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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

Immunity to ΦNM1γ6 in liquid culture is unaffected by the presence of a tolerated chromosomal targetGrowth curves of the indicated chromosomal insertion strains from Fig. 3 harboring either spacer 43T or pGG3 CRISPR plasmids, in the absence (dotted lines) or presence (solid lines) of ΦNM1γ6 addition at a MOI of 10. Black arrow denotes the time of phage addition; no ATc induction is utilized in this assay. The presence of a chromosomal target for spacer 43T has no discernable effect on culture growth during spacer 43T-mediated immunity to ΦNM1γ6 (compare solid green and blue lines).
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Figure 13: Immunity to ΦNM1γ6 in liquid culture is unaffected by the presence of a tolerated chromosomal targetGrowth curves of the indicated chromosomal insertion strains from Fig. 3 harboring either spacer 43T or pGG3 CRISPR plasmids, in the absence (dotted lines) or presence (solid lines) of ΦNM1γ6 addition at a MOI of 10. Black arrow denotes the time of phage addition; no ATc induction is utilized in this assay. The presence of a chromosomal target for spacer 43T has no discernable effect on culture growth during spacer 43T-mediated immunity to ΦNM1γ6 (compare solid green and blue lines).

Mentions: The requirement for transcription across target sequences during type III-A immunity contrasts with the transcription-independent targeting reported for type I (ref. 27) and type II (ref. 22,23) CRISPR-Cas systems. Given the temporal pattern of target transcription observed during the phage lytic cycle, it might be expected that CRISPR targeting of late genes would not provide immunity if the cell’s survival is already compromised at the onset of targeting. Indeed, we observed some differences in spacer effectiveness when infecting cells in liquid culture at very high MOI (~100): spacers targeting late genes were less protective (Extended Data Fig. 8). However, this effect was not pronounced at a MOI of 10, suggesting that the system is generally robust to delays in target transcription, in accordance with what we observed in efficiency of plaquing assays. Consistent with our findings at large, our survey of sequenced staphylococcal type III spacers showed that naturally acquired spacers with known target sequences produced crRNAs complementary to the non-template strand of predicted ORFs in 9/10 cases (Supplementary Table 2). This bias suggests negative selection of non-functional spacers targeting template strands. Alternatively, type III systems may utilize an unknown mechanism to discriminate template and non-template strands during spacer acquisition. The molecular details of the transcription-dependent targeting mechanism remain unclear. Preliminary experiments indicate that the presence of a transcript provided in trans is not sufficient to license DNA targeting (Extended Data Fig. 9). Consistent with this, induction of transcription across a plasmid-borne target results specifically in loss of the targeted plasmid (Extended Data Fig. 10). Hence, transcription in cis is probably required for DNA targeting. One possibility is that negative supercoiling generated in the wake of a passing transcription bubble could facilitate target DNA melting and improve crRNA recognition27. This hypothesis, however, would not account for the template and non-template strand asymmetry observed in our system. Another possibility is that exposure of the target non-template strand within the transcription bubble is required for annealing of a crRNA21. In this scenario, the observed asymmetry might be explained by occlusion of base-pairing to the template strand by either the nascent transcript or the RNA polymerase28. However, given the small size of the transcription bubble, this explanation may also be incomplete. Alternatively, transcription may be required to activate a targeting mechanism rather than facilitating target recognition or binding per se. In this case, effective targeting could require base pairing potential between the crRNA and the nascent transcript in cis, which would be absent for crRNAs with complementarity to the template strand. Previous reports29,30 have revealed specific RNA target cleavage activity for type III(−B/C) systems that could be relevant to the transcription-dependent DNA targeting observed in staphlyococci. Further experimentation will be needed to clarify these points. In summary, our work expands the repertoire of CRISPR-based immune functions to include a novel capacity for conditional tolerance of foreign elements, and establishes distinct genetic outcomes resulting from immunity to temperate phages via divergent CRISPR-Cas targeting mechanisms.


