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Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

Goldberg GW, Jiang W, Bikard D, Marraffini LA - Nature (2014)

Bottom Line: Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle.Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity.In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

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Related in: MedlinePlus

Type II CRISPR-Cas targeting in S. aureus prevents both lytic and lysogenic infectiona, Plaquing efficiency of ΦNM1 and ΦNM1γ6 on lawns of RN4220 harboring type II-A CRISPR-Cas plasmids as indicated. The parental vector, pDB184, serves as a non-targeting control. b, ΦNM1-ErmR lysogenization of RN4220 harboring either the spacer 43B-tII, 4B-tII, or non-targeting type II-A CRISPR plasmids. c, ΦNM2 sensitivity assay for seven randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (1–7). For comparison, a resistant non-lysogen harboring the spacer 43B-tII plasmid and a sensitive lysogen harboring the pDB184 plasmid were included as controls (respectively, C+ and C−). Picture represents a single experiment for 7 of 22 isolates. d, ΦNM2 plaquing efficiency on soft agar lawns for an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (8–13); a ΦNM1-ErmR lysogen harboring the pDB184 plasmid is also tested (−C/L). For comparison, plaquing efficiency of ΦNM2 on the non-lysogenic indicator strain harboring pDB184 or the targeting spacer 43B-tII plasmid are also shown (−C and +C, respectively). e, Agarose gel electrophoresis of plasmid DNA purified from isolates 8–13 and the parental spacer 43B-tII strain (C). +/− indicate the presence or absence of treatment with the BamHI restriction enzyme which produces 2 bands for the wild type spacer 43B-tII plasmid: 5367 bp and 3972 bp. Size markers correspond to 10 kb, 3 kb, and 0.5 kb bands of the 1kb DNA ladder from NEB. f, Colony PCR spanning the type II CRISPR array for isolates 8–13. Spacer 43B-tII plasmid DNA was used as a template for the control (C). 3 kb and 0.5 kb size markers are indicated. g, Colony PCR spanning the target region for isolates 8–13 and a ΦNM1-ErmR lysogen harboring the pDB184 control plasmid (C). Isolates # 10 and 11 harbor identical deletions within the prophage that remove the target region (see below). 3 kb and 0.5 kb size markers are indicated. The presence of attL and attR prophage integration arms was also verified independently for each isolate using PCR (data not shown). h, Location of the 16,985 bp deletion identified within the prophage harbored by isolates # 10 and 11 (shaded gray box). The location and orientation of the ermC insertion cassette is also shown (blue arrow). Deletion was mapped by primer walking. An ~9.1 kb product spanning the deletion was ultimately amplified using primers oGG6 and oGG241, and the deletion junction was sequenced by the Sanger method using oGG245. A perfect 14 bp direct repeat micro-homology flanks the deletion. i, Plaque-forming potential of overnight culture supernatants from isolates # 8, 10, and 11. Supernatants were plated by the soft agar method with RN4220 cells harboring the non-targeting pDB184 control plasmid as an indicator strain. Supernatants were also plated with spacer 43B-tII targeting lawns, yielding no detectable pfu. Isolate 8 appears to exhibit wild type levels of spontaneous prophage induction (compare to pGG3 control in Fig. 4a). No plaque-forming units were detected from the supernatants of isolates # 10 and 11 whatsoever, presumably resulting from their deletion of genes essential for prophage induction, including the ORF 43 major capsid protein. Dotted line represents the limit of detection for this assay. Error bars: mean ± s.d. (n=3). Panels e through g represent single experiments for 6 of 22 isolates.
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Figure 8: Type II CRISPR-Cas targeting in S. aureus prevents both lytic and lysogenic infectiona, Plaquing efficiency of ΦNM1 and ΦNM1γ6 on lawns of RN4220 harboring type II-A CRISPR-Cas plasmids as indicated. The parental vector, pDB184, serves as a non-targeting control. b, ΦNM1-ErmR lysogenization of RN4220 harboring either the spacer 43B-tII, 4B-tII, or non-targeting type II-A CRISPR plasmids. c, ΦNM2 sensitivity assay for seven randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (1–7). For comparison, a resistant non-lysogen harboring the spacer 43B-tII plasmid and a sensitive lysogen harboring the pDB184 plasmid were included as controls (respectively, C+ and C−). Picture represents a single experiment for 7 of 22 isolates. d, ΦNM2 plaquing efficiency on soft agar lawns for an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (8–13); a ΦNM1-ErmR lysogen harboring the pDB184 plasmid is also tested (−C/L). For comparison, plaquing efficiency of ΦNM2 on the non-lysogenic indicator strain harboring pDB184 or the targeting spacer 43B-tII plasmid are also shown (−C and +C, respectively). e, Agarose gel electrophoresis of plasmid DNA purified from isolates 8–13 and the parental spacer 43B-tII strain (C). +/− indicate the presence or absence of treatment with the BamHI restriction enzyme which produces 2 bands for the wild type spacer 43B-tII plasmid: 5367 bp and 3972 bp. Size markers correspond to 10 kb, 3 kb, and 0.5 kb bands of the 1kb DNA ladder from NEB. f, Colony PCR spanning the type II CRISPR array for isolates 8–13. Spacer 43B-tII plasmid DNA was used as a template for the control (C). 3 kb and 0.5 kb size markers are indicated. g, Colony PCR spanning the target region for isolates 8–13 and a ΦNM1-ErmR lysogen harboring the pDB184 control plasmid (C). Isolates # 10 and 11 harbor identical deletions within the prophage that remove the target region (see below). 3 kb and 0.5 kb size markers are indicated. The presence of attL and attR prophage integration arms was also verified independently for each isolate using PCR (data not shown). h, Location of the 16,985 bp deletion identified within the prophage harbored by isolates # 10 and 11 (shaded gray box). The location and orientation of the ermC insertion cassette is also shown (blue arrow). Deletion was mapped by primer walking. An ~9.1 kb product spanning the deletion was ultimately amplified using primers oGG6 and oGG241, and the deletion junction was sequenced by the Sanger method using oGG245. A perfect 14 bp direct repeat micro-homology flanks the deletion. i, Plaque-forming potential of overnight culture supernatants from isolates # 8, 10, and 11. Supernatants were plated by the soft agar method with RN4220 cells harboring the non-targeting pDB184 control plasmid as an indicator strain. Supernatants were also plated with spacer 43B-tII targeting lawns, yielding no detectable pfu. Isolate 8 appears to exhibit wild type levels of spontaneous prophage induction (compare to pGG3 control in Fig. 4a). No plaque-forming units were detected from the supernatants of isolates # 10 and 11 whatsoever, presumably resulting from their deletion of genes essential for prophage induction, including the ORF 43 major capsid protein. Dotted line represents the limit of detection for this assay. Error bars: mean ± s.d. (n=3). Panels e through g represent single experiments for 6 of 22 isolates.

