Title: <scp>BREX</scp> is a novel phage resistance system widespread in microbial genomes
Abstract: Article1 December 2014free access BREX is a novel phage resistance system widespread in microbial genomes Tamara Goldfarb Tamara Goldfarb Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hila Sberro Hila Sberro Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Eyal Weinstock Eyal Weinstock Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ofir Cohen Ofir Cohen Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Shany Doron Shany Doron Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yoav Charpak-Amikam Yoav Charpak-Amikam Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Shaked Afik Shaked Afik Computational Biology Graduate Group, University of California Berkeley, Berkeley, CA, USA Search for more papers by this author Gal Ofir Gal Ofir Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Rotem Sorek Corresponding Author Rotem Sorek Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tamara Goldfarb Tamara Goldfarb Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Hila Sberro Hila Sberro Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Eyal Weinstock Eyal Weinstock Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ofir Cohen Ofir Cohen Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Shany Doron Shany Doron Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yoav Charpak-Amikam Yoav Charpak-Amikam Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Shaked Afik Shaked Afik Computational Biology Graduate Group, University of California Berkeley, Berkeley, CA, USA Search for more papers by this author Gal Ofir Gal Ofir Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Rotem Sorek Corresponding Author Rotem Sorek Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Tamara Goldfarb1,‡, Hila Sberro1,‡, Eyal Weinstock1, Ofir Cohen1, Shany Doron1, Yoav Charpak-Amikam1, Shaked Afik2, Gal Ofir1 and Rotem Sorek 1 1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 2Computational Biology Graduate Group, University of California Berkeley, Berkeley, CA, USA ‡These authors contributed equally *Corresponding author. Tel: +972 8 934 6342; E-mail: [email protected] The EMBO Journal (2015)34:169-183https://doi.org/10.15252/embj.201489455 See also: R Barrangou & J van der Oost (January 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria. Synopsis BREX is a novel host DNA methylation-based defense system that protects B. cereus against a broad variety of phages. BREX-like systems can be found in 10% of sequenced bacterial genomes. BREX (Bacteriophage Exclusion) is a novel bacterial defense system that protects against a broad range of phages, both lytic and temperate. The system is present in 10% of all sequenced prokaryotic genomes and appears in 6 variants (subtypes). The system contains six genes, including ones coding for a protease, phosphatase, and methylase domain proteins. The system blocks phage DNA replication in a mechanism still undetermined. Introduction The ongoing arms race between bacteria and bacteriophages (phages) has led to the rapid evolution of extensive mechanisms to combat phage infection (Labrie et al, 2010; Stern & Sorek, 2011). Among these are restriction-modification systems (Tock & Dryden, 2005), abortive infection (Abi) mechanisms (Chopin et al, 2005), and the CRISPR-Cas adaptive defense system (Sorek et al, 2008; van der Oost et al, 2009; Deveau et al, 2010; Horvath & Barrangou, 2010). The relatively recent discovery of the complex and abundant CRISPR-Cas system highlights the fact that our knowledge of the arsenal of phage-defense mechanisms encoded in bacterial and archaeal genomes is incomplete. Indeed, accumulating evidence suggest that many additional phage resistance systems present in microbial genomes have yet to be discovered (Stern & Sorek, 2011; Makarova et al, 2013; Swarts et al, 2014). A recent study has reported that genes involved in phage resistance, such as restriction-modification enzymes and toxin–antitoxin systems, are non-randomly clustered to specific genomic locations in bacterial and archaeal genomes, forming genomic ‘defense islands’ (Makarova et al, 2011). One of the genes found enriched within defense islands is pglZ, a putative member of the alkaline phosphatase superfamily. This gene was previously reported as essential for a unique phage resistance phenotype in Streptomyces coelicolor A3(2), denoted