Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.

CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In Escherichia coli, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use FragSeq and biochemical approaches to determine enzymes involved in generation of defined prespacer ends. We demonstrate that RecJ is the main exonuclease trimming 5' ends of prespacer precursors, although its activity can be partially substituted by ExoVII. The RecBCD complex allows single strand-specific RecJ to process double-stranded regions flanking prespacers. Our results reveal intricate functional interactions of genome maintenance proteins with CRISPR interference and adaptation machineries during generation of prespacers capable of integration into CRISPR arrays.

Bacteriostatic antibiotics promote CRISPR-Cas adaptive immunity by enabling increased spacer acquisition.

Phages impose strong selection on bacteria to evolve resistance against viral predation. Bacteria can rapidly evolve phage resistance via receptor mutation or using their CRISPR-Cas adaptive immune systems. Acquisition of CRISPR immunity relies on the insertion of a phage-derived sequence into CRISPR arrays in the bacterial genome. Using Pseudomonas aeruginosa and its phage DMS3vir as a model, we demonstrate that conditions that reduce bacterial growth rates, such as exposure to bacteriostatic antibiotics (which inhibit cell growth without killing), promote the evolution of CRISPR immunity. We demonstrate that this is due to slower phage development under these conditions, which provides more time for cells to acquire phage-derived sequences and mount an immune response. Our data reveal that the speed of phage development is a key determinant of the evolution of CRISPR immunity and suggest that use of bacteriostatic antibiotics can trigger elevated levels of CRISPR immunity in human-associated and natural environments.

Genome Maintenance Proteins Modulate Autoimmunity Mediated Primed Adaptation by the Escherichia coli Type I-E CRISPR-Cas System.

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.