Characterising Host Factors Required for CRISPR Cas Adaptation in Escherichia coli

Edwards, Harry (2023) Characterising Host Factors Required for CRISPR Cas Adaptation in Escherichia coli. PhD thesis, University of Nottingham.

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The evolution of diverse prokaryotic immune systems has been driven by the constant threat of invasion by foreign genetic elements. Many prokaryotes contain the CRISPR-Cas system, which provides adaptive immunity through the acquisition of unique spacer sequences into the CRISPR array. These sequences guide the effector complexes to destroy the complementary invading genetic elements.

The function of the Cas proteins is well understood, due to their proximity to the CRISPR array. Other host factors have been discovered to play a role in spacer acquisition, such as IHF and DnaQ. However, aspects of spacer capture and integration by the Cas1 and Cas2 proteins remain elusive. This work aimed to determine and presents evidence for a broader collection of host factors required for CRISPR Cas adaptation in E. coli.

I demonstrate that the catalytic activity of the Cas1-Cas2 complex triggers activation of the SOS response and induces filamentation by sulA expression. This is due to non-specific activity across the genome, not just at the CRISPR array. Use of the lexA3 allele and overexpression of recX, which prevent induction of the SOS response, decreases the rate of spacer acquisition. Furthermore, induction of the SOS response, via growth in sub-inhibitory antibiotic concentrations, increases the rate of adaptation.

I also provide evidence for an equilibrium between the FtsH HflKC and FtsH-LapB complexes governing the level of adaptation. The deletion of hflC or hflK and overexpression of lapB decreases the rate of spacer acquisition, whilst overexpression of hflKC or ftsH increases the rate of adaptation. Furthermore, mutation of negative regulators of lapB reduces the rate of spacer acquisition. This suggests that HflKC and LapB compete to form complexes with FtsH, with the formation of FtsHHflKC complexes promoting adaptation.

Taken together, these results reveal the link between cell division and spacer acquisition.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Chalmers, Ronald
Keywords: immune systems, CRISPR-Cas, prokaryotes, bacteriophages
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 73413
Depositing User: Edwards, Mr Harry
Date Deposited: 31 Jul 2023 04:40
Last Modified: 31 Jul 2023 04:40

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