phage growth limitation (Pgl) (Chinenova et al, 1982). Streptomyces coelicolor strains carrying the Pgl system are sensitive to the first cycle of infection by phage ΦC31, but are resistant to phages emerging from this first cycle. Further studies mapped the Pgl phenotype to a cluster of four genes that were able to reconstitute the phenotype upon transfer to a new host (Sumby & Smith, 2002). These genes include pglZ, a putative phosphatase; pglW, a serine/threonine kinase domain-containing protein; pglX, a protein containing an adenine-specific DNA methyltransferase motif; and pglY, a protein containing a P-loop domain (Sumby & Smith, 2002). The Pgl system was active against ΦC31 and its homoimmune relatives, but not to any of the other phage that were tested (Laity et al, 1993). A molecular mechanism to explain the activity of the Pgl system was never deciphered. Based on the enrichment of pglZ-domain genes in genomic defense islands, it was suggested that genes containing this domain are involved in phage defense in multiple species (Makarova et al, 2011). In this work, we analyzed ~1,500 bacterial and archaeal genomes and found that pglZ-domain genes are present in about 10% of these genomes. Moreover, in more than half of the cases, the pglZ-domain gene was found embedded in a gene cluster composed of six genes, two of which (pglZ and pglX) display considerable sequence homology to genes in the Pgl system, and four additional genes that encode a putative protease, a protein with an ATPase domain, a predicted RNA-binding protein, and a gene of unknown function. We hypothesized that this gene cluster forms a novel phage-defense system, which we denote BREX (Bacteriophage Exclusion). Indeed, we show that transfer of this new system from Bacillus cereus into Bacillus subtilis provides B. subtilis with resistance to a broad range of Bacillus phages, both virulent and temperate phage. Our data show that phage adsorption occurs in BREX-containing strains, but that neither phage DNA replication nor lysogeny occurs, and that this system does not act via an abortive infection mechanism. This system does not display the Pgl phenotype and hence probably functions through a novel mode of action, different than that of the Pgl system. We provide further evidence that the system methylates chromosomal DNA at a specific motif and that this methylation is likely to be essential for the system's activity. Pan genomic and phylogenetic analyses further show that BREX undergoes extensive horizontal gene transfer and that pglZ-containing gene clusters can be divided into six coherent BREX subtypes in which the gene order and composition are conserved. Each BREX subtype contains 4–8 genes, some of which are core genes while others are subtype-specific. Finally, we found that a minority of phages escaped BREX defense, implying that these phages may have evolved anti-BREX mechanisms. Results BREX is abundant in bacteria and archaea Previous analyses of pglZ-domain genes demonstrated that this domain is enriched in defense islands of bacteria and archaea (Makarova et al, 2011), and documented a number of genes commonly associated with pglZ-domain genes (Makarova et al, 2011, 2013). To understand whether there is higher order organization among pglZ and its associated genes, we performed homology searches and found 144 occurrences of pglZ-domain genes in the 1,447 finished bacterial and archaeal genomes analyzed (Supplementary Table S1, 4). Remarkably, in 55% of the cases (79 of 144), the pglZ gene was embedded within a 6-gene cluster arranged in a highly conserved order in a diverse array of bacteria and archaea (Fig 1A; Supplementary Table S2). Subsequent searches conducted on 5,493 draft genome sequences deposited in NCBI showed that this 6-gene cluster is present in an additional 290 genomes (Supplementary Table S3). Figure 1. BREX confers resistance to phage A. The pglZ gene reproducibly appears within a six-gene cluster in various genomes. Representative appearances are shown. B. The BREX locus in B. cereus H3081.97. Coordinates below the genes denote the position along the NZ_ABDL01000007 contig in the draft genome of B. cereus H3081.97. Orange box within brxC represents the position of the ATPase P-loop motif. C. Operon organization of the B. cereus BREX system integrated in the B. subtilis genome. Expression throughout the operons was validated by RNA-seq. Positions of promoters and