期刊
JOURNAL OF BACTERIOLOGY
卷 194, 期 21, 页码 5728-5738出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.01184-12
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资金
- John H. Copenhaver, Jr., and William H. Thomas fellowship
- NIH [R01A1003256]
- NSF [MCB-9984521]
- CIHR Canada Graduate Scholarship Doctoral Award
- CIHR Emerging Team grant [XNE86943]
- [T32 AI007519]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1158229] Funding Source: National Science Foundation
Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer region of phage DMS3 to make it a target of resistance by the CRISPR/Cas system and screening for mutants that escape CRISPR/Cas-mediated resistance, we show that nucleotides within the PAM and seed sequence and across the non-seed-sequence regions are critical for the functioning of this CRISPR/Cas system. We also demonstrate that P. aeruginosa can acquire spacer content in response to lytic phage challenge, illustrating the adaptive nature of this CRISPR/Cas system. Finally, we demonstrate that the P. aeruginosa CRISPR/Cas system mediates a gradient of resistance to a phage based on the level of complementarity between CRISPR spacer RNA and phage protospacer target. This work introduces a new in vivo system to study CRISPR/Cas-mediated resistance and an additional set of tools for the elucidation of CRISPR/Cas function.
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