Acquisition of novel arrays via horizontal gene transfer rewires CRISPR-mediated defense in Pseudomonas aeruginosa

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the type I-F CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array unique to this system. Its core CRISPR-Cas system is composed of a cas operon flanked by two divergently organized arrays: CRISPR2 and CRISPR1. Meanwhile, the isolated CRISPR array, CRISPR3, was found ~1.3 million bp away from cas . The cas and three CRISPR arrays together function toward adaptive immunity, eliminating plasmids engineered with protospacer targets. If plasmids possessed an intact protospacer adjacent motif (PAM), hyperactive adaptation was stimulated in all CRISPR arrays of PA10145, whereas minimal to no adaptation was observed when the PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and cas -adjacent CRISPR2 have nearly identical leader sequences, with only 3 bp mismatches. Of the 1,196 P. aeruginosa genomes analyzed, all 281 isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Integrative conjugative elements and prophages only associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these highly transmissible mobile genetic elements. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas -adjacent arrays revealed that CRISPR3 is horizontally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories. IMPORTANCE Clustered regularly interspaced short palindromic repeat (CRISPR) arrays are typically located adjacent to cas genes, constituting the adaptive immune system. However, a substantial subset of CRISPR arrays, called isolated arrays, is found distantly located from their cognate cas operons. Despite their prevalence, the functional significance and evolutionary implications of isolated arrays have remained poorly understood. Here, we show that isolated arrays are active components of type I-F CRISPR-Cas systems in Pseudomonas aeruginosa . These arrays are continuously updated through hyperactive strand-biased primed adaptation and are transferred horizontally across P. aeruginosa , acting as conduits of immunological memory and proponents of CRISPR-mediated pan-immunity. We posit that bacterial populations mobilize these isolated arrays to maintain transferable memory in the pangenome while remaining compatible with ongoing horizontal gene transfer.