title: The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Cady
authors.forename: Joe
authors.initials: J
authors.lastname: Bondy-Denomy
authors.forename: Gary E
authors.initials: GE
authors.lastname: Heussler
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
authors.forename: George A
authors.initials: GA
authors.lastname: O'Toole
mesh_terms: D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D017105:Pseudomonas Phages / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000502:physiology* / Q000821:virology*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014779:Virus Replication
chemical_list: D004279:DNA, Viral