engleski

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

The CRISPR-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a crRNA-mediated interference mechanism. In Type IE CRISPR-Cas systems crRNA guided by a Cascade complex recognizes the matching target DNA and promotes R-loop formation, an RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. In E. coli, the CRISPR-Cas immunity against  phage was shown to be temperature dependent if CRISPR-Cas components were expressed from native promoters in cells lacking H-NS, the repressor of CRISPR locus and cas genes. Cas3 protein can also promote uncontrolled replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37 °C independently of the cell’s genotype. There was no increase of plasmid yields at 30 °C and similar results were observed with a helicase mutant at both temperatures. We also found that plasmid replication was strongly reduced by the hns mutation at 30 °C. Overall, our findings suggest that increased replication of ColE1 plasmids depends on change in ATPase/helicase activity of Cas3 protein which depends on the temperature of incubation.

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

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