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Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent

The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunitysystem of prokaryotes against mobile genetic elements using a CRISPR RNA (crRNA)-mediated interference mechanism. InType I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes anR-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complexwhere it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced inhnscells at 37◦C for unknownreasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled (‘runaway’) replication of ColE1plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to testwhether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature inhnsand/orhtpGmutants.We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37◦C, irrespective of the genotype of theanalysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reducedby thehnsmutation at 30◦C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.

CRISPR-Cas ; H-NS ; HtpG ; ColE1 ; temperature ; Escherichia coli

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