Skip to main content
Dryad logo

Strains and constructs for: A chimeric nuclease substitutes a phage CRISPR-Cas system to provide sequence specific immunity against subviral parasites

Citation

Barth, Zachary; Seed, Kimberley (2021), Strains and constructs for: A chimeric nuclease substitutes a phage CRISPR-Cas system to provide sequence specific immunity against subviral parasites, Dryad, Dataset, https://doi.org/10.6078/D1T704

Abstract

Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their hosts fitness. The PLE is a mobile element that integrates into the chromosome of Vibrio cholerae and parasitizes the bacteriophage ICP1 to move between cells. This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA binding domain that is highly similar to some PLE replication origin binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.

Methods

These files represent strains and constructs cloned and assayed for the production of the associated manuscript. Primer sequences are annotated within each file. Construct sequence was validated through Sanger sequencing.

Usage Notes

Files are named for the constructs they correspond to. Strains that contain each construct appear in parentheses after contruct name.

Funding

National Institute of Allergy and Infectious Diseases, Award: R01AI127652

National Institute of Allergy and Infectious Diseases, Award: R01AI153303