terminators were inferred by 5′ and 3′ RACE, respectively. D–G. Culture dynamics of phage-infected wild-type (black) versus BREX-containing (red) strains of B. subtilis BEST7003. Bacterial strains were exposed to phage at time = 0 h, and optical density measurements were read in a 96-well plate format. Each experiment was performed three times with three technical triplicates for each biological replicate. Error bars represent SEM. Shown are representative results for three of the ten phages tested; data for the remaining seven phages are found in Supplementary Fig S2. Download figure Download PowerPoint Two of the six genes found in this conserved cluster share homology with genes from the previously reported Pgl system (Sumby & Smith, 2002): pglZ, coding for a protein with a predicted alkaline phosphatase domain, and pglX, coding for a protein with a putative methylase domain. The four additional genes include (i) a Lon-like protease-domain gene, denoted here brxL; (ii) a gene coding for a protein with significant structural homology to the RNA-binding antitermination protein NusB (brxA, see Supplementary Fig S1); (iii) a gene of unknown function (brxB); and (iv) a large, ~1,200 amino acid protein with an ATP-binding motif (GXXXXGK[T/S]), which we denote brxC. Although this does not resemble any classical combination of genes currently known to be involved in phage defense, the preferential localization of this conserved gene cluster in the genomic vicinity of other defense genes suggests that it could form a novel phage-defense system. We denote this putative defense system the BREX (Bacteriophage Exclusion) system. BREX confers resistance to phage infection in Bacillus subtilis To determine whether the BREX system provides protection against phage infection, the complete BREX system from Bacillus cereus H3081.97 (Fig 1B) was cloned into a Bacillus subtilis BEST7003 strain lacking an endogenous BREX system. Proper integration of the intact system into the B. subtilis genome was verified by complete genome sequencing. We then verified, using RNA-seq, that the genes of the integrated system are transcribed in Bacillus subtilis when grown in exponential phase in rich medium. Furthermore, using 5′ and 3′ RACE, we determined that the system is transcribed as two operons with the first four genes, brxA-brxB-brxC-pglX, forming a single transcriptional unit, while the last two genes, pglZ-brxL, are co-expressed as a second transcriptional unit (Fig 1C, 4). The observation that the genes in the putative BREX system are co-transcribed as two long polycistronic mRNAs further supports that they work together as components of a functional system. Ten B. subtilis phages were selected for phage infection experiments, spanning a wide range of phage phylogeny, including Myoviridae (phages SPO1 and SP82G), lambda-like Siphoviridae phages (Φ105, rho10, rho14, and SPO2), and SPβ-like Siphoviridae phages (SPβ, Φ3T, SP16, and Zeta). Two of the phages are obligatory lytic (SPO1 and SP82G), while the remaining are temperate (Table 1). The phage sensitivity of B. subtilis strains either containing or lacking the BREX system was evaluated using both optical density measurements in a 96-well plate format, and double agar overlay plaque assays. Table 1. BREX protection against the phages used in this study Phage Genus Family Life cycle Infection blocked by BREX? Efficiency of BREX protectiona SPβ SPβ-like Siphoviridae Temperate Yes > 105 SP16 SPβ-like Siphoviridae Temperate Yes > 105 Zeta SPβ-like Siphoviridae Temperate Yes > 105 Φ3T SPβ-like Siphoviridae Temperate Yes > 105 SPO2 Lambda-like Siphoviridae Temperate Yes > 105 Φ105 Lambda-like Siphoviridae Temperate No 1 rho10 Lambda-like Siphoviridae Temperate No 1 rho14 Lambda-like Siphoviridae Temperate No 1 SPO1 SPO1-like Myoviridae Obligatory lytic Yes 8 × 102 ± 0.02 SP82G SPO1-like Myoviridae Obligatory lytic Yes 1.8 × 101 ± 0.08 a Protection efficiency was calculated as the ratio between the number of plaques formed on the BREX-lacking strain divided by the number of plaques formed on the BREX-containing strain with the same phage titer, using increasing titers. Standard deviation is calculated from a biological triplicate of the plaque experiment. Upon phage infection, the B. subtilis strain containing the BREX system showed resistance to seven of the ten phages tested (Table 1). Growth curves of BREX-containing bacteria infected with these