was sequenced (Table 6). The genomes of phages 2972 and DT1 were previously determined (28, 40). The annotation of phage 858 genome is presented in Table 6. As expected, it is highly related to other pac-type phages of S. thermophilus (28). The 858 and 2972 phage genomes share 90.9% nucleotide identity. Briefly, its linear double-stranded DNA contains 35,543 bp with an overall GC content of 39.8%. Only 5 of the 46 predicted open reading frames (ORFs) of phage 858 did not have close homologs in other S. thermophilus phages. In fact, three of them (ORF38, ORF39, and ORF40) were closer to deduced ORFs from the genome of Streptococcus suis 89/1591. Using the complete genomic sequences of phages 858, 2972, and DT1, we performed comparative analyses with the 39 newly added spacers found in the 30 analyzed S. thermophilus BIMs. The nucleotide sequence of 37 spacers out of 39 was 100% identical to a specific region found in the genome of at least one wild-type phage used in the challenge experiments (Table 5 and Fig. 3A). Spacers S2 and S26 had one mismatch with the proto-spacer in the phage genomes (Table 5). How- ever, the BIMs containing these two mismatched spacers had also acquired other spacers that were identical to a phage genomic region. Further analyses of the phage genomes indicated that all 39 new spacers analyzed in the present study correspond to a predicted coding region. Moreover, the spacer sequences cov- ered all phage modules as well as both strands. However, the new spacers originated most often from the coding strand than the noncoding strand (28 of 39 spacers from the coding strand [71.7%]), and about half of them were localized in the early expressed region of the phage genome (22 of 39 spacers [56.4%]), although this latter region corresponded to only 27 to 31% of the phage genome (17) (Table 5 and Fig. 3A). Interestingly, some spacers (S30, S36, and S37) were indepen- dently acquired by two BIM strains (Table 1). Comparative analyses of the regions flanking the proto-spac- ers in the phage genomes led to the identification of a specific sequence that was always located two nucleotides (NN) down- stream from the proto-spacers (Table 5). This CRISPR1-spe- cific sequence corresponds to the motif described recently (22). In fact, 34 of the 39 proto-spacers had the 3-flanking AGAAW motif. The other five proto-spacers (corresponding to spacers S2, S11, S13, S35, and S38) had one mismatched nucleotide in the AGAAW motif. However, these five spacers were found in BIMs that had acquired multiple spacers after the phage chal- lenge. To determine whether the strand and temporal expres- sion biases noted above could be explained by the presence of the AGAAW motif, the distribution of this conserved se- quence was analyzed in the genome of the three phages 2972, 858, and DT1 (Fig. 3B). The AGAAW motif was found almost three times more frequently on the coding strand than on the noncoding strand, with average values for the three S. ther- mophilus phages of 5.0 AGAAW/kb on the coding strand and 1.7 AGAAW/kb on the noncoding strand. These results sug- gest that spacer acquisition may not be random and that there may be a limited number of proto-spacers to be included in CRISPR1. On the other hand, between 36 and 40% of these motifs were found in the early expressed modules, while 56.4% of the acquired spacers correspond to this region. Thus, the proportion of the AGAAW motifs in the different transcrip- tion modules cannot totally explain the observed bias for the early expressed region. Phage response to the acquisition of a new spacer. As indi- cated elsewhere (2), phage mutants capable of infecting