Dy, R. L. V. | bioRxiv Science

bioRxiv2026

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

CRISPR-Cas systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the 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. PA10145 possesses a type I-F CRISPR-Cas composed of a cas operon flanked by two divergently organized CRISPRs. The isolated CRISPR array, CRISPR3, is located [~]1.3 million bp away from the cas loci. The cas and three CRISPR arrays are active. Plasmids with an engineered protospacer matching any of the three arrays were targeted and stimulated hyperactive adaptation in all CRISPR arrays of PA10145 if the plasmids possessed an intact protospacer adjacent motif (PAM), whereas minimal to no adaptation was observed when PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and the cas-adjacent CRISPR2 have nearly identical leader sequences with only 3 bp mismatches. From a survey of CRISPR loci in 1,198 P. aeruginosa genomes, isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Highly-transmissible mobile genetic elements (MGEs) associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these MGEs. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas-adjacent arrays revealed that CRISPR3 is laterally 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.

microbiology↗

bioRxiv2025

Random guide-independent DNA cleavage from the Argonaute of Exiguobacterium sp. AB2

BackgroundBacteria and bacteriophages (phages) are locked in a coevolutionary "arms race" to outcompete one another with novel systems and strategies. Regularly outnumbered tenfold by phages, bacteria have responded to the constant threat of phage predation by evolving a vast array of sophisticated defense systems. Among these, prokaryotic Argonautes (pAgos) are nucleic acid-guided endonucleases that target complementary sequences of invading mobile genetic elements (MGEs). However, as the preference for targeting MGE sequences has been demonstrated in only a limited number of pAgos, their precise physiological functions remain elusive. Here, we discovered a pAgo in Exiguobacterium sp. AB2, EsAgo, encoded in close proximity to other putative defense systems on the E. AB2 genome. Such clustering into genomic "defense islands" is a common phenomenon among prokaryotic defense systems, further implicating pAgos with a role in host defense. Accordingly, we had sought to characterize EsAgo as a nucleic acid-guided nucleic acid-targeting nuclease against MGEs for bacterial defense in this study. ResultsUsing sequence to structure homology tools, we show that the predicted model of EsAgo exhibits the structural characteristics typical of a full-length, catalytically active, DNA-guided pAgo. Akin to other pAgos, EsAgo uses a divalent cation cofactor to indiscriminately "chop" plasmids in vitro. Furthermore, a site-directed double mutant of EsAgo bearing two missense mutations at the catalytic site exhibited significantly reduced levels of this random plasmid-degrading activity. Lastly, when EsAgo was supplied with synthetic 5-P ssDNA guides, random nuclease activity was attenuated and may have resulted in the sequence-specific cleavage of dsDNA at 37{degrees}C. ConclusionsThese findings suggest that EsAgo functions as a DNA-interfering nuclease with or without DNA guides. Within the cell, it is possible that EsAgo utilizes this mechanism to screen and destroy foreign genetic elements. Moreover, the potential capacity for specific dsDNA cleavage at moderate temperatures gives rise to intriguing possibilities of repurposing EsAgo as a programmable nuclease for future biotechnological use.

microbiology↗