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

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

Immunity to ΦNM1γ6 in liquid culture is unaffected by the presence of a tolerated chromosomal targetGrowth curves of the indicated chromosomal insertion strains from Fig. 3 harboring either spacer 43T or pGG3 CRISPR plasmids, in the absence (dotted lines) or presence (solid lines) of ΦNM1γ6 addition at a MOI of 10. Black arrow denotes the time of phage addition; no ATc induction is utilized in this assay. The presence of a chromosomal target for spacer 43T has no discernable effect on culture growth during spacer 43T-mediated immunity to ΦNM1γ6 (compare solid green and blue lines).
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Related In: Results  -  Collection

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Figure 13: Immunity to ΦNM1γ6 in liquid culture is unaffected by the presence of a tolerated chromosomal targetGrowth curves of the indicated chromosomal insertion strains from Fig. 3 harboring either spacer 43T or pGG3 CRISPR plasmids, in the absence (dotted lines) or presence (solid lines) of ΦNM1γ6 addition at a MOI of 10. Black arrow denotes the time of phage addition; no ATc induction is utilized in this assay. The presence of a chromosomal target for spacer 43T has no discernable effect on culture growth during spacer 43T-mediated immunity to ΦNM1γ6 (compare solid green and blue lines).
Mentions: The requirement for transcription across target sequences during type III-A immunity contrasts with the transcription-independent targeting reported for type I (ref. 27) and type II (ref. 22,23) CRISPR-Cas systems. Given the temporal pattern of target transcription observed during the phage lytic cycle, it might be expected that CRISPR targeting of late genes would not provide immunity if the cell’s survival is already compromised at the onset of targeting. Indeed, we observed some differences in spacer effectiveness when infecting cells in liquid culture at very high MOI (~100): spacers targeting late genes were less protective (Extended Data Fig. 8). However, this effect was not pronounced at a MOI of 10, suggesting that the system is generally robust to delays in target transcription, in accordance with what we observed in efficiency of plaquing assays. Consistent with our findings at large, our survey of sequenced staphylococcal type III spacers showed that naturally acquired spacers with known target sequences produced crRNAs complementary to the non-template strand of predicted ORFs in 9/10 cases (Supplementary Table 2). This bias suggests negative selection of non-functional spacers targeting template strands. Alternatively, type III systems may utilize an unknown mechanism to discriminate template and non-template strands during spacer acquisition. The molecular details of the transcription-dependent targeting mechanism remain unclear. Preliminary experiments indicate that the presence of a transcript provided in trans is not sufficient to license DNA targeting (Extended Data Fig. 9). Consistent with this, induction of transcription across a plasmid-borne target results specifically in loss of the targeted plasmid (Extended Data Fig. 10). Hence, transcription in cis is probably required for DNA targeting. One possibility is that negative supercoiling generated in the wake of a passing transcription bubble could facilitate target DNA melting and improve crRNA recognition27. This hypothesis, however, would not account for the template and non-template strand asymmetry observed in our system. Another possibility is that exposure of the target non-template strand within the transcription bubble is required for annealing of a crRNA21. In this scenario, the observed asymmetry might be explained by occlusion of base-pairing to the template strand by either the nascent transcript or the RNA polymerase28. However, given the small size of the transcription bubble, this explanation may also be incomplete. Alternatively, transcription may be required to activate a targeting mechanism rather than facilitating target recognition or binding per se. In this case, effective targeting could require base pairing potential between the crRNA and the nascent transcript in cis, which would be absent for crRNAs with complementarity to the template strand. Previous reports29,30 have revealed specific RNA target cleavage activity for type III(−B/C) systems that could be relevant to the transcription-dependent DNA targeting observed in staphlyococci. Further experimentation will be needed to clarify these points. In summary, our work expands the repertoire of CRISPR-based immune functions to include a novel capacity for conditional tolerance of foreign elements, and establishes distinct genetic outcomes resulting from immunity to temperate phages via divergent CRISPR-Cas targeting mechanisms.

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