newly generated BIMs can be isolated under laboratory conditions. The characterization of phage mutants obtained through the selective pressure of resistance systems is particularly useful, since it has previously led to a better understanding of novel phage defense mechanisms (6, 25, 41). Using a similar ap- VOL. 190, 2008 CRISPR-ENCODED RESISTANCE IN S. THERMOPHILUS 1395 TABLE 5. List of new spacers found in CRISPR1 and the corresponding region in phages 2972, 858, and DT1 Spacer Phagea 5 positionb Spacer length (pb) Proto-spacer sequencec 3 flanking regiond Strand/modulee ORF/function in the genome of the phage used in the challenge S1 858 31378 30 CAACACATTCAACAGATTAATGAAGAATAC AAAGAAAAAA ()/E ORF40/primase S2 2972* 25432 30 TCCACTCACGTACAAATAGTGAGCGTACTC CTAAAAGGAT (–)/L ORF27/unknown S3 2972* 17202 30 TTACGTTTGAAAAGAATATCAAATCAATGA CGAGAAAGAT ()/L ORF20/receptor-binding protein S4 2972 31582 30 CTCAGTCGTTACTGGTGAACCAGTTTCAAT TGAGAAAAAA ()/E ORF38/primase S5 2972 22075 30 AGTTTCTTTGTCAGACTCTAACACAGCCGC TCAGAAAGTT ()/L ORF21/tail protein S6 2972* 34521 30 GCCCTTCTAATTGGATTACCTTCCGAGGTG TTAGAATTCC (–)/E ORF44/unknown S7 2972* 10299 30 AAGCAAGTTGATATATTTCTCTTTCTTTAT TAAGAAAACG (–)/L ORF17/unknown S8 2972 30016 29 CGTTTTCAGTCATTGGTGGTTTGTCAGCG AAAGAAATAA (–)/E ORF37/replication S9 2972* 7874 30 TTACTAGAGCGTGTCGTTAACCACTTTAAA TCAGAATATG ()/M ORF11/unknown S10 2972* 20650 30 TTCGTTAAAGTCACCTCGTGCTAGCGTTGC ATAGAAAGTT (–)/L ORF20/receptor-binding protein S11 2972* 8360 30 ATAACGGTAGCAAATATAAACCTGTTACTG TCAGAAGCTA ()/M ORF12/unknown S12 2972a 18998 30 GAAGTAGCCATACAAGAAGATGGATCAGCA CCAGAAATTG ()/L ORF20/receptor-binding protein S13 2972* 33602 30 GATGTCACTGAGTGTCTAAGCATTGCGTAC GAGGAAATCA ()/E ORF42/DNA binding S14 2972* 4830 30 TGAATAAGCAGTTCTTGACGACCAACCGAC ATAGAAAAGT (–)/M ORF6/capsid protein S15 2972* 34444 29 CAATTAACACAGCAATTAACACAGTATAT ACAGAAATTG ()/E ORF44/unknown S16 2972* 6799 30 ATGCCATTCTTTAAAGAGGCTTTACTCGTT AAAGAAAACG ()/M ORF9/capsid protein S17 2972 30547 30 GTTGGCGGACTACTCCTTCGAGGGGTTGAT CCAGAAATTA ()/E ORF37/replication S18 2972 30370 29 GAAGCACCTCTTGCGTTGATAAAAGTATT GCAGAAAATG ()/E ORF37/replication S19 2972 31709 29 ACATATCGACGTATCGTGATTATCCCATT CAAGAAAACA ()/E ORF38/primase S20 2972* 1113 30 TTATATCGAAGAACGACTGAAAGAGCTTGA GAAGAAAAAA ()/M ORF2/small terminase S21 2972* 19188 30 AAATCAACGTACATCCCGATATAGGCACGA TTAGAATCAG (–)/L ORF20/receptor-binding protein S22 2972 31708 30 GACATATCGACGTATCGTGATTATCCCATT CAAGAAAACA ()/E ORF38/primase S23 2972 26529 31 TGAAGTATTAGGTCTCTCAAAAGATGATATT GTAGAATACT ()/E ORF31/Cro-like repressor S24 2972 29923 30 AGTTGATTGCGTAATCAACCATCTCCATAA TTAGAATGGA (–)/E ORF37/replication S25 2972* 441 30 GCAACACTCAAACGTTGCAAACGCAAGCTT CGAGAATATC ()/E ORF1/unknown S26 2972 31606 31 CTCAGTCGTTACTGGTGAACCAGTT*TCAAT TGAGAAAAAA ()/E ORF38/primase S27 2972* 27032 30 TTTCATCGTCAATTTCCATGTTATAAATCT CTAGAAACTG (–)/E ORF33/unknown S28 2972 26530 30 GAAGTATTAGGTCTCTCAAAAGATGATATT GTAGAATACT ()/E ORF31/cro-like repressor S29 2972 32136 29 ATTGGCATGATTTCAATTTTAATTGGGAT GTAGAAAAAG ()/E ORF38/primase S30 2972* 33968 30 TCCAAGTTATTTGAGGAGTTATTAAGACAT GAAGAAATAT ()/E ORF43/unknown S31 2972 30803 30 TACCGAAACGACTGGTTTGAAAAATTCAAG GAAGAAAATC ()/E ORF38/primase S32 2972* 33044 30 ATTGTCTATTACGACAACATGGAAGATGAT GTAGAAATTT ()/E ORF41/unknown S33 858 30335 30 CTTCAAATGTACTGCAAGGCTGCAAAAGTA CCAGAAAATA ()/E ORF38/unknown S34 DT1 14535 30 GCTACTGAAAGCTACGAGGTTGGTAATCCT AAAGAATGGG ()/L ORF17/tail protein S35 DT1 13255 30 GTAGTTAGAGCGCTTGAAGCTAACGGTATA GAACCAAACA ()/L ORF15/tail protein S36 DT1 29132 30 TTAGATCTCATGAGTGGCGACAGTGAGCTT GTAGAATTAC ()/E ORF36/primase S37 DT1 20837 30 AACGATGAGGAACTCTTGGCAAAACTTACA