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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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Prophage induction is limited by type III-A CRISPR immunity in tolerant lysogensa, Plaque-forming potential (measured in pfu/ml) of supernatants from overnight cultures of ΦNM1 lysogens carrying the tolerant spacer 32T or 43T CRISPR plasmids, or the pGG3 non-targeting control (C). b, Growth curve of ΦNM1-ErmR lysogens or a non-lysogen control harboring the pGG3 or spacer 43T CRISPR plasmids as indicated, with or without the addition of the prophage-inducing agent mitomycin C (MMC) at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).
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Figure 4: Prophage induction is limited by type III-A CRISPR immunity in tolerant lysogensa, Plaque-forming potential (measured in pfu/ml) of supernatants from overnight cultures of ΦNM1 lysogens carrying the tolerant spacer 32T or 43T CRISPR plasmids, or the pGG3 non-targeting control (C). b, Growth curve of ΦNM1-ErmR lysogens or a non-lysogen control harboring the pGG3 or spacer 43T CRISPR plasmids as indicated, with or without the addition of the prophage-inducing agent mitomycin C (MMC) at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).

Mentions: In order to definitively demonstrate that transcription-dependent targeting offers a biological mechanism for conditional tolerance, we integrated the ΦNM1 target sequence for spacers 43T and 43B into the chromosome of S. aureus RN4220 under the control of a tightly regulated tetracycline-inducible promoter, thus emulating target lysogenization. The target was placed in both orientations with respect to the inducible promoter (Fig. 3c) and with respect to the chromosomal origin of replication (Extended Data Fig. 7a). The resulting strains were then transformed with the spacer 43T or 43B plasmids in a reverse CRISPR immunity assay, and plated in the absence or presence of the inducer. CRISPR immunity was only achieved when transcription across the target was induced with anhydrotetracycline in the presence of an antisense crRNA, regardless of the target’s orientation (Fig. 3d and Extended Data Fig. 7a). Once again, we confirmed this finding to be a type III-specific phenomenon by transforming the strains from Fig. 3c with the spacer 43B-tII type II-A CRISPR plasmid targeting the same region (Extended Data Fig. 7b). We corroborated this result by following the growth of spacer 43T transformants in liquid media (Fig. 3e). Upon addition of the inducer, growth was only inhibited for cells with the target in the forward orientation for which spacer 43T produces an antisense crRNA. Importantly, tolerance achieved in the absence of the inducer did not appear to affect growth (Fig. 3e, dotted lines). Finally, having established that type III-A CRISPR-Cas systems can block lytic infection but tolerate lysogenization, we examined the effect of tolerant spacers on prophage induction of ΦNM1 lysogens in culture. Compared to a spacerless lysogen control, the phage titer resulting from spontaneous induction of overnight cultures was significantly lower for lysogens harboring a tolerant spacer (Fig. 4a). We next followed the growth of cultures induced directly with the DNA-damaging agent, mitomycin C (Fig. 4b, solid lines). While the spacerless lysogen control cultures succumbed to prophage induction, the presence of a tolerant spacer prevented lysis.


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

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

Prophage induction is limited by type III-A CRISPR immunity in tolerant lysogensa, Plaque-forming potential (measured in pfu/ml) of supernatants from overnight cultures of ΦNM1 lysogens carrying the tolerant spacer 32T or 43T CRISPR plasmids, or the pGG3 non-targeting control (C). b, Growth curve of ΦNM1-ErmR lysogens or a non-lysogen control harboring the pGG3 or spacer 43T CRISPR plasmids as indicated, with or without the addition of the prophage-inducing agent mitomycin C (MMC) at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).
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Related In: Results  -  Collection

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Figure 4: Prophage induction is limited by type III-A CRISPR immunity in tolerant lysogensa, Plaque-forming potential (measured in pfu/ml) of supernatants from overnight cultures of ΦNM1 lysogens carrying the tolerant spacer 32T or 43T CRISPR plasmids, or the pGG3 non-targeting control (C). b, Growth curve of ΦNM1-ErmR lysogens or a non-lysogen control harboring the pGG3 or spacer 43T CRISPR plasmids as indicated, with or without the addition of the prophage-inducing agent mitomycin C (MMC) at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).
Mentions: In order to definitively demonstrate that transcription-dependent targeting offers a biological mechanism for conditional tolerance, we integrated the ΦNM1 target sequence for spacers 43T and 43B into the chromosome of S. aureus RN4220 under the control of a tightly regulated tetracycline-inducible promoter, thus emulating target lysogenization. The target was placed in both orientations with respect to the inducible promoter (Fig. 3c) and with respect to the chromosomal origin of replication (Extended Data Fig. 7a). The resulting strains were then transformed with the spacer 43T or 43B plasmids in a reverse CRISPR immunity assay, and plated in the absence or presence of the inducer. CRISPR immunity was only achieved when transcription across the target was induced with anhydrotetracycline in the presence of an antisense crRNA, regardless of the target’s orientation (Fig. 3d and Extended Data Fig. 7a). Once again, we confirmed this finding to be a type III-specific phenomenon by transforming the strains from Fig. 3c with the spacer 43B-tII type II-A CRISPR plasmid targeting the same region (Extended Data Fig. 7b). We corroborated this result by following the growth of spacer 43T transformants in liquid media (Fig. 3e). Upon addition of the inducer, growth was only inhibited for cells with the target in the forward orientation for which spacer 43T produces an antisense crRNA. Importantly, tolerance achieved in the absence of the inducer did not appear to affect growth (Fig. 3e, dotted lines). Finally, having established that type III-A CRISPR-Cas systems can block lytic infection but tolerate lysogenization, we examined the effect of tolerant spacers on prophage induction of ΦNM1 lysogens in culture. Compared to a spacerless lysogen control, the phage titer resulting from spontaneous induction of overnight cultures was significantly lower for lysogens harboring a tolerant spacer (Fig. 4a). We next followed the growth of cultures induced directly with the DNA-damaging agent, mitomycin C (Fig. 4b, solid lines). While the spacerless lysogen control cultures succumbed to prophage induction, the presence of a tolerant spacer prevented lysis.

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