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.

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.

Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

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

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.

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.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

Isolation and characterization of Septuagintavirus; a novel clade of Escherichia coli phages within the subfamily Vequintavirinae.

Escherichia coli 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 E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus, within the subfamily Vequintavirinae. 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, Certrevirus, while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli 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 Vequintavirinae 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 Vequintavirinae, contributing to the pool of knowledge currently known about these phages. IMPORTANCE: Escherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus, within the subfamily Vequintavirinae. We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the existing pool of knowledge about these phages.

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

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.

Y-Complex Proteins Show RNA-Dependent Binding Events at the Cell Membrane and Distinct Single-Molecule Dynamics.

Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis, RNase Y in turn interacts with the so-called "Y-complex" consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.

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.

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

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.