CAAGAATAGC ()/L ORF22/unknown S38 DT1 9893 30 GCATTCATGGTTTGTTGGTATTTAACGTAT TCGGAACTGG (–)/L ORF15/tail protein S39 DT1 2603 30 TATTTTATCAGTCATCATGGCGTCATAGCC GAAGAAAACG (–)/M ORF4/Large terminase a *, DNA regions that are 100% identical between phages 858 and 2972. b That is, the 5 position of the proto-spacer in the phage genome. c Underlined and italicized nucleotides indicate a mismatch between the phage and the spacer. An asterisk indicates a deletion. d That is, the 3 flanking sequence in the phage genome. A mismatch in the AGAAW motif is boldfaced. e Transcription module: E, early expressed genes; M, middle expressed genes; L, late expressed genes. 1396 DEVEAU ET AL. J. BACTERIOL. proach, 20 phage mutants that infect S. thermophilus BIMs DGCC7710858 S3 (7 mutants of 2972), DGCC77102972 S4 (4 mutants of 2972), DGCC77102972 S6 (4 mutants of 2972), DGCC77102972 S4858S32 (4 mutants of 858), and DGCC77102972 S62972.S6BS20 (1 mutant of 2972) were fur- ther characterized (Fig. 2). All of the phage mutants had the same restriction profiles as the wild-type phages (data not shown). The proto-spacers, as well as their flanking regions (approximately 100 pb upstream and downstream), were se- quenced in the mutants. Four distinct types of mutations were observed in these mutants: (i) a single-nucleotide mutation directly within the proto-spacer (8 of 20 mutant phages), (ii) a two-nucleotide mutation directly in the proto-spacer (3 of 20 mutant phages), (iii) a single nucleotide mutation in the AGAA flanking sequence (7 of 20 mutant phages), and (iv) a deletion in the proto-spacer (2 of 20 mutant phages). In 14 cases where a nucleotide mutation occurred, the de- duced amino acid was changed. These mutations had appar- ently no effect (besides enabling infection of the BIM) on the completion of the phage lytic cycle. In 6 other cases, the amino acid was not changed, but this silent mutation generated a change of codon. Again, these mutations did not prevent the FIG. 3. Schematic representation of the S. thermophilus bacteriophage genomes used in the present study. (A) Distribution of the sequences corresponding to the new spacers in the three phage genomes. The spacers above the genome correspond to the positive strand, while those indicated in lower part correspond to the negative strand. Spacers indicated by an asterisk contain one mismatch with the phage sequence. ORFs connected by a gray box possess more than 70% identity at the amino acid level. (B) Distribution of the AGAAW motif on both strands for the three phages. VOL. 190, 2008 CRISPR-ENCODED RESISTANCE IN S. THERMOPHILUS 1397 TABLE 6. ORFs deduced from the genome of S. thermophilus phage 858 and their predicted functions ORF Start Stop Size (aa)a Mass (kDa) pI Putative ribosome-binding site and start codon (AAAGGAGGTGA) Best matchesb (% amino acid identity) Sizec (aa) Putative function 1 319 525 68 8.1 10.5 Not identified ATG gp68 phage Sfi11 67/68 (98) 68 2 414 827 137 16.2 8.7 CAGAGAGGTTAgtaca ATG ORF1 phage 2972 137/137 (100) 137 3 1009 1461 150 16.8 6.7 AAAATATTTGCggatgaatac TTG ORF2 phage 2972 145/150 (96) 150 Terminase small subunit 4 1448 2119 223 25.3 9.0 AAAGGAGCTGTaagcg ATG ORF3 phage 2972 223/223 (100) 223 Terminase large subunit 5 2412 2999 195 22.7 4.8 AGAGCCCTTGCgataacaaata ATG ORF4 phage 2972 194/195 (99) 195 Terminase large subunit 6 3008 4513 501 57.6 5.0 GTAGGAGGAATg ATG ORF5 phage 2972 501/501 (100) 501 Portal protein 7 4510 5403 297 34.4 8.8 TTGAGAGGGAAtatga ATG ORF6 phage 2972 294/297 (98) 297 Capsid protein 8 5591 6172 193 21.3 4.8 TAGATAGGAGAaata ATG ORF7 phage 2972 193/193 (100) 193 Scaffold protein 9 6192 6551 119 12.8 