seven phages at a multiplicity of infection (MOI) of 10−3–10−4 were similar to the uninfected bacteria, while declines in optical density measurements were observed for the control strain lacking the BREX system, indicating lysis of the infected cells (Fig 1D–F; Supplementary Fig S2). These results confirm that BREX is a phage-defense system that provides protection against a wide array of phages, including both virulent and temperate ones. In contrast to the protection from phage infection observed with the first seven phages tested, phage resistance was not observed upon infection with phage Φ105 and its close relatives, rho10 and rho14. Similar kinetics of cell lysis were observed for strains either containing or lacking the BREX system (Fig 1G; Supplementary Fig S2). Considering that phage Φ105 is estimated to share high (83–97%) genome homology with rho10 and rho14 (Rudinski & Dean, 1979), the inability of the BREX system to protect against these three phages could indicate that this phage family has evolved strategies to counteract the BREX defense, as has been observed with other bacterial defense systems (Bondy-Denomy et al, 2013). If such strategies exist, their identification could provide insight into the mechanism of action of the BREX system. Alternatively, the resistance of phage Φ105 and its relatives to the BREX system could also stem from intrinsic differences in the infection cycle of these phages making them immune to BREX-mediated defense, or because they do not encode a target for the BREX activity. To further evaluate the level of protection provided by the BREX system against the phages that were tested, plaque assays were performed using increasing phage concentrations. For five of the phages, no plaques were observed when the BREX-containing strain was challenged even with the highest phage concentrations, indicating that the BREX system provides at least 105-fold protection against cell lysis upon infection (Table 1). Plaque assays also confirmed that phage Φ105 and its relatives evade BREX defense, because similar efficiencies of plating and plaque morphology were observed in both BREX-containing and wild-type control strains (Table 1). Interestingly, for two of the phages tested, SPO1 and SP82G, plaque assays showed only a 101-fold reduction in plaque numbers in BREX-containing strains (Table 1). These results were consistent with the observation that incubation of the BREX-containing strain with these two phages for extended periods of time (> 20 h) often resulted in an eventual culture decline occurring at apparently stochastic points in time (Fig 2A). To gain further insight into the nature of the incomplete BREX defense against these phages, we performed a one-step phage growth curve assay (Carlson, 2005) with SPO1. Briefly, this experiment involves mixing SPO1-infected cells with a SPO1-sensitive B. subtilis cells and plating them together using an agar overlay method. Phage bursts from successful infections are visualized as a single plaque on a lawn from the SPO1-sensitive B. subtilis strain, enabling an evaluation of the number of phages that have adsorbed and completed a successful infection cycle (4). Enumeration of plaques during the first 45 min of the time course infection indicated that the SPO1 phage was able to complete the lytic cycle only in 9 ± 4% of the initially infected cells (Fig 2B). A delay in the kinetics of the phage cycle was also observed, with phage bursts observed 75 and 105 min following infection of BREX-lacking and BREX-containing cells, respectively (Fig 2B). Together, these results suggest that the BREX system provides significant, but not complete, protection from infection by phages SPO1 and SP82G. Figure 2. Phage infection dynamics in BREX-containing cells Culture dynamics over an extended period (> 30 h) for SPO1-infected wild-type (black) versus BREX-containing (red) strains of B. subtilis BEST7003. Each curve represents a single technical replicate grown in a single well on a 96-well plate. Culture decline is temporally reproducible for the BREX-lacking strain but occurs later, at apparently stochastic time points, for the BREX-containing strain. Re-growth following culture crash represents phage-resistant mutants. Phage production during a one-step phage growth curve experiment with wild-type (black) and BREX-containing (red) strains of B. subtilis BEST7003 infected with SPO1. Error bars represent