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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

Characterization of spacer 32T isolates lysogenized with ΦNM1-ErmRa, ΦNM2 sensitivity assay. Eight randomly selected ΦNM1-ErmR lysogen clones were re-streaked through the indicated ΦNM2-seeded region from top to bottom (1–8); C, sensitive ΦNM1-ErmR lysogen harboring the pGG3 control plasmid. b, PCR amplification of the CRISPR array (upper panel) and spacer 32T target region (lower panel) for the strains tested in a. The pGG3 control lysogen (C) lacks a phage-targeting spacer in its CRISPR array. 1 kb and 0.5 kb size markers are indicated. All 8 PCR products for the target region were sequenced by the Sanger method and no mutations were found (data not shown). c, Plaque-forming potential of filtered supernatants from spacer 32T lysogen overnight cultures inoculated in triplicate. Plaque-forming units (pfu) were enumerated on soft agar lawns of RN4220 harboring either the pGG3 control (C) or spacer 32T CRISPR plasmids. Dotted line represents the limit of detection for this assay. d, ΦNM2 plaquing efficiency on soft agar lawns of an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 32T (9–14); a ΦNM1-ErmR lysogen harboring the pGG3 control plasmid was also tested (−C/L). Plaquing efficiency on the non-lysogenic indicator strain harboring pGG3 is shown for comparison (−C). Error bars: mean ± s.d. (n=3). Panels a and b represent single experiments performed for 8 of 32 isolates.
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Figure 5: Characterization of spacer 32T isolates lysogenized with ΦNM1-ErmRa, ΦNM2 sensitivity assay. Eight randomly selected ΦNM1-ErmR lysogen clones were re-streaked through the indicated ΦNM2-seeded region from top to bottom (1–8); C, sensitive ΦNM1-ErmR lysogen harboring the pGG3 control plasmid. b, PCR amplification of the CRISPR array (upper panel) and spacer 32T target region (lower panel) for the strains tested in a. The pGG3 control lysogen (C) lacks a phage-targeting spacer in its CRISPR array. 1 kb and 0.5 kb size markers are indicated. All 8 PCR products for the target region were sequenced by the Sanger method and no mutations were found (data not shown). c, Plaque-forming potential of filtered supernatants from spacer 32T lysogen overnight cultures inoculated in triplicate. Plaque-forming units (pfu) were enumerated on soft agar lawns of RN4220 harboring either the pGG3 control (C) or spacer 32T CRISPR plasmids. Dotted line represents the limit of detection for this assay. d, ΦNM2 plaquing efficiency on soft agar lawns of an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 32T (9–14); a ΦNM1-ErmR lysogen harboring the pGG3 control plasmid was also tested (−C/L). Plaquing efficiency on the non-lysogenic indicator strain harboring pGG3 is shown for comparison (−C). Error bars: mean ± s.d. (n=3). Panels a and b represent single experiments performed for 8 of 32 isolates.

Mentions: In order to investigate the behavior of type III CRISPR immunity during temperate phage infection, we introduced pGG3, a plasmid carrying the type III-A CRISPR-Cas system of Staphylococcus epidermidis RP62a (ref. 17), into Staphylococcus aureus RN4220 (ref. 18). This strain is sensitive to the lambda-like temperate phages of S. aureus Newman, a clinical isolate harboring four heteroimmune prophages (ΦNM1-4) which carry genes that enhance the pathogenicity of their host19. We also identified a spacer in one of the CRISPR loci of S. aureus MSHR1132 (ref. 20) with near-perfect identity to a conserved target sequence present in ΦNM1 (Fig. 1a), ΦNM2, and ΦNM4. This spacer, referred to as 32T (Supplementary Table 1), was added to the CRISPR locus of pGG3. Using ΦNM1, we first established that this spacer prevents lytic infection by showing that plaquing efficiency is reduced approximately seven orders of magnitude when compared to a strain carrying the pGG3 plasmid without the ΦNM1-targeting spacer (Fig. 1b). We then introduced an erythromycin resistance gene (ermC) into ΦNM1 to facilitate quantification of lysogens which have stably integrated a chromosomal prophage (creating ΦNM1-ErmR). Using this system, we expected to find results consistent with a report describing CRISPR-mediated immunity to lysogenization by phage lambda in E. coli6. Surprisingly, we obtained the same efficiency of lysogenization compared to the control strain lacking spacer 32T (Fig. 1c). To test whether the presence of mismatches between the 32T crRNA and its target was influencing this phenomenon, we engineered spacer 32T* with a perfect match to its target, but obtained the same results (Fig. 1b and c). We next sought to determine whether genetic CRISPR-Cas inactivation is responsible for the apparent tolerance of these lysogens by testing them for sensitivity to ΦNM2. All 14 clones maintained resistance to ΦNM2 mediated by spacer 32T (Extended Data Fig. 1a, d). Finally, we demonstrated that spacer 32T tolerance does not result from genetic alteration of the target phage (Extended Data Fig. 1b, c). Tolerance was also observed for ΦNM4 (Extended Data Fig. 2), demonstrating that the tolerance phenomenon is not specific for the ΦNM1-ErmR phage or its integration locus. These results demonstrate that type III-A CRISPR immunity can block lytic infection but tolerate lysogenization without concomitant genetic CRISPR-Cas inactivation or alteration of the phage genome.