8.8 AAAGGAAATTTtaa ATG ORF8 phage 2972 115/119 (96) 119 Capsid protein 10 6570 7616 348 37.5 4.9 AGAGGAGGAACattaaaac ATG ORF9 phage 2972 346/348 (99) 348 Capsid protein 11 7628 7789 53 6.0 9.3 TTAAGAGGTACtgat ATG ORF10 phage 2972 52/53 (98) 53 12 7801 8142 113 13.0 4.6 TAGTGAGGTATggc ATG ORF11 phage 2972 113/113 (100) 113 13 8139 8453 104 11.5 9.5 AAAGAGGGAGAggtgttatttct ATG ORF12 phage 2972 104/104 (100) 104 14 8455 8793 112 12.5 8.9 CAAGGTGGTGAaataac ATG ORF13 phage 2972 112/112 (100) 112 15 8795 9181 128 14.6 5.0 AATGGCTAAGTgggaataag ATG ORF14 phage 2972 127/128 (99) 128 16 9195 9704 169 18.5 4.8 TCAGGAGGAAAaa ATG ORF15 phage 2972 168/169 (99) 168 Tail protein 17 9782 10135 117 13.2 4.7 ATAGGAGTTAAaaaca ATG gp117 phage Sfi11 101/117 (86) 117 18 10186 10503 105 12.6 9.9 CGAGGAATTAAtcactaatgct ATG ORF17 phage 2972 104/105 (99) 105 19 10493 15046 1517 153.4 9.6 AGAGGGGCTTGctag ATG ORF18 phage 2972 1511/1517(99) 1517 Tail protein 20 15046 16581 511 57.7 5.3 ATGAGAGGTATtaaata ATG ORF19 phage 2972 508/511 (99) 511 21 16581 21434 1617 178.5 5.3 ATTTGAGGAGAgatatatata ATG ORF20 phage 2972 1599/1617(98) 1605 Receptor-binding protein 22 21435 23456 673 74.2 5.7 GTAGGAGGTTTttaa TTG ORF21 phage 2972 659/673 (97) 673 Structural protein 23 23473 23859 128 14.6 4.7 AAGAAAGGAAAaatat ATG ORF22 phage 2972 126/128 (98) 128 24 23885 24028 47 5.5 6.6 TTAGGAGGAAGaaca ATG ORF23 phage 2972 47/47 (100) 47 25 23991 24278 95 11.0 8.7 CAAGAGCTTGTaggcttgtctc ATG T. parva 22/87 (25) 497 26 24275 24409 44 5.0 10.7 AAGCGAGGTTGataa ATG S. pyogenes MGAS5005 19/35 (54) 61 27 24437 24691 84 9.3 9.5 GAAACTTGAGAgg ATG ORF87 phage Sfi21 68/79 (86) 87 Holin 28 24693 25295 200 21.8 4.5 TAAGGAAGGAAaatagt ATG ORF25 phage DT1 180/200 (90) 200 Endolysin 29 25341 25463 40 4.6 8.8 AAACAAAGCGGtgtc ATG ORF27 phage 2972 40/40 (100) 40 30 25502 26263 253 30.0 9.7 AAAGATGGTGTcataag ATG Phage J1 248/253 (98) 253 Endonuclease 31 26325 26552 75 8.6 3.9 AATCCCGGTTAca ATG Phage J1 70/75 (93) 186 Endolysin 32 26720 26851 43 5.3 8.9 AAACGAGGTGAaaaca ATG ORF30 phage 2972 43/43 (100) 43 33 26952 27161 69 7.9 7.9 AAAGGAGAACTta ATG ORF31 phage 2972 68/69 (98) 69 Cro-like repressor 34 27178 27300 40 5.0 8.0 AAAGGTATTTAaa ATG ORF32 phage 2972 40/40 (100) 40 35 27557 28030 157 18.1 6.2 TAGGGAGGGTAggaattaaat ATG ORF33 phage 2972 157/157 (100) 157 36 28027 28728 233 26.2 6.6 AAAGGAGAAACcttaacataag ATG ORF34 phage 2972 233/233 (100) 233 37 28685 30097 470 54.2 8.6 AAAGGGGTGTAaggtag ATG ORF35 phage 2972 309/473 (65) 445 Helicase 38 30104 30577 157 18.2 4.8 AGATTTGGAGAtaaaaaaac ATG S. suis 89/1591 110/157 (70) 156 39 30582 31397 271 30.7 7.8 Not identified ATG S. suis 89/1591 180/263 (68) 268 40 31366 32910 514 60.0 8.0 AAAGGAGTTAGatactaaac ATG S. suis 89/1591 363/508 (71) 517 Primase 41 33167 33487 106 12.1 9.9 AAAGAAAGGCAactttcaa GTG ORF39 phage 2972 102/106 (96) 106 42 33698 33871 57 6.6 8.1 GAAAGAGATGAtagaact ATG ORF17 phage O1205 44/57 (77) 57 43 33868 34023 51 6.3 5.6 GTAGGAGATTAgtagagtt ATG ORF41 phage 2972 51/51 (100) 51 44 34024 34536 170 19.6 6.3 TGAGGTGGAATag ATG ORF42 phage 2972 170/170 (100) 170 DNA-binding protein 45 34505 34831 108 12.1 9.2 ATAGGAAAGGAaagatggtaa ATG ORF43 phage 2972 108/108 (100) 108 46 34835 35542 235 27.7 9.1 TGAGGAGTTATtaagac ATG ORF44 phage 2972 234/235 (99) 235 a That is, the number of amino acids (aa) of the predicted protein. b If the host species is not mentioned, the phage infects a S. thermophilus strain. c That is, the size in amino acids (aa) of the best-matched protein. 