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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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Transcription of target sequences is required for type III-A CRISPR immunitya, Schematic diagram of the ΦNM1 genome and the position of targets used in this study. T, crRNA anneals to the top strand; B, bottom strand. Gray arrows represent the ΦNM1 central promoter driving divergent transcription. b, Immunity against ΦNM1 infection provided by spacers targeting the phage regions shown in a. Dotted line indicates the limit of detection for the assay. c, Immunity against ΦNM1γ6 infection. Inset; comparison of lysis phenotypes for ΦNM1 (turbid) and ΦNM1γ6 (clear), representative of four technical replicates. d, Leftward promoter consensus sequences at the ΦNM1 and ΦNM1γ6 central promoter. The ΦNM1γ6 mutation in the −10 element is shown in red. The putative transcription start site is noted (+1). e, Comparison of phage transcription profiles from cells infected with ΦNM1 (gray line) or ΦNM1γ6 (red line), 15 minutes post-infection. Phage-derived transcripts are plotted in reads per million total-mapped reads (RPM) relative to their position on the genome; arrows indicate the direction of transcription plotted in each graph; the vertical dotted line marks the position of the central promoter. Error bars: mean ± s.d. (n=3).
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Figure 2: Transcription of target sequences is required for type III-A CRISPR immunitya, Schematic diagram of the ΦNM1 genome and the position of targets used in this study. T, crRNA anneals to the top strand; B, bottom strand. Gray arrows represent the ΦNM1 central promoter driving divergent transcription. b, Immunity against ΦNM1 infection provided by spacers targeting the phage regions shown in a. Dotted line indicates the limit of detection for the assay. c, Immunity against ΦNM1γ6 infection. Inset; comparison of lysis phenotypes for ΦNM1 (turbid) and ΦNM1γ6 (clear), representative of four technical replicates. d, Leftward promoter consensus sequences at the ΦNM1 and ΦNM1γ6 central promoter. The ΦNM1γ6 mutation in the −10 element is shown in red. The putative transcription start site is noted (+1). e, Comparison of phage transcription profiles from cells infected with ΦNM1 (gray line) or ΦNM1γ6 (red line), 15 minutes post-infection. Phage-derived transcripts are plotted in reads per million total-mapped reads (RPM) relative to their position on the genome; arrows indicate the direction of transcription plotted in each graph; the vertical dotted line marks the position of the central promoter. Error bars: mean ± s.d. (n=3).

Mentions: To determine whether prophage tolerance is a spacer-specific phenomenon, we designed a variety of spacers with 100% target identity, targeting different regions of the ΦNM1 genome on both strands (Fig. 2a). We first tested the ability of each spacer to prevent lytic infection (Fig. 2b). Surprisingly, spacer functionality varied with the predicted transcriptional context of each target sequence. Spacers matching putative lytic genes to the right of the central promoter which are predicted to be unidirectionally transcribed were only effective when they targeted the predicted non-template strand (top strand according to our spacer nomenclature). Meanwhile, transcription is predicted to be bi-directional to the left of the central promoter19. Spacers targeting this region prevented plaque formation regardless of the strand targeted. This resembled the activity reported for the type III-B CRISPR-Cas system of the archaeon, Sulfolobus islandicus REY15A, where immunity to plasmid transformation depended on the presence of promoters flanking a target sequence21. We thus reasoned that transcription-dependent targeting could explain the discrepancies in spacer functionality. Indeed, ΦNM1 transcription profiles assessed by RNA-sequencing of RN4220 cultures 6, 15, 30, and 45 min post infection revealed predominantly unidirectional transcription to the right of the central promoter, while bi-directional transcription was detected to the left of the central promoter (Extended Data Fig. 3).


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

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

Transcription of target sequences is required for type III-A CRISPR immunitya, Schematic diagram of the ΦNM1 genome and the position of targets used in this study. T, crRNA anneals to the top strand; B, bottom strand. Gray arrows represent the ΦNM1 central promoter driving divergent transcription. b, Immunity against ΦNM1 infection provided by spacers targeting the phage regions shown in a. Dotted line indicates the limit of detection for the assay. c, Immunity against ΦNM1γ6 infection. Inset; comparison of lysis phenotypes for ΦNM1 (turbid) and ΦNM1γ6 (clear), representative of four technical replicates. d, Leftward promoter consensus sequences at the ΦNM1 and ΦNM1γ6 central promoter. The ΦNM1γ6 mutation in the −10 element is shown in red. The putative transcription start site is noted (+1). e, Comparison of phage transcription profiles from cells infected with ΦNM1 (gray line) or ΦNM1γ6 (red line), 15 minutes post-infection. Phage-derived transcripts are plotted in reads per million total-mapped reads (RPM) relative to their position on the genome; arrows indicate the direction of transcription plotted in each graph; the vertical dotted line marks the position of the central promoter. Error bars: mean ± s.d. (n=3).
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Related In: Results  -  Collection

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Figure 2: Transcription of target sequences is required for type III-A CRISPR immunitya, Schematic diagram of the ΦNM1 genome and the position of targets used in this study. T, crRNA anneals to the top strand; B, bottom strand. Gray arrows represent the ΦNM1 central promoter driving divergent transcription. b, Immunity against ΦNM1 infection provided by spacers targeting the phage regions shown in a. Dotted line indicates the limit of detection for the assay. c, Immunity against ΦNM1γ6 infection. Inset; comparison of lysis phenotypes for ΦNM1 (turbid) and ΦNM1γ6 (clear), representative of four technical replicates. d, Leftward promoter consensus sequences at the ΦNM1 and ΦNM1γ6 central promoter. The ΦNM1γ6 mutation in the −10 element is shown in red. The putative transcription start site is noted (+1). e, Comparison of phage transcription profiles from cells infected with ΦNM1 (gray line) or ΦNM1γ6 (red line), 15 minutes post-infection. Phage-derived transcripts are plotted in reads per million total-mapped reads (RPM) relative to their position on the genome; arrows indicate the direction of transcription plotted in each graph; the vertical dotted line marks the position of the central promoter. Error bars: mean ± s.d. (n=3).
Mentions: To determine whether prophage tolerance is a spacer-specific phenomenon, we designed a variety of spacers with 100% target identity, targeting different regions of the ΦNM1 genome on both strands (Fig. 2a). We first tested the ability of each spacer to prevent lytic infection (Fig. 2b). Surprisingly, spacer functionality varied with the predicted transcriptional context of each target sequence. Spacers matching putative lytic genes to the right of the central promoter which are predicted to be unidirectionally transcribed were only effective when they targeted the predicted non-template strand (top strand according to our spacer nomenclature). Meanwhile, transcription is predicted to be bi-directional to the left of the central promoter19. Spacers targeting this region prevented plaque formation regardless of the strand targeted. This resembled the activity reported for the type III-B CRISPR-Cas system of the archaeon, Sulfolobus islandicus REY15A, where immunity to plasmid transformation depended on the presence of promoters flanking a target sequence21. We thus reasoned that transcription-dependent targeting could explain the discrepancies in spacer functionality. Indeed, ΦNM1 transcription profiles assessed by RNA-sequencing of RN4220 cultures 6, 15, 30, and 45 min post infection revealed predominantly unidirectional transcription to the right of the central promoter, while bi-directional transcription was detected to the left of the central promoter (Extended Data Fig. 3).

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.

Show MeSH
Related in: MedlinePlus