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

Abstract

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.

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

“…In agreement with our previous study (34), compared to the wildtype self-targeting strain (wt), we observed a decrease in primed adaptation efficiency by ~90% in recJ and by ~99% in the recB recJ derivative strains. We also detected a less prominent ~36% decrease in the ∆recD strain (which retains helicase but not nuclease activity) and ~70% decreases in both recB and recC strains (which lack both activities) (fig.…”

“…S1, A and B, and tables S2 and S3). In the previous work (34), no differences in primed adaptation efficiency between the wt and either ∆recD, ∆recB, or ∆recC strains were observed. This discrepancy is likely explained by a more sensitive and quantitative method used to detect spacer acquisition in the current work (high-throughput sequencing versus quantification of agarose gel band intensity used previously).…”

“…Raw sequencing data obtained in this study are available in Sequence Read Archive (BioProject Accession: PRJNA862875). Spacer alignments are from Kurilovich et al (34)…”

“…To study primed adaptation in the type I-E system of E. coli, we previously developed an inducible self-targeting experimental system, in which a spacer matching a sequence elsewhere in the genome is inserted into the CRISPR array ( 24 ). In a separate study, we demonstrated that inactivation of the host single strand–specific 5′→3′ exonuclease RecJ alone or especially together with RecBCD helicase-nuclease severely decreased primed adaptation efficiency in self-targeting cells ( 34 ). Here, we study the effect of various E. coli exonucleases on generation of prespacers using the self-targeting model and in vitro approaches.…”

Prespacers formed during primed adaptation associate with the Cas1–Cas2 adaptation complex and the Cas3 interference nuclease–helicase

“…The initial Cas3 cleavage in the nontarget strand of PPS generates a 3′ end ( 50 ). Next, Cas3 moves along a nicked strand and directs Cas1–Cas2 to the unwound target strand to seek 3′-TTC-5′ (AAG PAM complement) sequences that are processed by cellular nucleases ( 30 , 60 ) with the formation of ∼33 nt fragments associated with 5′-AAG-3′ PAM and complementary ∼37 nt fragments containing single-stranded 3′-NNTTC-5′ extensions. Complementary ∼33/∼37 fragments form partially double-stranded DNA prespacers that stay bound to Cas1/Cas2 and are delivered to the CRISPR array.…”