Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications.

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

The enigmatic epitranscriptome of bacteriophages: putative RNA modifications in viral infections.

RNA modifications play essential roles in modulating RNA function, stability, and fate across all kingdoms of life. The entirety of the RNA modifications within a cell is defined as the epitranscriptome. While eukaryotic RNA modifications are intensively studied, understanding bacterial RNA modifications remains limited, and knowledge about bacteriophage RNA modifications is almost nonexistent. In this review, we shed light on known mechanisms of bacterial RNA modifications and propose how this knowledge might be extended to bacteriophages. We build hypotheses on enzymes potentially responsible for regulating the epitranscriptome of bacteriophages and their host. This review highlights the exciting prospects of uncovering the unexplored field of bacteriophage epitranscriptomics and its potential role to shape bacteriophage-host interactions.

A viral ADP-ribosyltransferase attaches RNA chains to host proteins.

The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate1,2. NAD has previously been identified as a 5' modification of cellular RNAs3-5. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections6, so RNAylation may have far-reaching implications.

NAD-capped RNAs - a redox cofactor meets RNA.

RNA modifications immensely expand the diversity of the transcriptome, thereby influencing the function, localization, and stability of RNA. One prominent example of an RNA modification is the eukaryotic cap located at the 5' terminus of mRNAs. Interestingly, the redox cofactor NAD can be incorporated into RNA by RNA polymerase in vitro. The existence of NAD-modified RNAs in vivo was confirmed using liquid chromatography and mass spectrometry (LC-MS). In the past few years novel technologies and methods have characterized NAD as a cap-like RNA structure and enabled the investigation of NAD-capped RNAs (NAD-RNAs) in a physiological context. We highlight the identification of NAD-RNAs as well as the regulation and functions of this epitranscriptomic mark in all domains of life.

Integrated Omics Reveal Time-Resolved Insights into T4 Phage Infection of E. coli on Proteome and Transcriptome Levels.

Bacteriophages are highly abundant viruses of bacteria. The major role of phages in shaping bacterial communities and their emerging medical potential as antibacterial agents has triggered a rebirth of phage research. To understand the molecular mechanisms by which phages hijack their host, omics technologies can provide novel insights into the organization of transcriptional and translational events occurring during the infection process. In this study, we apply transcriptomics and proteomics to characterize the temporal patterns of transcription and protein synthesis during the T4 phage infection of E. coli. We investigated the stability of E. coli-originated transcripts and proteins in the course of infection, identifying the degradation of E. coli transcripts and the preservation of the host proteome. Moreover, the correlation between the phage transcriptome and proteome reveals specific T4 phage mRNAs and proteins that are temporally decoupled, suggesting post-transcriptional and translational regulation mechanisms. This study provides the first comprehensive insights into the molecular takeover of E. coli by bacteriophage T4. This data set represents a valuable resource for future studies seeking to study molecular and regulatory events during infection. We created a user-friendly online tool, POTATO4, which is available to the scientific community and allows access to gene expression patterns for E. coli and T4 genes.