-
Nature Aug 2023The 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...
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.
Topics: ADP Ribose Transferases; Bacteriophage T4; Escherichia coli; NAD; Ribosomal Proteins; Viral Proteins; Escherichia coli Proteins; RNA; Protein Biosynthesis; Gene Expression Regulation, Bacterial; Protein Processing, Post-Translational
PubMed: 37587340
DOI: 10.1038/s41586-023-06429-2 -
Nature Communications Jul 2023The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is...
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
Topics: Bacteriophage T4; DNA Primase; DNA Helicases; DNA Replication; DNA Primers; DNA, Viral
PubMed: 37474605
DOI: 10.1038/s41467-023-40106-2 -
Nature Communications May 2023Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we...
Designing artificial viral vectors (AVVs) programmed with biomolecules that can enter human cells and carry out molecular repairs will have broad applications. Here, we describe an assembly-line approach to build AVVs by engineering the well-characterized structural components of bacteriophage T4. Starting with a 120 × 86 nm capsid shell that can accommodate 171-Kbp DNA and thousands of protein copies, various combinations of biomolecules, including DNAs, proteins, RNAs, and ribonucleoproteins, are externally and internally incorporated. The nanoparticles are then coated with cationic lipid to enable efficient entry into human cells. As proof of concept, we assemble a series of AVVs designed to deliver full-length dystrophin gene or perform various molecular operations to remodel human genome, including genome editing, gene recombination, gene replacement, gene expression, and gene silencing. These large capacity, customizable, multiplex, and all-in-one phage-based AVVs represent an additional category of nanomaterial that could potentially transform gene therapies and personalized medicine.
Topics: Humans; Bacteriophage T4; Genome, Human; Genetic Vectors; Capsid Proteins; Capsid; DNA, Viral
PubMed: 37253769
DOI: 10.1038/s41467-023-38364-1 -
Journal of Virology Jun 2023Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of...
Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.
Topics: Bacteriophage T4; CRISPR-Cas Systems; Escherichia coli; Genome, Viral; DNA Restriction-Modification Enzymes
PubMed: 37306585
DOI: 10.1128/jvi.00599-23 -
Nature May 2024Bacteria have adapted to phage predation by evolving a vast assortment of defence systems. Although anti-phage immunity genes can be identified using bioinformatic...
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Topics: Escherichia coli; DNA Glycosylases; Bacteriophage T4; Virus Replication; T-Phages; Genome, Viral; Soil Microbiology; Metagenomics; Phylogeny
PubMed: 38632404
DOI: 10.1038/s41586-024-07329-9 -
Microbiome Aug 2023There is an increasing interest in investigating the human gut virome for its influence on the gut bacterial community and its putative influence on the trajectory...
BACKGROUND
There is an increasing interest in investigating the human gut virome for its influence on the gut bacterial community and its putative influence on the trajectory towards health or disease. Most gut virome studies are based on sequencing of stored fecal samples. However, relatively little is known about how conventional storage buffers and storage conditions affect the infectivity of bacteriophages and influence the downstream metavirome sequencing.
RESULTS
We demonstrate that the infectivity and genome recovery rate of different spiked bacteriophages (T4, c2 and Phi X174) are variable and highly dependent on storage buffers. Regardless of the storage temperature and timespan, all tested phages immediately lost 100% (DNA/RNA Shield) or more than 90% (StayRNA and RNAlater) of their infectivity. Generally, in SM buffer at 4 °C phage infectivity was preserved for up to 30 days and phage DNA integrity was maintained for up to 100 days. While in CANVAX, the most effective buffer, all spiked phage genomes were preserved for at least 100 days. Prolonged storage time (500 days) at - 80 °C impacted viral diversity differently in the different buffers. Samples stored in CANVAX or DNA/RNA Shield buffer had the least shifts in metavirome composition, after prolonged storage, but they yielded more contigs classified as "uncharacterised". Moreover, in contrast to the SM buffer, these storage buffers yielded a higher fraction of bacterial DNA in metavirome-sequencing libraries. We demonstrated that the latter was due to inactivation of the DNases employed to remove extra-cellular DNA during virome extraction. The latter could be partly avoided by employing additional washing steps prior to virome extraction.
CONCLUSION
Fecal sample storage buffers and storage conditions (time and temperature) strongly influence bacteriophage infectivity and viral composition as determined by plaque assay and metavirome sequencing. The choice of buffer had a larger effect than storage temperature and storage time on the quality of the viral sequences and analyses. Based on these results, we recommend storage of fecal virome samples at in SM buffer at 4 °C for the isolation of viruses and at - 80 °C for metagenomic applications if practically feasible (i.e., access to cold storage). For fecal samples stored in other buffers, samples should be cleared of these buffers before viral extraction and sequencing. Video Abstract.
Topics: Humans; Bacteriophages; DNA, Bacterial; Feces; Metagenome; RNA
PubMed: 37635262
DOI: 10.1186/s40168-023-01632-9 -
Viruses Aug 2023The preservative qualities of individual ionic compounds impacting the infectivity of T4 virions were elucidated. T4 virions were immersed in quasi-pure ionic solutions...
