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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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Visualization of ΦNM1 transcription profiles 6, 15, 30, and 45 min post infection (MOI 20)Rightward and leftward expression values are plotted as blue and fuchsia lines, respectively, in reads per million (RPM). Position of relevant spacer targets are indicated with vertical solid lines. The dotted line with arrowheads marks the position of the central promoter. To improve readability, all curves were smoothened by plotting the average RPM values over a 500 bp sliding-window. To the left of the central promoter, rightward expression is comparable to leftward expression by 30 min post infection, consistent with the strand-independent targeting observed for this region.
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Figure 7: Visualization of ΦNM1 transcription profiles 6, 15, 30, and 45 min post infection (MOI 20)Rightward and leftward expression values are plotted as blue and fuchsia lines, respectively, in reads per million (RPM). Position of relevant spacer targets are indicated with vertical solid lines. The dotted line with arrowheads marks the position of the central promoter. To improve readability, all curves were smoothened by plotting the average RPM values over a 500 bp sliding-window. To the left of the central promoter, rightward expression is comparable to leftward expression by 30 min post infection, consistent with the strand-independent targeting observed for this region.

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)

Visualization of ΦNM1 transcription profiles 6, 15, 30, and 45 min post infection (MOI 20)Rightward and leftward expression values are plotted as blue and fuchsia lines, respectively, in reads per million (RPM). Position of relevant spacer targets are indicated with vertical solid lines. The dotted line with arrowheads marks the position of the central promoter. To improve readability, all curves were smoothened by plotting the average RPM values over a 500 bp sliding-window. To the left of the central promoter, rightward expression is comparable to leftward expression by 30 min post infection, consistent with the strand-independent targeting observed for this region.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214910&req=5

Figure 7: Visualization of ΦNM1 transcription profiles 6, 15, 30, and 45 min post infection (MOI 20)Rightward and leftward expression values are plotted as blue and fuchsia lines, respectively, in reads per million (RPM). Position of relevant spacer targets are indicated with vertical solid lines. The dotted line with arrowheads marks the position of the central promoter. To improve readability, all curves were smoothened by plotting the average RPM values over a 500 bp sliding-window. To the left of the central promoter, rightward expression is comparable to leftward expression by 30 min post infection, consistent with the strand-independent targeting observed for this region.
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