Abstract
Bacteria can harbour multiple defence systems within their genomes to counteract phage infection, including both innate and adaptive immune systems. CRISPR-Cas systems constitute the adaptive immune response of bacteria and multiple CRISPR-Cas systems can exist in a single strain – such is the case for Serratia which contains three CRISPR-Cas systems: type I-E, I-F and III-A. To evade CRISPR-Cas immunity phages have also evolved multiple strategies, such as point mutations and anti-CRISPR proteins. Although these phage-encoded mechanisms are effective against CRISPR-Cas targeting and widespread amongst phages, it is possible that phages encode other strategies to circumvent the host defence systems.
In this thesis, we isolated and characterized three Serratia phages and examined their interaction with the CRISPR-Cas systems in the bacterial host. We show that a Serratia siphovirus can alter bacterial metabolism upon infection, but infection can be arrested by type I-E and type I-F CRISPR-Cas immunity. Although the infection is cleared in anti-phage strains, phage infection produces major physiological changes in the host.
Furthermore, in this study, we examined two phage-encoded strategies to evade CRISPR-Cas systems. We isolated a jumbo phage that evades CRISPR-Cas targeting by the formation of a proteinaceous shell that shields the phage DNA and excludes CRISPR-complexes to the bacterial cytoplasm. Although this nucleus-like structure can prevent DNA targeting, our results show that the type III-A CRISPR-Cas systems can target phage mRNA in the bacterial cytoplasm and arrest the infection.
Moreover, we investigated the mechanism by which a T4-related phage escapes the three CRISPR-Cas systems in Serratia. Our results reveal that the phage possesses all the enzymes necessary to produce a type of DNA hypermodification that could hinder DNA targeting by the type I CRISPR-Cas systems in Serratia.
Overall, this study broadens our knowledge of the diverse phage interactions with CRISPR-Cas systems during infection. Our results provide insight into the effects of phage infection and CRISPR-Cas targeting on host cell physiology. Moreover, here we examine the advantages of maintaining different defence systems to fight infection against diverse phages. Finally, our study unravels a new phage-encoded mechanism to evade CRISPR-Cas systems.