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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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Conditional tolerance is achieved via transcription-dependent CRISPR-Cas targetinga, ΦNM1-ErmR lysogenization for additional spacers. C, pGG3 non-targeting control. b, Transformation of ΦNM1-lysogenic competent cells with CRISPR-Cas plasmids containing different spacers (transformation efficiency is measured as cfu/ng of plasmid DNA). C, pGG3 non-targeting control. c, Integration of the 43T/B ΦNM1 target region into the chromosome of S. aureus. Target sequences (inserted in both forward and reverse orientations) are under the control of the tetracycline-inducible promoter Pxyl/tet*. The 43T/B crRNAs are shown annealing to either the top or bottom strands. d, Transformation of both strains shown in c, as well as an isogenic control strain lacking the target insertion, with CRISPR-Cas plasmids containing spacers 43T or 43B. Transformants were plated on selective plates with or without anhydrotetracycline (ATc) for induction of the Pxyl/tet* promoter. e, Growth curve of strains shown in d expressing the spacer 43T CRISPR-Cas system, in the presence or absence of ATc addition at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).
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Figure 3: Conditional tolerance is achieved via transcription-dependent CRISPR-Cas targetinga, ΦNM1-ErmR lysogenization for additional spacers. C, pGG3 non-targeting control. b, Transformation of ΦNM1-lysogenic competent cells with CRISPR-Cas plasmids containing different spacers (transformation efficiency is measured as cfu/ng of plasmid DNA). C, pGG3 non-targeting control. c, Integration of the 43T/B ΦNM1 target region into the chromosome of S. aureus. Target sequences (inserted in both forward and reverse orientations) are under the control of the tetracycline-inducible promoter Pxyl/tet*. The 43T/B crRNAs are shown annealing to either the top or bottom strands. d, Transformation of both strains shown in c, as well as an isogenic control strain lacking the target insertion, with CRISPR-Cas plasmids containing spacers 43T or 43B. Transformants were plated on selective plates with or without anhydrotetracycline (ATc) for induction of the Pxyl/tet* promoter. e, Growth curve of strains shown in d expressing the spacer 43T CRISPR-Cas system, in the presence or absence of ATc addition at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).

Mentions: Given that temperate phages silence transcription of their lytic genes during lysogeny15, we hypothesized that transcription-dependent targeting would allow a variety of otherwise effective spacers to tolerate prophage target sequences. The corollary to this prediction is that targets which are constitutively transcribed during lysogeny (e.g., leftward from the central promoter, Extended Data Fig. 5b) should not be tolerated. Five additional spacers were tested for their ability to tolerate lysogenization by ΦNM1-ErmR (Fig. 3a). As expected, lysogenization was tolerated by spacers 17T, 43T and 61T-1 targeting lytic genes to the right of the central promoter, but not by spacers 2B and 17B targeting genes constitutively expressed leftward during lysogeny. Consistent with the notion that type III-A tolerance results from differences in transcription at the target, we did not observe tolerance with the transcription-independent type II-A CRISPR-Cas system, even when targeting a lytic gene (Extended Data Fig. 4b–h). In order to rule out the possibility that type III-A tolerance is influenced by processes which occur during phage infection, we corroborated these results using a “reverse” CRISPR immunity assay by electroporating CRISPR plasmids into pre-existing lysogens. In this experiment, transformation with a targeting CRISPR-Cas system results in attack of chromosomal prophage sequences and an inability to form colonies14. Again, results varied with the transcriptional context of target sequences in a manner consistent with our previous findings (Fig. 3b). Collectively, these results demonstrate that prophages are not intrinsically tolerated during CRISPR immunity in staphylococci, and suggest rather that type III-A tolerance is only achieved during lysogeny under the condition that transcription is silenced at target sequences.


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

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

Conditional tolerance is achieved via transcription-dependent CRISPR-Cas targetinga, ΦNM1-ErmR lysogenization for additional spacers. C, pGG3 non-targeting control. b, Transformation of ΦNM1-lysogenic competent cells with CRISPR-Cas plasmids containing different spacers (transformation efficiency is measured as cfu/ng of plasmid DNA). C, pGG3 non-targeting control. c, Integration of the 43T/B ΦNM1 target region into the chromosome of S. aureus. Target sequences (inserted in both forward and reverse orientations) are under the control of the tetracycline-inducible promoter Pxyl/tet*. The 43T/B crRNAs are shown annealing to either the top or bottom strands. d, Transformation of both strains shown in c, as well as an isogenic control strain lacking the target insertion, with CRISPR-Cas plasmids containing spacers 43T or 43B. Transformants were plated on selective plates with or without anhydrotetracycline (ATc) for induction of the Pxyl/tet* promoter. e, Growth curve of strains shown in d expressing the spacer 43T CRISPR-Cas system, in the presence or absence of ATc addition 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 3: Conditional tolerance is achieved via transcription-dependent CRISPR-Cas targetinga, ΦNM1-ErmR lysogenization for additional spacers. C, pGG3 non-targeting control. b, Transformation of ΦNM1-lysogenic competent cells with CRISPR-Cas plasmids containing different spacers (transformation efficiency is measured as cfu/ng of plasmid DNA). C, pGG3 non-targeting control. c, Integration of the 43T/B ΦNM1 target region into the chromosome of S. aureus. Target sequences (inserted in both forward and reverse orientations) are under the control of the tetracycline-inducible promoter Pxyl/tet*. The 43T/B crRNAs are shown annealing to either the top or bottom strands. d, Transformation of both strains shown in c, as well as an isogenic control strain lacking the target insertion, with CRISPR-Cas plasmids containing spacers 43T or 43B. Transformants were plated on selective plates with or without anhydrotetracycline (ATc) for induction of the Pxyl/tet* promoter. e, Growth curve of strains shown in d expressing the spacer 43T CRISPR-Cas system, in the presence or absence of ATc addition at the indicated timepoint (black arrow). Error bars: mean ± s.d. (n=3).
Mentions: Given that temperate phages silence transcription of their lytic genes during lysogeny15, we hypothesized that transcription-dependent targeting would allow a variety of otherwise effective spacers to tolerate prophage target sequences. The corollary to this prediction is that targets which are constitutively transcribed during lysogeny (e.g., leftward from the central promoter, Extended Data Fig. 5b) should not be tolerated. Five additional spacers were tested for their ability to tolerate lysogenization by ΦNM1-ErmR (Fig. 3a). As expected, lysogenization was tolerated by spacers 17T, 43T and 61T-1 targeting lytic genes to the right of the central promoter, but not by spacers 2B and 17B targeting genes constitutively expressed leftward during lysogeny. Consistent with the notion that type III-A tolerance results from differences in transcription at the target, we did not observe tolerance with the transcription-independent type II-A CRISPR-Cas system, even when targeting a lytic gene (Extended Data Fig. 4b–h). In order to rule out the possibility that type III-A tolerance is influenced by processes which occur during phage infection, we corroborated these results using a “reverse” CRISPR immunity assay by electroporating CRISPR plasmids into pre-existing lysogens. In this experiment, transformation with a targeting CRISPR-Cas system results in attack of chromosomal prophage sequences and an inability to form colonies14. Again, results varied with the transcriptional context of target sequences in a manner consistent with our previous findings (Fig. 3b). Collectively, these results demonstrate that prophages are not intrinsically tolerated during CRISPR immunity in staphylococci, and suggest rather that type III-A tolerance is only achieved during lysogeny under the condition that transcription is silenced at target sequences.

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