SD. Y-axis represents absolute phage concentrations. Black and red arrows point to the time point of maximal burst for BREX-lacking and BREX-containing strains, respectively. Phage production during a one-step phage growth curve experiment with wild-type (black) and BREX-containing (red) strains of B. subtilis BEST7003 infected with Φ3T. Error bars represent SD. Y-axis represents relative phage concentrations normalized to the value at the beginning of the infection. Multiplex PCR assay showing lysogeny during a phage infection time course in the strain lacking BREX (black), but not in BREX-containing strain (red), or uninfected (U) strains. Amplicons for the bacterial DNA, phage DNA, and lysogen-specific DNA are 293, 485, and 1,218 bp, respectively. Download figure Download PowerPoint The mode of action of BREX is different than that of the Pgl system BREX is an apparently complex system with proteins that are predicted to have multiple biochemical activities (e.g., protease, phosphatase, methylase). Fully deciphering its mechanism of action and understanding the role of each of its six proteins in phage defense is expected to be non-trivial and would probably require multiple genetic, biochemical, and structural studies. Here, we set out to provide initial insights into the function of the BREX system. Due to the homology of a subset of the genes in the BREX system to genes in the Pgl system, we first examined whether BREX also functions through the Pgl mode of action. The Pgl phenotype observed in S. coelicolor A3 predicts that the first infection cycle by the phage would be successful, producing viable phage progeny. We used one-step phage growth curve assays to examine the first infection cycle of phage Φ3T in BREX-containing cells. While wild-type control strains displayed phage burst sizes of 61.5 ± 10.2 particles per infected cell (Fig 2C), there was no production of Φ3T phage during infections of BREX-containing cells under similar conditions. To exclude the possibility that productive phage infection could occur, but at later times, experiments were extended to 120 min (three infection cycles in wild-type strains) in BREX-containing cells. Plaques were not observed, even at later times (Fig 2C). These results demonstrate that unlike the S. coelicolor Pgl system, the BREX system halts Φ3T production prior to the first round of infection. Previous experiments with the S. coelicolor Pgl system demonstrated that although the Pgl defense system prevents continued propagation of the temperate phage ΦC31, it does not block lysogeny of the phage (Chinenova et al, 1982). To determine whether BREX also permits lysogeny, we examined phage Φ3T integration into the B. subtilis genome during infection using a PCR assay. In wild-type control strains, lysogeny was first detected 10 min following phage infection (Fig 2D). However, no evidence for phage integration into the host genome was found in BREX-containing cells. Evaluation of lysogeny in bacterial colonies that survived the phage infection also indicated that none of the surviving BREX-containing colonies were lysogens, while all surviving colonies tested in strains lacking the BREX system were lysogenic for phage Φ3T. Together, these results suggest that although BREX and Pgl share a subset of genes, the two systems probably exert their defense through different modes of action. BREX is not an abortive infection system One of the common forms of phage defense is abortive infection (Abi), where infected cells commit ‘suicide’ before phage progeny are produced, thus protecting the culture from phage propagation (Chopin et al, 2005). Such a phenotype predicts that with a high MOI, where nearly all bacteria are infected in the first cycle, massive cell death will be observed in the culture. To test whether the BREX system works through an Abi mechanism, we infected the BREX-containing B. subtilis strain with increasing concentrations of the Φ3T phage against which the BREX was shown to provide resistance. Even at an MOI > 1, no significant growth arrest or culture decline was found in the liquid culture, suggesting that the BREX is not an Abi system (Fig 3A). Figure 3. Initial characterization of BREX activity BREX does not work via an abortive infection mechanism. Increasing the MOI of the Φ3T infection from 0.05 to 5 shortens the time to culture crash for the BREX-lacking strain, but does not result in culture decline for