Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3\' overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3\'-5\' exonucleases DnaQ and ExoT can trim long 3\' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3\'-5\' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

Bacteria and archaea use CRISPR-Cas adaptive immunity systems to interfere with viruses, plasmids, and other mobile genetic elements. During the process of adaptation, CRISPR-Cas systems acquire immunity by incorporating short fragments of invaders\' genomes into CRISPR arrays. The acquisition of fragments of host genomes leads to autoimmunity and may drive chromosomal rearrangements, negative cell selection, and influence bacterial evolution. In this study, we investigated the role of proteins involved in genome stability maintenance in spacer acquisition by the Escherichia coli type I-E CRISPR-Cas system targeting its own genome. We show here, that the deletion of recJ decreases adaptation efficiency and affects accuracy of spacers incorporation into CRISPR array. Primed adaptation efficiency is also dramatically inhibited in double mutants lacking recB and sbcD but not in single mutants suggesting independent involvement and redundancy of RecBCD and SbcCD pathways in spacer acquisition. While the presence of at least one of two complexes is crucial for efficient primed adaptation, RecBCD and SbcCD affect the pattern of acquired spacers. Overall, our data suggest distinct roles of the RecBCD and SbcCD complexes and of RecJ in spacer precursor selection and insertion into CRISPR array and highlight the functional interplay between CRISPR-Cas systems and host genome maintenance mechanisms.

CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a \"directionality\" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency of most systems against bona fide targets. As such, most primed adaptation to date has been studied within the context of imperfect sequence complementarity between spacers and targets. Here, we take advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust primed adaptation even within the context of a perfectly matched target. Using next-generation sequencing to survey acquired spacers, we observe strand bias and positional preference that are consistent with a 3\'-5\' translocation of the adaptation machinery. We show that spacer acquisition happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that predominates irrespective of the priming strand. We systematically characterize protospacer sequence constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we observe increased interference efficiency, which, when coupled with forced maintenance of a targeted plasmid, provides a useful experimental system to study spacer loss. Based on these observations, we propose that the Legionella pneumophila type I-C system represents a powerful model to study primed adaptation and the interplay between CRISPR interference and adaptation.

Prokaryotic type I CRISPR-Cas systems respond to the presence of mobile genetic elements such as plasmids and phages in two different ways. CRISPR interference efficiently destroys foreign DNA harboring protospacers fully matching CRISPR RNA spacers. In contrast, even a single mismatch between a spacer and a protospacer can render CRISPR interference ineffective but causes primed adaptation-efficient and specific acquisition of additional spacers from foreign DNA into the CRISPR array of the host. It has been proposed that the interference and primed adaptation pathways are mediated by structurally different complexes formed by the effector Cascade complex on matching and mismatched protospacers. Here, we present experimental evidence and present a simple mathematical model that shows that when plasmid copy number maintenance/phage genome replication is taken into account, the two apparently different outcomes of the CRISPR-Cas response can be accounted for by just one kind of effector complex on both targets. The results underscore the importance of consideration of targeted genome biology when considering consequences of CRISPR-Cas systems action.