precise-phage-mutagenesis-with-ngtet-assisted-crispr-cas-systems

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating ...

Max Planck Institute for Terrestrial Microbiology

Delft University of Technology

All domains of life utilize a diverse set of modified ribonucleotides that can impact the sequence, structure, function, stability, and the fate of RNAs, as well as their interactions with other molecules. Today, more than 160 different RNA modifications are known that decorate the RNA at the 5'-terminus or internal RNA positions. The boost of next-generation sequencing technologies sets the foundation to identify and study the functional role of RNA modifications. The recent advances in the field of RNA modifications reveal a novel regulatory layer between RNA modifications and proteins, which is central to developing a novel concept called "epitranscriptomics." The majority of RNA modifications studies focus on the eukaryotic epitranscriptome. In contrast, RNA modifications in prokaryotes are poorly characterized. This review outlines the current knowledge of the prokaryotic epitranscriptome focusing on mRNA modifications. Here, it is described that several internal and 5'-terminal RNA modifications either present or likely present in prokaryotic mRNA. Thereby, the individual techniques to identify these epitranscriptomic modifications, their writers, readers and erasers, and their proposed functions are explored. Besides that, still unanswered questions in the field of prokaryotic epitranscriptomics are pointed out, and its future perspectives in the dawn of next-generation sequencing technologies are outlined.

Shaping the Bacterial Epitranscriptome-5'-Terminal and Internal RNA Modifications.

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 . We investigated the stability of -originated transcripts and proteins in the course of infection, identifying the degradation of  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  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  and T4 genes.

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

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 substrate. NAD has previously been identified as a 5' modification of cellular RNAs. 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 infections, so RNAylation may have far-reaching implications.

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

The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage 'Arash' from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel 'Arashvirus' genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent.

Isolation and molecular characterization of the Salmonella Typhimurium orphan phage Arash.

NAD is a coenzyme central to metabolism that also serves as a 5'-terminal cap for bacterial and eukaryotic transcripts. Thermal degradation of NAD can generate nicotinamide and ADP-ribose (ADPR). Here, we use LC-MS/MS and NAD captureSeq to detect and identify NAD-RNAs in the thermophilic model archaeon Sulfolobus acidocaldarius and in the halophilic mesophile Haloferax volcanii. None of the four Nudix proteins of S. acidocaldarius catalyze NAD-RNA decapping in vitro, but one of the proteins (Saci_NudT5) promotes ADPR-RNA decapping. NAD-RNAs are converted into ADPR-RNAs, which we detect in S. acidocaldarius total RNA. Deletion of the gene encoding the 5'-3' exonuclease Saci-aCPSF2 leads to a 4.5-fold increase in NAD-RNA levels. We propose that the incorporation of NAD into RNA acts as a degradation marker for Saci-aCPSF2. In contrast, ADPR-RNA is processed by Saci_NudT5 into 5'-p-RNAs, providing another layer of regulation for RNA turnover in archaeal cells.

Identification of NAD-RNA species and ADPR-RNA decapping in Archaea.

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.

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

 is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified  infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, , within the subfamily . Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, , while displaying limited homology to other genera of the  family. Host range analysis showed that these phages have limited activity against a panel of  strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of  family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily , contributing to the pool of knowledge currently known about these phages.

Isolation and characterization of ; a novel clade of  phages within the subfamily .

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.

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

Phage engineering is an emerging technology due to the promising potential application of phages in medical and biotechnological settings. Targeted phage mutagenesis tools are required to customize the phages for a specific application and generate, in addition to that, so-called designer phages. CRISPR-Cas technique is used in various organisms to perform targeted mutagenesis. Yet, its efficacy is notably limited for phage mutagenesis due to the highly abundant phage DNA modifications. Addressing this challenge, we have developed a novel approach that involves the temporal removal of phage DNA cytosine modifications, allowing for effective CRISPR-Cas targeting and subsequent introduction of mutations into the phage genome. The removal of cytosine modification relies on the catalytic activity of a eukaryotic ten-eleven translocation methylcytosine (TET) dioxygenase. TET enzymes iteratively de-modify methylated or hydroxymethylated cytosines on phage DNA. The temporal removal of cytosine modification ultimately enables efficient DNA cleavage by Cas enzymes and facilitates mutagenesis. To streamline the application of the coupled TET-CRISPR-Cas system, we use Golden Gate cloning for fast and efficient assembly of a vector that comprises a TET oxidase and a donor DNA required for scarless site-specific phage mutagenesis. Our approach significantly advances the engineering of modified phage genomes, enabling the efficient generation of customized phages for specific applications.

Article	Year
Shaping the Bacterial Epitranscriptome-5'-Terminal and Internal RNA Modifications. Advanced biology\| PubMed ID: 34121369	2021
Integrated Omics Reveal Time-Resolved Insights into T4 Phage Infection of on Proteome and Transcriptome Levels. Viruses\| PubMed ID: 36423111	2022
A viral ADP-ribosyltransferase attaches RNA chains to host proteins. Nature\| PubMed ID: 37587340	2023
Isolation and molecular characterization of the Salmonella Typhimurium orphan phage Arash. BMC microbiology\| PubMed ID: 37858092	2023
Identification of NAD-RNA species and ADPR-RNA decapping in Archaea. Nature communications\| PubMed ID: 37989750	2023
The enigmatic epitranscriptome of bacteriophages: putative RNA modifications in viral infections. Current opinion in microbiology\| PubMed ID: 38217927	2024
Isolation and characterization of ; a novel clade of phages within the subfamily . Microbiology spectrum\| PubMed ID: 39101714	2024
Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications. PLoS genetics\| PubMed ID: 39231196	2024
Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 39363080	2025

Nadiia Pozhydaieva

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