RecA status determines SOS- and RecBCD-dependent outcomes in CRISPR–Cas adaptation
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CRISPR–Cas immunity depends on integrating DNA fragments from invading elements, yet how this process is tuned by host physiology remains poorly understood. Here, we investigate the relationship between CRISPR adaptation and the bacterial SOS DNA damage response in Escherichia coli using a highly sensitive colony-based adaptation assay. Blocking SOS with a lexA3 allele suppresses adaptation, while paradoxically, deleting recA enhances it. These findings indicate that RecA contributes positively to adaptation indirectly, through LexA cleavage and induction of SOS-regulated functions, while complete loss of RecA is associated with a RecBCD-dependent DNA-processing state that favours successful adaptation. The RecA inhibitors RecX and PsiB did not phenocopy Δ recA , indicating that inhibitor-mediated perturbation of RecA in RecA-proficient cells and complete loss of RecA have different consequences for adaptation. Genome-wide maps of recovered spacers show that RecA and LexA reshape the relative distribution of chromosomal and plasmid-derived spacers, linking CRISPR adaptation to replication and DNA repair dynamics. Although Cas1–Cas2 catalyses spacer integration autonomously in vitro, our findings suggest that successful adaptation in vivo emerges from interactions between spacer acquisition, DNA repair, and bacterial stress physiology, embedding CRISPR adaptation within cellular networks that balance immune protection with the risk of autoimmunity.
Author summary
Bacteria can protect themselves from viruses using CRISPR systems, which store fragments of viral DNA as a genetic memory of past infections. However, this process is dangerous because the same machinery can accidentally capture fragments of the bacterium’s own DNA, potentially causing self-destruction. How bacteria balance these risks is still poorly understood.
In this study, we examined how CRISPR activity is linked to bacterial DNA damage responses using the model organism Escherichia coli . We used a sensitive colony-based assay that allows rare events in which bacteria acquire new CRISPR memories to be seen directly as small blue outgrowths within bacterial colonies. We found that a major stress-response pathway helps this process succeed, but that deleting one of its central proteins, RecA, unexpectedly increased adaptation.
We also found that stress-response genes influence which parts of the genome are used as sources of new CRISPR memories. Together, our results show that CRISPR immunity is closely connected to broader stress and DNA repair systems inside the cell, rather than operating as an isolated immune mechanism.
