Abstract
Mobile genetic elements (MGEs), such as viruses and plasmids, facilitate the transfer of genetic material between cells, allowing bacteria and archaea to adapt to their environments. These MGEs can provide beneficial traits to their hosts but also come with a fitness cost, leading to the evolution of host defences against them. In turn, MGEs have developed mechanisms to evade these defences, resulting in an ongoing arms race between the two. This PhD Thesis is divided into three main parts, and the red thread that connects them is an in-depth examination of this arms race, focusing on the interactions between MGEs and their hosts and the immune defences and counter-defences that arise as a result. The ultimate goal of this work is to gain a better understanding of fundamental biological principles and lay the foundation for future biotechnological developments.
Part I presents a general introduction to MGEs as the main drivers of microbial evolution and the current knowledge on prokaryotic defence systems and anti-defence strategies. In Manuscript 1, I provide an overview of the broad diversity and abundance of immune system mechanisms in prokaryotes, including degradation of foreign nucleic acids, inhibition of replication and transcription, and abortive infection (Abi). In Manuscript 2, I review the large diversity of counter-defence strategies evolved by MGEs in response to prokaryotic immune systems, the different approaches for their discovery, and how immune inhibitors can aid in the development and improvement of genome engineering tools, therapeutics and diagnostics.
Part II focuses on the diversity and molecular mechanisms underlying CRISPR–Cas immunity, one of the most widespread defence mechanisms. The classification and identification of CRISPR–Cas systems are challenged by their dynamic nature and expanding diversity. In Manuscript 3, we developed CRISPRCasTyper, a convenient tool to facilitate the automated identification and typing of CRISPR–Cas systems present in prokaryotic sequences based on the latest classification and nomenclature. The mechanisms of action of the different CRISPR–Cas types are very diverse, being able to recognise different type of substrates. Therefore, the simultaneous presence of multiple CRISPR–Cas types in one cell increases the chances of survival by broadening the range of targeted parasites. For example, we previously showed that DNA-targeting CRISPR–Cas systems (e.g. type I) could not defend Serratia against a jumbo phage that protects its genome within a nucleus-like shell. In contrast, the Serratia RNA-targeting CRISPR–Cas system (type III) provides immunity to jumbo phages, yet the molecular mechanisms of defence were unknown. In Manuscript 4, we uncover the mechanistic basis underlying such protection, showing that the recognition of jumbo phage mRNA activates the accessory DNase NucC, which degrades the bacterial DNA, triggering cell death (i.e. abortive infection, or Abi) to prevent phage replication. Importantly, Abi protects the clonal population by suppressing the viral epidemic. Despite the selective advantage provided by immune systems, their associated fitness costs have led to the evolution of regulatory mechanisms to control their activity. In Manuscript 5, we investigate the role of Hfq in the modulation of three CRISPR–Cas systems in Serratia and show that upregulation occurs by direct interaction with cas transcripts, stimulating their translation and stability. The post-transcriptional control of cas transcripts consequently affects immunity by enhancing adaptation and interference. Overall, this provides a novel mode of regulation ruled by post-transcriptional control of expression.
Part III examines the presence of CRISPR–Cas components in MGEs and the emergence of functions alternative to host immunity. In Manuscript 6, we systematically investigate the distribution, prevalence, diversity, and putative functions of plasmid-encoded CRISPR–Cas systems. Our analyses revealed a broad diversity of plasmid-encoded CRISPR–Cas systems widespread across taxa and primarily involved in plasmid-plasmid competition. In Manuscript 7, we investigate the inhibition of CRISPR–Cas systems by phage-encoded small, non-coding RNAs that mimic the sequence and secondary structure of CRISPR repeats. This constitutes a novel mode of CRISPR–Cas inhibition via RNA molecules, potentially enabling the custom design of more controllable CRISPR–Cas biotechnologies.
A variety of techniques were used in this research, including phage microbiology, genetic manipulations, molecular and biochemical assays, deep sequencing, bioinformatics, flow cytometry and confocal imaging. Overall, this PhD Thesis broadens our understanding of the complex interactions that arise in the arms race between prokaryotes and MGEs. The research presented lays the basis for further research into how CRISPR–Cas systems and counter-defences evolved and are repurposed for alternative functions.