title: Molecular strategies applied by bacteriophage T4 for efficient hijacking of Escherichia coli
title: Molekulare Strategien des Bakteriophagen T4 zur effizienten Übernahme der Kontrolle über Escherichia coli
titleType: AlternativeTitle
Creators: name: Pozhydaieva, Nadiia
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: RNA modifications
Multiomics
RNaylation
Phagen-Wirt-Interaktion
Phagenmutagenese
Chemistry &amp; allied sciences
Chemie
Bakteriophagen
RNAylation
Bacteriophage
Phage-Host Interaction
Engineering of phages
RNA-Modifikationen
Phage-Bakterium Interaktion

description: Bacteriophages are bacterial predators that serve as excellent models to study host-pathogen interactions and hold significant potential for industrial and medical applications. These include the utilization of bacteriophages as alternatives to antibiotics in combating multi-resistant bacterial strains, controlling microbial communities, and engineering phages for specific diagnostics purposes. To fully exploit the potential of phages, a comprehensive understanding of phage infection mechanisms and the bacterial countermeasures is crucial. The T4 phage stands out as one of the model bacteriophages and its infection of Escherichia coli is one of the best-studied bacterium-bacteriophage interactions. Research on the molecular mechanisms of T4 phage infection has strongly shaped our understanding of the fundamental principles in molecular biology. Fundamental concepts like DNA being a blueprint of life, principles in molecular genetics, phage evolutionary mechanisms, and beyond were discovered upon research on T4 phage. Additionally, many T4 phage proteins became indispensable tools in molecular biology. 
However, despite the considerable knowledge gained from studying the T4 phage, numerous aspects of its infection remain unresolved. While approximately half of the T4 proteins have been associated with specific functions, the roles of the remaining 45% of T4 phage proteins are still unexplored. Therefore, this knowledge gap alone makes it evident that our molecular understanding of the T4 phage infection and the strategies the phage employs to execute an efficient infection is far from complete. This thesis aimed to enhance our knowledge of the T4 phage infection at the molecular level and to uncover previously unexplored mechanisms used by the T4 phage to carry out infection efficiently.
Chapter II describes a multi-omics study designed to provide a temporal resolution of an E. coli infection with T4 phage at the molecular level. The transcriptome and proteome of E. coli and T4 phage were analyzed throughout the infection. This enabled the identification of temporal gene expression patterns for T4 phage transcripts. Even more, a decoupling of transcription and translation processes was observed for certain T4 phage genes. The transcriptome and proteome analysis of E. coli revealed a general degradation of host transcripts and preservation of the host proteins. This study presents the molecular kinetics of T4 phage infection for the first time. The results strongly suggest the existence of additional, unexplored regulatory mechanisms that allow differential degradation of host and phage transcripts and decoupling of transcription and translation for specific phage genes.
A possible explanation for the differential RNA degradation upon infection and decoupling of transcription and translation observed for some T4 phage genes, could be the presence of RNA modifications. RNA modifications may provide the molecular basis for the discrimination between bacterial and viral transcripts during T4 phage infection. Chapter III summarizes the current knowledge on the bacterial epitranscriptome, emphasizing mRNA modifications. The known writers, readers, and erasers that regulate RNA modifications, and techniques to identify and study specific RNA modifications are discussed. This work demonstrates a significant knowledge gap regarding RNA modifications on bacterial mRNA, their modulators, and their biological significance.
While some initial insights into the bacterial epitranscriptome already exist, the epitranscriptome of bacteriophages remains unexplored to date. In Chapter IV, the current knowledge of RNA modifications in bacteria is used to hypothesize how some bacterial and phage enzymes may shape and modulate the epitranscriptome of bacteriophages during infection.
To investigate the biological role of potential T4 phage-derived infection regulators, it is necessary to study how their absence or inactivity affects phage infection. However, this requires efficient tools for phage mutagenesis. CRISPR-Cas is a powerful tool for precise genome engineering, but its effectiveness for T4 phage mutagenesis is severely hampered by the highly abundant modifications of T4 DNA. Chapter V outlines an approach for temporal reduction of T4 phage DNA modifications. This enables efficient and scarless CRISPR-Cas-based mutagenesis of T4 phage DNA. This system not only facilitates T4 phage mutagenesis but also allows the study of the role of DNA modifications in phage infection and has the potential to be extended to other phages beyond T4 phage.
Chapter VI focuses on the T4 ADP-ribosyltransferase ModB and reveals that ModB not only accepts NAD as a substrate to perform ADP-ribosylation but also NAD-RNA to perform RNAylation – a novel post-translational modification. This modification was shown to be introduced by ModB in vitro and in vivo. Furthermore, it was demonstrated that ModB RNAylates several E. coli proteins, including ribosomal proteins S1 and L2. The biological role and molecular mechanisms of RNAylation were investigated. Additionally, a T4 phage mutant with a catalytically inactive ModB was generated to explore the impact of ModB activity on phage infection and phage phenotype.
This thesis contributes to an enhanced understanding of the T4 phage infection of E. coli by providing insights into the molecular organization of infection, showing the impact of phage DNA modifications on phage phenotype and mutagenesis efficiency, and through the discovery of RNAylation – a novel post-translational protein modification. Apart from deepening our understanding of the T4 phage infection and its regulation, the knowledge gained in this thesis also lays the groundwork for its translation into application. Particularly, understanding the molecular organization of the phage infection and its gene expression patterns is essential for designing a synthetic phage or tailoring an existing phage for specific needs. To tailor the T4 phage, the mutagenesis strategy reported here can be efficiently applied. Furthermore, the discovery of RNAylation expands the arsenal of T4 phage-derived molecular tools, as RNAylation can find potential applications in synthetic biology for the development of novel artificial cellular RNA-protein constructs and opens up new possibilities for the design of next-generation RNA-based therapeutics.
Taken together, this study expands our understanding of the molecular mechanisms underlying efficient T4 phage infection and underscores that the discovery potential based on T4 phage research is far from being fully exploited.