Mentions: Importantly, the two complementary spacers (2T and 4T) targeting the opposite strand of spacers 2B and 4B were not escaped by ΦNM1γ6, indicating that the 2B/4B escape phenotype did not result from changes to the target DNA per se. Consistent with this, we did not observe differences in the ΦNM1 and ΦNM1γ6 plaquing efficiency when targeting the 4B region via Cas9-mediated type II-A CRISPR immunity (Extended Data Fig. 4a), which was shown to cleave dsDNA even in the absence of target transcription22,23. We thus reasoned that the ΦNM1γ6 type III-A CRISPR-escape and clear-plaque phenotypes could result from a localized, unidirectional reduction in transcription, e.g., leftward from the central promoters. Indeed, de novo sequencing of ΦNM1γ6 revealed a single nucleotide polymorphism in a crucial residue of the central promoters’ leftward −10 element (Fig. 2d), immediately upstream of the SAPPVI_g4 cI-like repressor gene required for lysogenic establishment, and ~1700 bp away from the 2B target sequence. Encouraged by this result, we directly assessed ΦNM1γ6 transcription profiles using RNA-seq, 6 and 15 min post infection (Extended Data Fig. 5a). Consistent with our hypothesis, leftward transcription (Fig. 2e, lower panel) of the lysogenization operon 15 min post infection was strongly reduced, while rightward transcription (Fig. 2e, upper panel) in this region was relatively unchanged. Taken together, these findings suggest that transcription across target sequences is a requirement for type III-A CRISPR immunity. Previously reported24 strand-independent immunity against plasmids in S. epidermidis may also follow this rule, as bi-directional transcription was detected across targets (Extended Data Fig. 6).


Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

Goldberg GW, Jiang W, Bikard D, Marraffini LA - Nature (2014)

Type II CRISPR-Cas targeting in S. aureus prevents both lytic and lysogenic infectiona, Plaquing efficiency of ΦNM1 and ΦNM1γ6 on lawns of RN4220 harboring type II-A CRISPR-Cas plasmids as indicated. The parental vector, pDB184, serves as a non-targeting control. b, ΦNM1-ErmR lysogenization of RN4220 harboring either the spacer 43B-tII, 4B-tII, or non-targeting type II-A CRISPR plasmids. c, ΦNM2 sensitivity assay for seven randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (1–7). For comparison, a resistant non-lysogen harboring the spacer 43B-tII plasmid and a sensitive lysogen harboring the pDB184 plasmid were included as controls (respectively, C+ and C−). Picture represents a single experiment for 7 of 22 isolates. d, ΦNM2 plaquing efficiency on soft agar lawns for an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (8–13); a ΦNM1-ErmR lysogen harboring the pDB184 plasmid is also tested (−C/L). For comparison, plaquing efficiency of ΦNM2 on the non-lysogenic indicator strain harboring pDB184 or the targeting spacer 43B-tII plasmid are also shown (−C and +C, respectively). e, Agarose gel electrophoresis of plasmid DNA purified from isolates 8–13 and the parental spacer 43B-tII strain (C). +/− indicate the presence or absence of treatment with the BamHI restriction enzyme which produces 2 bands for the wild type spacer 43B-tII plasmid: 5367 bp and 3972 bp. Size markers correspond to 10 kb, 3 kb, and 0.5 kb bands of the 1kb DNA ladder from NEB. f, Colony PCR spanning the type II CRISPR array for isolates 8–13. Spacer 43B-tII plasmid DNA was used as a template for the control (C). 3 kb and 0.5 kb size markers are indicated. g, Colony PCR spanning the target region for isolates 8–13 and a ΦNM1-ErmR lysogen harboring the pDB184 control plasmid (C). Isolates # 10 and 11 harbor identical deletions within the prophage that remove the target region (see below). 3 kb and 0.5 kb size markers are indicated. The presence of attL and attR prophage integration arms was also verified independently for each isolate using PCR (data not shown). h, Location of the 16,985 bp deletion identified within the prophage harbored by isolates # 10 and 11 (shaded gray box). The location and orientation of the ermC insertion cassette is also shown (blue arrow). Deletion was mapped by primer walking. An ~9.1 kb product spanning the deletion was ultimately amplified using primers oGG6 and oGG241, and the deletion junction was sequenced by the Sanger method using oGG245. A perfect 14 bp direct repeat micro-homology flanks the deletion. i, Plaque-forming potential of overnight culture supernatants from isolates # 8, 10, and 11. Supernatants were plated by the soft agar method with RN4220 cells harboring the non-targeting pDB184 control plasmid as an indicator strain. Supernatants were also plated with spacer 43B-tII targeting lawns, yielding no detectable pfu. Isolate 8 appears to exhibit wild type levels of spontaneous prophage induction (compare to pGG3 control in Fig. 4a). No plaque-forming units were detected from the supernatants of isolates # 10 and 11 whatsoever, presumably resulting from their deletion of genes essential for prophage induction, including the ORF 43 major capsid protein. Dotted line represents the limit of detection for this assay. Error bars: mean ± s.d. (n=3). Panels e through g represent single experiments for 6 of 22 isolates.
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Figure 8: Type II CRISPR-Cas targeting in S. aureus prevents both lytic and lysogenic infectiona, Plaquing efficiency of ΦNM1 and ΦNM1γ6 on lawns of RN4220 harboring type II-A CRISPR-Cas plasmids as indicated. The parental vector, pDB184, serves as a non-targeting control. b, ΦNM1-ErmR lysogenization of RN4220 harboring either the spacer 43B-tII, 4B-tII, or non-targeting type II-A CRISPR plasmids. c, ΦNM2 sensitivity assay for seven randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (1–7). For comparison, a resistant non-lysogen harboring the spacer 43B-tII plasmid and a sensitive lysogen harboring the pDB184 plasmid were included as controls (respectively, C+ and C−). Picture represents a single experiment for 7 of 22 isolates. d, ΦNM2 plaquing efficiency on soft agar lawns for an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 43B-tII (8–13); a ΦNM1-ErmR lysogen harboring the pDB184 plasmid is also