Abstract
Bacteria fight a constant battle against their parasites – bacteriophages (phages) and other mobile genetic elements – which has led to the evolution of various defence strategies. Bacterial defence can be considered innate or adaptive. Restriction–modification (RM) systems provide innate immunity by discriminating self-DNA modified by a methyltransferase (MTase) from unmethylated invading DNA, which is cleaved by a restriction endonuclease (REase). CRISPR–Cas systems are adaptive because they acquire an infection memory that can be used to recognize a threat upon repeated attack. In response, phages have evolved various anti-CRISPR (Acr) proteins that can inhibit CRISPR–Cas defence.
The present thesis discusses topics from both the innate and the adaptive fronts of the battle between bacteria and phages, with a focus on different regulatory strategies involved in defence and counter-defence. Our characterization of an RM system in Pectobacterium carotovorum led to the discovery that the MTase of this RM system suppresses an additional defence mechanism and affects the expression of various other genes. These findings highlight conflicts between different defence systems and provide evidence for their involvement in cellular regulatory circuits.
In addition, this work investigated the regulation of Acr production by anti-CRISPR-associated (Aca) proteins. Acrs and Acas are typically encoded in an operon such as acrIF8–aca2 in P. carotovorum phage ZF40. We show that Aca2 inhibits expression of this operon in multiple ways. Aca2 represses transcription by binding one of two similar inverted repeats in the operon promoter. The second inverted repeat can be bound in the transcript, which leads to blockage of ribosome access and inhibition of translation. Both modes of repression require the helix-turn-helix domain of Aca2. These results demonstrate the need for sophisticated regulatory mechanisms in the deployment of counter-defence strategies.
Overall, this thesis broadens our understanding of the complex regulation involved in the arms race between bacteria and phages. We present evidence for non-canonical roles of RM systems and genetic conflicts between different defence mechanisms. Importantly, by demonstrating Aca-mediated anti-CRISPR regulation, we lay the foundation for further research into how phages control the availability of these CRISPR–Cas inhibitors.