Author:

Abstract:

Bacteriophages, or phages, are viruses that specifically prey on bacteria. Ever since their discovery more than a century ago, early phage research has played a pivotal role in shaping the field of molecular biology and transformed our understanding of genetics and fundamental cellular processes. Now, in an era where the global increase in multidrug-resistant bacteria poses a significant threat, the need for alternative therapeutic approaches has sparked a renewed interest in the study of phage biology. Over the past decade, this research has been bolstered by progress in high-throughput sequencing methods, leading to a continuously growing repository of phage genomic sequences. However, while genomics unveiled the remarkable molecular diversity of phages and offered insights in their taxonomy and genome organizations, it also laid bare our lack of understanding on the transcriptomic features that orchestrate the expression of the genes encoded within. Yet, the elucidation of phage gene expression regulation mechanisms is crucial to grasp all the complex layers involved in phage-host interplay, which is critically important for the development of phage-based applications in medicine, biotechnology and synthetic biology. Recently, the phage field embraced bulk RNA-sequencing (RNA-seq) methods, enabling global profiling of gene expression levels of the phage and the host during different stages of infection. However, despite providing major insights in host responses and temporal gene expression patterns, underlying regulatory mechanisms and key phage-encoded transcriptional signals, such as transcription start sites (TSS), termination sites (TTS) and operon structures, often remain unexplored in classic RNA-seq studies. This can mainly be attributed to the inability of RNA-seq to discriminate between primary and processed transcripts, and the inherent loss of information on transcript continuity due to the short-read nature of the sequencing platform. To this end, in the first part of this dissertation, we developed a specialized transcriptomics approach to unlock the transcriptomic architectures of non-model phages and annotate their primary transcriptional features on a genome-wide scale. We applied ONT-cappable-seq on a diverse set of eight phages that target the opportunistic human pathogen Pseudomonas aeruginosa (LUZ7, LUZ100, LUZ19, LUZ24, phiKZ, YuA, 14-1, and PAK_P3) to uncover their TSSs, TTSs and operons. In the second part of this dissertation we showed that ONT-cappable-seq can be extended to chart the transcriptional landscape of phages infecting bacteria beyond the Pseudomonas genus, as illustrated for Thermus thermophilus phage P23-45. Finally, as nature's ancient bioengineers, the ingenious and elegant transcriptional regulation mechanisms that phages use to take control of their bacterial hosts can be leveraged as a source of inspiration for synthetic biology applications. To this end, guided by ONT-cappable-seq data, we mined the genomes of Pseudomonas phages in search of tailored genetic parts that can be exploited to modulate non-model SynBio host Pseudomonas.