The preservative qualities of individual ionic compounds impacting the infectivity of T4 virions were elucidated. T4 virions were immersed in quasi-pure ionic solutions prior to the adsorption process, and the plaque forming unit (pfu) values of these were measured following the conventional method. In neutral ionic solutions, the minimum and the optimum concentrations of preservative qualities corresponded with the results obtained from the multi-ionic media/buffers. In acid and alkali solutions, phages show tolerances at a pH range of 5-11 in multi-ionic media/buffers. T4 virions show no tolerance in quasi-pure acid, neutral, and weak alkaline conditions. The preservative quality of T4 virions increased in over 10 mM OH solution, equivalent to a pH value over 10, which corresponds to the pKa of the deprotonation of the DNA bases G and T. Infectivity was lost below 10 mM OH and higher than 10 mM OH. These results imply that maintaining infectivity of a virion may need the flexibility of the intra-capsid DNA by deprotonation.
Topics: Adsorption; Bacteriophage T4; Capsid; Capsid Proteins
PubMed: 37632079
DOI: 10.3390/v15081737 -
Nature Communications Jul 2023E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole...
E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole E217 virion before and after DNA ejection at 3.1 Å and 4.5 Å resolution, respectively, determined using cryogenic electron microscopy (cryo-EM). We identify and build de novo structures for 19 unique E217 gene products, resolve the tail genome-ejection machine in both extended and contracted states, and decipher the complete architecture of the baseplate formed by 66 polypeptide chains. We also determine that E217 recognizes the host O-antigen as a receptor, and we resolve the N-terminal portion of the O-antigen-binding tail fiber. We propose that E217 design principles presented in this paper are conserved across PB1-like Myoviridae phages of the Pbunavirus genus that encode a ~1.4 MDa baseplate, dramatically smaller than the coliphage T4.
Topics: Pseudomonas Phages; Cryoelectron Microscopy; O Antigens; Microscopy, Electron; Myoviridae; Bacteriophage T4
PubMed: 37422479
DOI: 10.1038/s41467-023-39756-z -
ISME Communications Sep 2023The interactions between bacteria and bacteriophage have important roles in the global ecosystem; in turn changes in environmental parameters affect the interactions...
The interactions between bacteria and bacteriophage have important roles in the global ecosystem; in turn changes in environmental parameters affect the interactions between bacteria and phage. However, there is a lack of knowledge on whether clonal bacterial populations harbour different phenotypes that respond to phage in distinct ways and whether the abundance of such phenotypes within bacterial populations is affected by variations in environmental parameters. Here we study the impact of variations in nutrient availability, bacterial growth rate and phage abundance on the interactions between the phage T4 and individual Escherichia coli cells confined in spatial refuges. Surprisingly, we found that fast growing bacteria survive together with all of their clonal kin cells, whereas slow growing bacteria survive in isolation. We also discovered that the number of bacteria that survive in isolation decreases at increasing phage doses possibly due to lysis inhibition in the presence of secondary adsorptions. We further show that these changes in the phenotypic composition of the E. coli population have important consequences on the bacterial and phage population dynamics and should therefore be considered when investigating bacteria-phage interactions in ecological, health or food production settings in structured environments.
PubMed: 37684358
DOI: 10.1038/s43705-023-00299-5 -
Frontiers in Microbiology 2023Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage-a...
Antiviral drug development is important for human health, and the emergence of novel COVID-19 variants has seriously affected human lives and safety. A bacteriophage-a bacterial virus with a small and simple structure-is an ideal experimental candidate for studying the interactions between viruses and their hosts. In this study, the effects and mechanisms of catecholamines on phages were explored, and dopamine (DA) was found to have general and efficient anti-infection effects. A clear dose-dependent effect was observed when different phages were treated with DA, with higher DA concentrations exhibiting stronger anti-phage activity. The half maximal inhibitory concentration values of DA for vB-EcoS-IME167, T4 Phage, and VMY22 were determined as 0.26, 0.12, and 0.73 mg mL, respectively. The anti-phage effect of DA increased with treatment duration. In addition, the anti-infection activities of DA against vB-EcoS-IME167, T4 Phage, and VMY22 were increased by 10, 10, and 10 folds compared to that of the control. This ability of DA was observed only in phages and not in the host bacteria. Morphological changes of phages were observed under transmission electron microscopy following their treatment with DA, and considerable changes in adsorption were confirmed quantitative reverse transcription polymerase chain reaction. These results suggest that the anti-phage effect of DA is primarily due to the destruction of the external structure of the phage. This study, to the best of our knowledge, is the first to report the universal anti-phage infection effect of dopamine, which provides novel information regarding DA and forms a basis for further research and development of antiviral drugs. Moreover, it provides a new perspective for the research about the defense and counter-defense of bacteria and bacteriophages.
PubMed: 38029195
DOI: 10.3389/fmicb.2023.1272447