Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity
- Laboratory of Bacteriology, The Rockefeller University, United States
- Howard Hughes Medical Institute, The Rockefeller University, United States
Figures
Figure 1 with 1 supplement
Bacteriophage-resistant colonies display two modes of spacer acquisition, determined by the sequence of the founder spacer.
Agarose gel electrophoresis of the products of the amplification of the pCRISPR array present in staphylococci infected with ϕNM4γ4, using plasmid DNA templates extracted from: (A) liquid cultures …
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Figure 1—source data 1
Spacer sequences from the PCR products shown in Figure 1, Figure 1—figure supplement 1BC, Figure 2B, Figure 3B, Figure 4B–C, Figure 5A–B, Figure 5—figure supplement 1D–G, Figure 7A and Figure 7CD.
- https://cdn.elifesciences.org/articles/53078/elife-53078-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
PCR analysis of the CRISPR array of re-streaked cells.
(A) New spacers integrate into the CRISPR array in a polarized manner, between the leader sequence (gray box, L), an AT-rich sequence that specifies the site of integration, and the first repeat …
Figure 2
CRISPR expansion likely involves priming by the first acquired spacer.
(A) Agarose gel electrophoresis of the products of the amplification of the pCRISPR array present in staphylococci infected in semi-solid media with ϕNM4γ4, using plasmid DNA templates extracted …
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Figure 2—source data 1
Spacer sequences, their number of reads and their position in the phage genome; obtained from next-generation sequencing of the 26 colonies reported in Figure 2B.
- https://cdn.elifesciences.org/articles/53078/elife-53078-fig2-data1-v1.xlsx
Figure 3 with 2 supplements
The ability of phage to escape targeting by the founding spacer determines colony heterogeneity.
(A) Efficiency of plaquing (EOP), calculated as the number of ϕNM4γ4 plaques on the test strain relative to the total number of phage particles in the stock. Different mono-spacer (red) and …
Figure 3—figure supplement 1
Target sequences of different escaper phages.
DNA from 2 to 4 escaper plaques obtained in the experiment described in Figure 3A was isolated and the corresponding target region was amplified with target-specific primers and sequenced as …
Figure 3—figure supplement 2
Quantification of escaper phage within spacer F founder colonies.
(A) Phage was isolates from surviving colonies obtained in the experiment described by Figure 1F and 0-fold serial dilutions were spotted on lawns of sensitive staphylococci [CRISPR(-)], or …
Figure 4
Multi-spacer colonies have impaired growth.
(A) Enumeration of colony forming units (CFU) obtained after infection of staphylococci carrying pCRISPR or pCRISPR(Δcas2) plasmids (clear or dashed pattern bars), containing spacer C or F (red or …
Figure 5 with 2 supplements
Multi-spacer colonies display sectored morphologies.
(A) Image of a smooth colony (containing the founder spacer UU) as well as the gel agarose analysis of PCR products obtained after amplification of its pCRISPR array; ‘c’ shows amplification of …
Figure 5—figure supplement 1
Analysis of additional smooth and sectored colonies.
(A) Image of a plate containing staphylococci carrying pCRISPR that survive ϕNM4γ4 infection in solid media. (B) Image of two smooth colonies (containing the founder spacers AB and AC) from the …
Figure 5—figure supplement 2
Requirement of spacer acquisition for the formation of sectored colonies.
(A) Images of a plate grown for 48 hr after top agar infection of wild-type or Δcas2 mono-spacer founder F cells, with ϕNM4γ4 containing or not additional spacer F escaper phage. (B) Images of …
Figure 6
Host-phage co-evolution within multi-spacer colonies leads to increased resistance.
(A) Resistance score of staphylococci carrying the pCRISPR programmed with founder spacer F to the stock ϕNM4γ4 phage (time 0) or the phages isolated from colonies that survived 24, 36 and 48 hr …
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Figure 6—source data 1
Raw data used for the plots shown in Figure 6.
- https://cdn.elifesciences.org/articles/53078/elife-53078-fig6-data1-v1.xlsx
Figure 7
Analysis of S. thermophilus phage-resistant colonies.
(A) Phage-resistant colonies obtained 24 or 72 hr after infection of S. thermophilus DGCC7710 with ϕ2972. Satellite colonies further analyzed are labeled (α, β and γ). (B) Agarose gel …
Additional files
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Supplementary file 1
List of oligonucleotide primers used in this study.
- https://cdn.elifesciences.org/articles/53078/elife-53078-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53078/elife-53078-transrepform-v1.docx