1398 DEVEAU ET AL. J. BACTERIOL. phage to complete the infection process. In mutant phages 858.S32C and 2972.S6D, 75-nucleotide and 1-nucleotide dele- tions, respectively, occurred. The 75-nucleotide deletion in phage 858.S32C targeted the end of ORF42 and the beginning of ORF43 of phage 858 (Table 6). No putative function could be assigned to either ORF42 (57 amino acids) or ORF43 (51 amino acids) (Table 6). Interestingly, the deletion led to the formation of an ORF42-ORF43 fusion product, but no func- tion could be assigned to the deduced fusion protein (83 amino acids). Phage mutant 2972.S6D had a one-base deletion, which led to a frameshift and, consequently, the presence of several stop codons in the ORF44 sequence for which no putative function could been assigned (Table 6). Taken altogether, these data confirm that a newly added spacer must be identical to the proto-spacer to be fully effective and that the CRISPR1-specific sequence (NNAGAAW) is also important for the phage resistance phenotype. DISCUSSION The remarkable diversity and metabolic capabilities of bac- teria allow them to grow and prosper in every ecosystem where life forms have been found (18). Similarly, bacteriophages are present in these same ecosystems, including manufactured eco- logical niches such as food fermentation vats. It is now believed that phages represent the most abundant biological entities on the planet (7). Thus, it is not surprising to observe that bacteria have devised a number of strategies to defend against these prolific invaders. CRISPRs and their associated cas genes con- stitute the latest defense mechanism unveiled in prokaryotes (2). In the present study, we show that CRISPR-mediated phage resistance is indeed a novel antiphage system since its general mode of action is distinct from the previously known systems. Our results also demonstrate that CRISPR-mediated phage resistance protects S. thermophilus against the two main groups of phage known to infect this bacterial species. Thus, this antiphage system is exceptionally broad and effective. This wide-ranging efficacy against phages is in agreement with the fact that CRISPRs have been found in a wide range of bacte- rial genomes (19, 22, 30). The isolation and characterization of BIMs obtained through iterative phage challenges have revealed that one spacer will typically be added to the CRISPR1 locus. However, multiple spacers can also be acquired by CRISPR1, providing enhanced resistance to phages. The iterative addition of spac- ers is particularly interesting and separates the CRISPR-me- diated phage resistance from other natural antiphage defense systems. With the other four systems (adsorption inhibition, DNA ejection inhibition, restriction-modification systems, and Abi), it is not possible to generate new phage-resistant deriv- atives (when phage mutants have emerged) without any fitness cost to the host at each generation. In contrast, CRISPR- mediated phage resistance allows the acquisition of a new spacer specific to the phage mutants without an obvious fitness cost associated with it. Thus, it is possible to create multiresis- tant S. thermophilus strains by successive challenges using dif- ferent phages. Because new spacers were almost always inserted at the leader end of the CRISPR1 locus, it is tempting to hypothesize that spacer position could serve as a memory of previous phage encounters by a strain. Although this may be true for many BIMs, spacer deletion did occur in some of the BIMs. Thus, this presumed historical perspective, albeit interesting, may be of limited value in some cases. Similarly, the reason for the prevalence of new spacer acquisitions at the leader end of the repeat arrays is unknown, although a putative role of the leader could explain this phenomenon. Nonetheless, it is