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

Abstract

The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a CRISPR RNA (crRNA)-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled (‘runaway’) replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.

Cas3 Protein—A Review of a Multi-Tasking Machine

“…This unusual property of Cas3 to stimulate uncontrolled plasmid replication has been shown to be temperature-dependent, increased at 37 • C and decreased at 30 • C (Figure 3). Reduced or inhibited ATPase/helicase activity of Cas3 at 30 • C was found responsible for this effect, and ∆hns mutation was found to indirectly decrease plasmid copy number at 30 • C [78].…”

Synchrotron Radiation Circular Dichroism, a New Tool to Probe Interactions between Nucleic Acids Involved in the Control of ColE1-Type Plasmid Replication

“…This so-called runaway replication causes non-controlled overproduction of plasmid DNA in cells leading to adverse effects, including cell death [51]. Such a phenomenon was also observed in E. coli cells bearing ColE1-type plasmids, though the final effect was a decreased viability of bacteria, from about 2-fold [52] to about 10-fold [53], rather than the complete elimination of living cells. Therefore, we suggest that less efficient negative control of plasmid replication initiation by Hfq, observed in E. coli hfq mutants [17,18], might result in runaway replication of ColE1-like plasmid and impaired viability of transformed bacteria.…”