Hyper-stimulation of Pyrococcus furiosus CRISPR DNA uptake by a self-transmissible plasmid

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Pyrococcus furiosus possesses three distinct CRISPR-Cas effector complexes (Type I-A, I-B, III-B).
• These complexes utilize CRISPR RNA (crRNA) guides derived from seven CRISPR arrays for adaptive immunity.
• CRISPR adaptation involves integrating new DNA sequences (spacers) into CRISPR arrays to recognize foreign genetic elements.

Purpose of the Study:

• To investigate the CRISPR adaptation response of Pyrococcus furiosus to a novel, self-transmissible plasmid, pT33.3.
• To elucidate the mechanisms underlying enhanced spacer integration observed in response to this plasmid.
• To determine the role of specific CRISPR-Cas systems and pre-existing spacers in this adaptation process.

Main Methods:

• Generating transconjugant strains of Pyrococcus furiosus harboring the pT33.3 plasmid.
• Employing high-throughput sequencing to analyze spacer acquisition in CRISPR arrays.
• Comparing spacer integration patterns between strains exposed to the novel plasmid and a laboratory control plasmid.
• Investigating the dependence of the adaptation phenotype on specific CRISPR arrays and effector complexes.

Main Results:

• Transconjugant strains showed significantly higher new spacer integration levels compared to control strains.
• Newly acquired spacers were preferentially derived from a specific region of the pT33.3 plasmid.
• Spacer acquisition exhibited a bi-directional strand bias, characteristic of Type I CRISPR systems' primed adaptation.
• A single pre-existing spacer in a CRISPR array was crucial for targeting the plasmid, alongside the I-B effector complex.

Conclusions:

• Pyrococcus furiosus exhibits a robust, primed adaptation response to the conjugative plasmid pT33.3.
• This response is dependent on a specific matching spacer and the Type I-B CRISPR effector complex.
• The findings suggest a history of natural encounters between Pyrococcus furiosus and this plasmid or related mobile genetic elements, leading to an enhanced defense mechanism.