tested (−C/L). For comparison, plaquing efficiency of ΦNM2 on the non-lysogenic indicator strain harboring pDB184 or the targeting spacer 43B-tII plasmid are also shown (−C and +C, respectively). e, Agarose gel electrophoresis of plasmid DNA purified from isolates 8–13 and the parental spacer 43B-tII strain (C). +/− indicate the presence or absence of treatment with the BamHI restriction enzyme which produces 2 bands for the wild type spacer 43B-tII plasmid: 5367 bp and 3972 bp. Size markers correspond to 10 kb, 3 kb, and 0.5 kb bands of the 1kb DNA ladder from NEB. f, Colony PCR spanning the type II CRISPR array for isolates 8–13. Spacer 43B-tII plasmid DNA was used as a template for the control (C). 3 kb and 0.5 kb size markers are indicated. g, Colony PCR spanning the target region for isolates 8–13 and a ΦNM1-ErmR lysogen harboring the pDB184 control plasmid (C). Isolates # 10 and 11 harbor identical deletions within the prophage that remove the target region (see below). 3 kb and 0.5 kb size markers are indicated. The presence of attL and attR prophage integration arms was also verified independently for each isolate using PCR (data not shown). h, Location of the 16,985 bp deletion identified within the prophage harbored by isolates # 10 and 11 (shaded gray box). The location and orientation of the ermC insertion cassette is also shown (blue arrow). Deletion was mapped by primer walking. An ~9.1 kb product spanning the deletion was ultimately amplified using primers oGG6 and oGG241, and the deletion junction was sequenced by the Sanger method using oGG245. A perfect 14 bp direct repeat micro-homology flanks the deletion. i, Plaque-forming potential of overnight culture supernatants from isolates # 8, 10, and 11. Supernatants were plated by the soft agar method with RN4220 cells harboring the non-targeting pDB184 control plasmid as an indicator strain. Supernatants were also plated with spacer 43B-tII targeting lawns, yielding no detectable pfu. Isolate 8 appears to exhibit wild type levels of spontaneous prophage induction (compare to pGG3 control in Fig. 4a). No plaque-forming units were detected from the supernatants of isolates # 10 and 11 whatsoever, presumably resulting from their deletion of genes essential for prophage induction, including the ORF 43 major capsid protein. Dotted line represents the limit of detection for this assay. Error bars: mean ± s.d. (n=3). Panels e through g represent single experiments for 6 of 22 isolates.
Mentions: Importantly, the two complementary spacers (2T and 4T) targeting the opposite strand of spacers 2B and 4B were not escaped by ΦNM1γ6, indicating that the 2B/4B escape phenotype did not result from changes to the target DNA per se. Consistent with this, we did not observe differences in the ΦNM1 and ΦNM1γ6 plaquing efficiency when targeting the 4B region via Cas9-mediated type II-A CRISPR immunity (Extended Data Fig. 4a), which was shown to cleave dsDNA even in the absence of target transcription22,23. We thus reasoned that the ΦNM1γ6 type III-A CRISPR-escape and clear-plaque phenotypes could result from a localized, unidirectional reduction in transcription, e.g., leftward from the central promoters. Indeed, de novo sequencing of ΦNM1γ6 revealed a single nucleotide polymorphism in a crucial residue of the central promoters’ leftward −10 element (Fig. 2d), immediately upstream of the SAPPVI_g4 cI-like repressor gene required for lysogenic establishment, and ~1700 bp away from the 2B target sequence. Encouraged by this result, we directly assessed ΦNM1γ6 transcription profiles using RNA-seq, 6 and 15 min post infection (Extended Data Fig. 5a). Consistent with our hypothesis, leftward transcription (Fig. 2e, lower panel) of the lysogenization operon 15 min post infection was strongly reduced, while rightward transcription (Fig. 2e, upper panel) in this region was relatively unchanged. Taken together, these findings suggest that transcription across target sequences is a requirement for type III-A CRISPR immunity. Previously reported24 strand-independent immunity against plasmids in S. epidermidis may also follow this rule, as bi-directional transcription was detected across targets (Extended Data Fig. 6).

Bottom Line: Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle.Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity.In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity.

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Related in: MedlinePlus