worth mentioning that when spacer deletion was observed in a BIM, a new spacer always occurred in the vicinity of the deleted region. It is possible that the spacer deletion occurs by homol- ogous recombination between CRISPR direct repeats. A key feature in CRISPR-mediated phage resistance is that the newly acquired spacer (between 29 and 31 nucleotides in size) must be identical to the phage genomic sequence to provide resistance. Only 2 of the newly acquired spacers (of 39) described here were not identical (one mismatch) to a known phage sequence. However, these two spacers were found in BIMs that had acquired more than one spacer, and the other associated spacers had a perfect match to a phage genomic sequence. Moreover, most of the analyzed phage mutants that were able to infect these BIMs had a mutation in the proto- spacer. Interestingly, 7 of the 20 mutated phages analyzed had no mutation in the proto-spacer but had a mutation in the AGAA flanking sequence. This sequence appears to play a critical role in CRISPR1-mediated phage resistance in S. ther- mophilus because a mutation within this sequence allows the phage to escape the CRISPR1-mediated resistance. This strongly suggests that CRISPR1 and/or the cas-associated pro- teins may be involved in a nucleotide recognition mechanism. The importance of the NNAGAAW motif was recently con- firmed by their presence in proto-spacer region corresponding to the CRISPR1 spacers from several different S. thermophilus strains (22). All of the phage genomic sequences matching the acquired spacers in the S. thermophilus BIMs were found in a coding region, and the coding strand was three times more frequently targeted. We believe that the frequency of the NNAGAAW motif in the phage genome (2.9 on the coding strand for 1 on the noncoding strand) is responsible for this bias. In addition, early transcribed modules of the phage genome appear to be more frequently targeted for the acquisition of new CRISPR1 spacers. It has been previously hypothesized that CRISPR may play a role in an RNA interference system (30). Thus, it is tempting to speculate that the early transcribed phage mRNAs would be a preferential target for a mechanism such as RNA interference. Rapidly silencing the phage infection may allow the cell to recover thereby, increasing cell survival. The acquisition of a spacer from a coding sequence also suggests that the targeted gene is important for phage devel- opment. However, this observation is debatable as most of the phage genome (89.6% for DT1 and 93.8% for 2972) is coding. Furthermore, phage mutants were still able to propagate effi- ciently despite the apparent gene inactivation. As indicated above, it is possible that the CRISPR-mediated resistance somehow targets the mRNA. Knowing that many S. thermophi- lus phage genes are transcribed as part of a polycistronic mRNA (26, 42), inactivating larger transcripts may prevent the translation of essential phage proteins. In conclusion, the CRISPR/cas system clearly represents a VOL. 190, 2008 CRISPR-ENCODED RESISTANCE IN S. THERMOPHILUS 1399 novel and interesting avenue for the development of phage- resistant bacterial strains for fermentation and biotechnologi- cal processes. Moreover, because of the widespread distribu- tion of phages in various ecosystems, CRISPRs likely play a significant role in prokaryotic evolution and ecology (2). The identification of a nucleotide motif in the phage genome that is important for the phage resistance phenotype is another clue toward the elucidation of the molecular mode of action of the CRISPR1 mechanism. 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