RecA status determines SOS- and RecBCD-dependent outcomes in CRISPR-Cas adaptation

Abstract

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 {triangleup}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.