BREX-containing strain. Error bars represent SD of technical triplicates. The system does not interfere with phage adsorption to bacterial cells. Strains either containing (red) or lacking (black) the BREX system were infected with phage Φ3T and then chloroform-treated 15 min following infection. The culture was plated on Φ3T-sensitive B. subtilis cells and plaques, representing extracellular, unadsorbed phages, were counted. Phage DNA replication does not occur in BREX-containing cells (red), but is observed in the BREX-lacking strain (black). Y-axis represents relative phage concentrations normalized to the value at the beginning of the infection, as measured by Illumina sequencing. Download figure Download PowerPoint BREX allows phage adsorption but blocks phage DNA replication To gain further insight into the stage at which the infection cycle is blocked by BREX, we asked whether phage adsorption is prevented. Adsorption assays showed that Φ3T efficiently adsorbs to both BREX-containing and BREX-lacking cells, indicating that BREX does not block adsorption (Fig 3B). We then assayed whether BREX allows phage DNA replication within infected cells. For this, we extracted total cellular DNA (including chromosomal DNA and intracellular phage DNA) at successive time points following a high-MOI infection by Φ3T and submitted the extracted DNA to Illumina sequencing. Since host DNA is not degraded following Φ3T infection (Supplementary Fig S3), mapping the sequenced reads to the reference B. subtilis and Φ3T genomes allowed quantification of the number of Φ3T genome equivalents per infected cell at each time point. In wild-type control cells, phage DNA replication began between 10 and 15 min following infection, and after 30 min, phage DNA levels were elevated 81-fold relative to that observed at the 10-min time point (Fig 3C). In contrast, no increase in phage DNA levels was observed in BREX-containing cells (Fig 3C). These results indicate that phage DNA replication does not occur in BREX-containing cells and that this system exerts its function at the early stages of the infection cycle. BREX methylates bacterial DNA but does not degrade phage DNA The presence of a predicted m6A DNA adenine methylase (the pglX gene) in the BREX system prompted us to examine whether either bacterial or phage DNA are methylated in a BREX-dependent manner. To test this, we used the PacBio sequencing platform that directly detects m6A modifications in sequenced DNA (Murray et al, 2012). In DNA extracted from BREX-containing cells, the PacBio platform clearly detected m6A methylation on the 5th position of the non-palindromic hexamer TAGGAG (Fig 4A). While nearly all TAGGAG motifs were methylated in BREX-containing B. subtilis cells (Fig 4B), no methylation on this motif was observed in the strain lacking the BREX system. These results suggest that BREX drives motif-specific methylation on the genomic DNA of the bacteria in which it resides. Figure 4. Methylation activity of BREX A. Consensus sequence around m6A modified bases in the BREX-containing B. subtilis genome. The modified base is marked by an arrow. Modifications were directly detected by DNA sequencing using the PacBio platform. B. Statistics of modified motifs in the BREX-containing B. subtilis genome. C, D. Culture dynamics of non-infected (C) or phage-infected (D) cultures. Curves depict culture dynamics of strains lacking BREX (black) and BREX-containing (red) strains of B. subtilis BEST7003, as well as a BREX-containing strain where the pglX methylase was deleted (green). Axes and error bars are as in Fig 1. E. Southern blot analysis of phage Φ3T genome during infection. Numbers indicate time (in min) following infection; U, uninfected. Probe was designed to match positions 94,645–95,416 in the phage genome. Each lane contains 200 ng total DNA. Download figure Download PowerPoint To examine whether BREX also methylates the invading phage DNA, we extracted total cellular DNA (including chromosomal DNA and intracellular phage DNA) at 10 and 15 min following a high-MOI infection by Φ3T and subjected the extracted DNA to PacBio sequencing. As in the previous assay, we found that TAGGAG motifs in the bacterial genome were methylated throughout the infection. However, none of the 43 TAGGAG motifs present on the phage genome was found to be methylated at any of the time points sampled during infection. The presence of bacterial-specific