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

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

Characterization of spacer 32T isolates lysogenized with ΦNM1-ErmRa, ΦNM2 sensitivity assay. Eight randomly selected ΦNM1-ErmR lysogen clones were re-streaked through the indicated ΦNM2-seeded region from top to bottom (1–8); C, sensitive ΦNM1-ErmR lysogen harboring the pGG3 control plasmid. b, PCR amplification of the CRISPR array (upper panel) and spacer 32T target region (lower panel) for the strains tested in a. The pGG3 control lysogen (C) lacks a phage-targeting spacer in its CRISPR array. 1 kb and 0.5 kb size markers are indicated. All 8 PCR products for the target region were sequenced by the Sanger method and no mutations were found (data not shown). c, Plaque-forming potential of filtered supernatants from spacer 32T lysogen overnight cultures inoculated in triplicate. Plaque-forming units (pfu) were enumerated on soft agar lawns of RN4220 harboring either the pGG3 control (C) or spacer 32T CRISPR plasmids. Dotted line represents the limit of detection for this assay. d, ΦNM2 plaquing efficiency on soft agar lawns of an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 32T (9–14); a ΦNM1-ErmR lysogen harboring the pGG3 control plasmid was also tested (−C/L). Plaquing efficiency on the non-lysogenic indicator strain harboring pGG3 is shown for comparison (−C). Error bars: mean ± s.d. (n=3). Panels a and b represent single experiments performed for 8 of 32 isolates.
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Related In: Results  -  Collection

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Figure 5: Characterization of spacer 32T isolates lysogenized with ΦNM1-ErmRa, ΦNM2 sensitivity assay. Eight randomly selected ΦNM1-ErmR lysogen clones were re-streaked through the indicated ΦNM2-seeded region from top to bottom (1–8); C, sensitive ΦNM1-ErmR lysogen harboring the pGG3 control plasmid. b, PCR amplification of the CRISPR array (upper panel) and spacer 32T target region (lower panel) for the strains tested in a. The pGG3 control lysogen (C) lacks a phage-targeting spacer in its CRISPR array. 1 kb and 0.5 kb size markers are indicated. All 8 PCR products for the target region were sequenced by the Sanger method and no mutations were found (data not shown). c, Plaque-forming potential of filtered supernatants from spacer 32T lysogen overnight cultures inoculated in triplicate. Plaque-forming units (pfu) were enumerated on soft agar lawns of RN4220 harboring either the pGG3 control (C) or spacer 32T CRISPR plasmids. Dotted line represents the limit of detection for this assay. d, ΦNM2 plaquing efficiency on soft agar lawns of an additional six randomly selected ΦNM1-ErmR lysogen clones isolated during infection of RN4220/spacer 32T (9–14); a ΦNM1-ErmR lysogen harboring the pGG3 control plasmid was also tested (−C/L). Plaquing efficiency on the non-lysogenic indicator strain harboring pGG3 is shown for comparison (−C). Error bars: mean ± s.d. (n=3). Panels a and b represent single experiments performed for 8 of 32 isolates.
Mentions: In order to investigate the behavior of type III CRISPR immunity during temperate phage infection, we introduced pGG3, a plasmid carrying the type III-A CRISPR-Cas system of Staphylococcus epidermidis RP62a (ref. 17), into Staphylococcus aureus RN4220 (ref. 18). This strain is sensitive to the lambda-like temperate phages of S. aureus Newman, a clinical isolate harboring four heteroimmune prophages (ΦNM1-4) which carry genes that enhance the pathogenicity of their host19. We also identified a spacer in one of the CRISPR loci of S. aureus MSHR1132 (ref. 20) with near-perfect identity to a conserved target sequence present in ΦNM1 (Fig. 1a), ΦNM2, and ΦNM4. This spacer, referred to as 32T (Supplementary Table 1), was added to the CRISPR locus of pGG3. Using ΦNM1, we first established that this spacer prevents lytic infection by showing that plaquing efficiency is reduced approximately seven orders of magnitude when compared to a strain carrying the pGG3 plasmid without the ΦNM1-targeting spacer (Fig. 1b). We then introduced an erythromycin resistance gene (ermC) into ΦNM1 to facilitate quantification of lysogens which have stably integrated a chromosomal prophage (creating ΦNM1-ErmR). Using this system, we expected to find results consistent with a report describing CRISPR-mediated immunity to lysogenization by phage lambda in E. coli6. Surprisingly, we obtained the same efficiency of lysogenization compared to the control strain lacking spacer 32T (Fig. 1c). To test whether the presence of mismatches between the 32T crRNA and its target was influencing this phenomenon, we engineered spacer 32T* with a perfect match to its target, but obtained the same results (Fig. 1b and c). We next sought to determine whether genetic CRISPR-Cas inactivation is responsible for the apparent tolerance of these lysogens by testing them for sensitivity to ΦNM2. All 14 clones maintained resistance to ΦNM2 mediated by spacer 32T (Extended Data Fig. 1a, d). Finally, we demonstrated that spacer 32T tolerance does not result from genetic alteration of the target phage (Extended Data Fig. 1b, c). Tolerance was also observed for ΦNM4 (Extended Data Fig. 2), demonstrating that the tolerance phenomenon is not specific for the ΦNM1-ErmR phage or its integration locus. These results demonstrate that type III-A CRISPR immunity can block lytic infection but tolerate lysogenization without concomitant genetic CRISPR-Cas inactivation or alteration of the phage genome.

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