Oxidative stress elicited by phage infection induces Staphylococcal type III-A CRISPR–Cas system

Figures

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  Figure 1.. The cas1 gene sequence is essential for type III-A CRISPR–Cas immunity against phage infection and plasmid invasion. (A) Schematic of the type III-A CRISPR–Cas system in S. aureus TZ0912. The promoter of CRISPR1, CRISPR2, and cas genes are indicated as arrows; n = number of spacers. (B) Schematic of the pMini-CRISPR construct. The pMini-CRISPR plasmid carries the CRISPR1 leader and two repeats interrupted with a spacer targeting phage genes. (C) A 10-fold serial dilution of phage phiIPLA-RODI was spotted on lawns of S. aureus WT and mutant strains all carrying the pMini-CRISPR plasmid that expresses an additional crRNA for enhanced immunity. Plaquing experiments were replicated three times with similar results and representative data are shown. (D) Retention of the untargeted plasmid pRMC2 and targeted plasmid pCR1SP1 in the WT and mutant strains. The strains containing the untargeted or targeted plasmid were grown in TSB with ATc to induce transcription of the protospacer for 10 h followed by plating on TSA with or without chloramphenicol. % CFUs containing plasmid was scored as the number of CmR CFU/the total number of CFU. All data are means ± standard deviation of three…

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  Figure 2.. The cas1 gene sequence is crucial for cas10–csm genes expression. (A) RNA-seq reads were mapped to the WT, Δcas1, Δcas2, and ΔPcas genome to determine the relative abundance of cas10–csm gene expression by IGV software. (B) qRT-PCR analysis of cas10 and csm3 expression in the WT, Δcas1, and Δcas2 mutant strains, respectively. (C) Western blot and densitometry analysis of Csm3 protein level in WT, Δcas1, and Δcas2 mutant strains. The expression level of Csm3 was determined using that of RpoB as the internal control. (D) The nucleotide sequence of the Pcas promoter. Transcriptional start site predicted by RNA-seq is shaded with yellow boxes. The -10 and -35 regions are shaded with green boxes. Nucleotides highlighted in red were subjected to point mutations. (E) As in panel (C), but in WT and ΔPcas mutant strains. (F) qRT-PCR analysis of Pcas, cas10, and csm3 in the WT infected by phage phiIPLA-RODI for 4 h. All data are means ± standard deviation of three independent experiments. A two-tailed unpaired Student’s t-test was used to calculate P-values; *P< 0.05, ***P< 0.001; ns, not significant.

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  Figure 3.. Pcas function is divergent among Staphylococcus type III-A CRISPR–Cas systems. (Aand D) qRT-PCR analysis of cas10 and csm3 in the WT and ΔPcas mutant from S. aureus 08BA02186 or S. argenteus CQ0721. (B and E) Retention of the untargeted plasmid pRMC2 and targeted plasmid pCR1SP1 in the WT and ΔPcas mutant from S. aureus 08BA02186 or S. argenteus CQ0721. % CFUs containing plasmid was scored as the number of CmR CFU/the total number of CFU. (Cand F) A tenfold serial dilution of phage phiIPLA-RODI was spotted on lawns of the WT and ΔPcas mutant from S. aureus 08BA02186 or S. argenteus CQ0721 strains carrying the gp036 target pCRISPR plasmid. Plaquing experiments were replicated three times with similar results and representative data are shown. (G) qRT-PCR analysis of cas10 in the WT, ΔPcas and seven mutants containing different Pcas point mutations. (H) qRT-PCR analysis of csm3 in the WT, ΔPcas, and seven mutants containing different Pcas point mutations. (I) Western blot and densitometry analysis of Csm3 protein level in the WT, ΔPcas, and seven mutants containing different Pcas point mutations. The expression level of Csm3 was determined using that of RpoB as the internal…

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  Figure 4.. MgrA directly represses type III-A CRISPR–Cas system by binding to Pcas promoter. (A) qRT-PCR analysis of cas10 and csm3 expression in the WT and ΔmgrA strains, respectively. (B) EMSA of the Pcas promoter with the indicated concentration of purified MgrA protein. DNA–protein complexes are indicated by an asterisk. (C) Retention of the untargeted plasmid pRMC2 and targeted plasmid pCR1SP1 in the WT and ΔmgrA strains. The strains containing the untargeted or targeted plasmid were grown in TSB with ATc to induce transcription of the protospacer for 10 h followed by plating on TSA with or without chloramphenicol. % CFUs containing plasmid was scored as the number of CmR CFU/the total number of CFU. (D) Growth curve of WT and ΔmgrA strains either uninfected or infected with phage phiIPLA-RODI at a MOI of 0.01. (E) Enumeration of PFU from cultures shown in panel (D), 0, 4, and 8 h after infection. (F) Generation of ROS in WT infected with or without phage phiIPLA-RODI. The PBS buffer was used as a blank control. (G and H) Competitive EMSAs. Pcas promoter was incubated with an increasing purified MgrA (G) or MgrAC12S (H) protein in the absence or presence of H2O2. DNA–protein complexes…

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  Figure 5.. Model of the MgrA-mediated regulation of type III-A CRISPR–Cas immunity. In S. aureus type III-A CRISPR–Cas system, cas1 harbors an internal Pcas promoter, which drives the transcription of cas10-csm genes encoding the interference complex. This promoter is repressed by MgrA in the absence of phage infection, facilitating the acquisition of MGEs and preventingautoimmunity. However, phage infection can induce ROS production, which in turn oxidizes MgrA, causing its dissociation from the Pcas promoter. This release of MgrA-mediated inhibition restores CRISPR–Cas activity, thereby enhancing the defense against phage infection.