An Endogenous Staphylococcus aureus CRISPR-Cas System Limits Phage Proliferation and Is Efficiently Excised from the Genome as Part of the SCC mec Cassette

Figures

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  FIG 1. Overview of the type III-A CRISPR-Cas systems examined in S. aureus. (a) Graphic representation of the type III-A CRISPR-Cas system, including leader sequences (yellow), cas and csm genes (red), and CRISPR array 1 (green/gray) and 2 (brown/gray). (b) Spacer content in CRISPR array 1 and 2 in 69 Danish clinical isolates and 1 reference strain (08BA02176). Green and brown squares symbolize the presence of the spacer in CRISPR array 1 and 2, respectively. STs are designated by color, and the number of isolates that carry identical CRISPR arrays is indicated in bold (the absence of a number indicates 1 strain). The spacers are presented in the order in which they occur in the array.

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  FIG 2. The SCCmec type V(5C2&5), harboring type III-A CRISPR-Cas, is shared between staphylococcal species. (a) Phylogenetic core genome SNP tree based on hits from a BLASTn search of the cas gene sequence (cas1 to cas6) of strain 110900. cas gene percent identity, SCC subtype, and numbers of S. aureus spacers are indicated at the bottom. a, sum of spacers homologous to the S. aureus spacers (1.1 to 2.3) found in staphylococcal isolates. (b) Graphic presentation of SCCmec type V(5C2&5) with the highly conserved region (gray shading).

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  FIG 3. Excision of SCCmec. (a) The SCCmec type V(5C2&5) of S. aureus strain 1109000 with predicted circularizable fragments. The primers and location of DRs are shown below the SCCmec. (b) Excision frequencies of the 38- and 59-kb SCCmec fragments compared to chromosomal DNA quantities (n = 3). The primer pairs criF/circR and arsF/circR were used for detecting the 38-kb and 59-kb fragments, respectively, as shown in panel a. (c) Alignment of DR1 and DR2, with mismatches highlighted in bold and underlined in the DR2 sequence.

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  FIG 4. CRISPR-Cas activity against phage phiIPLA-RODI during liquid infection. Cultures (OD600, 0.15) of 110900 WT (green) or the ΔCRISPR mutant (brown) were grown with (closed symbols) or without (open symbols) increasing MOIs of phiIPLA-RODI phage in 1:1 TSB-phage buffer, with OD600 being monitored every 20 min for 24 h (n = 3; 5 technical replicates).

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  FIG 5. CRISPR-Cas reduces phiIPLA-RODI burst size. (a) One-step growth curves of phiIPLA-RODI using WT 110900 or the ΔCRISPR mutant as a propagative strain determined from 5 biological replicates (standard errors of the means [SEM] are shown) and compared with a paired two-tailed t test (****, P < 0.0001). The nonlinear regression sigmoidal curves were fitted using GraphPad Prism with least-squares regression. Burst size was calculated as the top plateau average divided by the bottom plateau average. (b) Plaque formation of phiIPLA-RODI using either WT 110900 or the ΔCRISPR mutant as the recipient. Data are mean log transformed PFU per milliliter for 4 biological replicates, with SD. **, P = 0.0087 (unpaired t test). The EOP of phiIPLA-RODI on 110900 WT was 29% of that on the ΔCRISPR mutant.