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Virology Journal Dec 2010Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least... (Review)
Review
Bacteriophage T4 initiates DNA replication from specialized structures that form in its genome. Immediately after infection, RNA-DNA hybrids (R-loops) occur on (at least some) replication origins, with the annealed RNA serving as a primer for leading-strand synthesis in one direction. As the infection progresses, replication initiation becomes dependent on recombination proteins in a process called recombination-dependent replication (RDR). RDR occurs when the replication machinery is assembled onto D-loop recombination intermediates, and in this case, the invading 3' DNA end is used as a primer for leading strand synthesis. Over the last 15 years, these two modes of T4 DNA replication initiation have been studied in vivo using a variety of approaches, including replication of plasmids with segments of the T4 genome, analysis of replication intermediates by two-dimensional gel electrophoresis, and genomic approaches that measure DNA copy number as the infection progresses. In addition, biochemical approaches have reconstituted replication from origin R-loop structures and have clarified some detailed roles of both replication and recombination proteins in the process of RDR and related pathways. We will also discuss the parallels between T4 DNA replication modes and similar events in cellular and eukaryotic organelle DNA replication, and close with some current questions of interest concerning the mechanisms of replication, recombination and repair in phage T4.
Topics: Bacteriophage T4; DNA Replication; DNA, Viral; Models, Biological; Recombination, Genetic; Replication Origin; Viral Proteins; Virus Replication
PubMed: 21129203
DOI: 10.1186/1743-422X-7-358 -
Proceedings of the National Academy of... Mar 2016Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate...
Bacteriophage T4 consists of a head for protecting its genome and a sheathed tail for inserting its genome into a host. The tail terminates with a multiprotein baseplate that changes its conformation from a "high-energy" dome-shaped to a "low-energy" star-shaped structure during infection. Although these two structures represent different minima in the total energy landscape of the baseplate assembly, as the dome-shaped structure readily changes to the star-shaped structure when the virus infects a host bacterium, the dome-shaped structure must have more energy than the star-shaped structure. Here we describe the electron microscopy structure of a 3.3-MDa in vitro-assembled star-shaped baseplate with a resolution of 3.8 Å. This structure, together with other genetic and structural data, shows why the high-energy baseplate is formed in the presence of the central hub and how the baseplate changes to the low-energy structure, via two steps during infection. Thus, the presence of the central hub is required to initiate the assembly of metastable, high-energy structures. If the high-energy structure is formed and stabilized faster than the low-energy structure, there will be insufficient components to assemble the low-energy structure.
Topics: Bacteria; Bacteriophage T4; Cryoelectron Microscopy; Crystallography, X-Ray; Kinetics; Models, Molecular; Protein Structure, Secondary; Protein Structure, Tertiary; Videotape Recording; Viral Proteins; Virion; Virus Assembly
PubMed: 26929357
DOI: 10.1073/pnas.1601654113 -
Viruses Jun 2018The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the host. However, the functions of many of these proteins are not known. In this...
The lytic bacteriophage T4 employs multiple phage-encoded early proteins to takeover the host. However, the functions of many of these proteins are not known. In this study, we have characterized the T4 early gene , located in a dispensable region of the T4 genome. We show that heterologous production of MotB is highly toxic to , resulting in cell death or growth arrest depending on the strain and that the presence of increases T4 burst size 2-fold. Previous work suggested that affects middle gene expression, but our transcriptome analyses of T4 vs. T4 wt infections reveal that only a few late genes are mildly impaired at 5 min post-infection, and expression of early and middle genes is unaffected. We find that MotB is a DNA-binding protein that binds both unmodified host and T4 modified [(glucosylated, hydroxymethylated-5 cytosine, (GHme-C)] DNA with no detectable sequence specificity. Interestingly, MotB copurifies with the host histone-like proteins, H-NS and StpA, either directly or through cobinding to DNA. We show that H-NS also binds modified T4 DNA and speculate that MotB may alter how H-NS interacts with T4 DNA, host DNA, or both, thereby improving the growth of the phage.
Topics: Bacteriophage T4; DNA, Viral; DNA-Binding Proteins; Escherichia coli; Gene Expression Profiling; Genetic Fitness; Mutation; Promoter Regions, Genetic; Sequence Analysis, RNA; Transcription, Genetic; Viral Proteins
PubMed: 29949907
DOI: 10.3390/v10070343 -
Proceedings of the National Academy of... Jul 2001Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on... (Review)
Review
Double-strand break (DSB) repair and DNA replication are tightly linked in the life cycle of bacteriophage T4. Indeed, the major mode of phage DNA replication depends on recombination proteins and can be stimulated by DSBs. DSB-stimulated DNA replication is dramatically demonstrated when T4 infects cells carrying two plasmids that share homology. A DSB on one plasmid triggered extensive replication of the second plasmid, providing a useful model for T4 recombination-dependent replication (RDR). This system also provides a view of DSB repair in T4-infected cells and revealed that the DSB repair products had been replicated in their entirety by the T4 replication machinery. We analyzed the detailed structure of these products, which do not fit the simple predictions of any of three models for DSB repair. We also present evidence that the T4 RDR system functions to restart stalled or inactivated replication forks. First, we review experiments involving antitumor drug-stabilized topoisomerase cleavage complexes. The results suggest that forks blocked at cleavage complexes are resolved by recombinational repair, likely involving RDR. Second, we show here that the presence of a T4 replication origin on one plasmid substantially stimulated recombination events between it and a homologous second plasmid that did not contain a T4 origin. Furthermore, replication of the second plasmid was increased when the first plasmid contained the T4 origin. Our interpretation is that origin-initiated forks become inactivated at some frequency during replication of the first plasmid and are then restarted via RDR on the second plasmid.
Topics: Bacteriophage T4; DNA Damage; DNA Repair; DNA Replication; DNA Topoisomerases, Type I; DNA, Viral; Models, Genetic; Plasmids; Replication Origin; Viral Proteins
PubMed: 11459966
DOI: 10.1073/pnas.131007598 -
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 -
Viruses Nov 2022Bacteriophages are highly abundant viruses of bacteria. The major role of phages in shaping bacterial communities and their emerging medical potential as antibacterial...
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.
Topics: Bacteriophage T4; Proteome; Transcriptome; Escherichia coli; Protein Biosynthesis
PubMed: 36423111
DOI: 10.3390/v14112502 -
Virology Apr 2020An essential step in the morphogenesis of tailed bacteriophages is the joining of heads and tails to form infectious virions. Our understanding of the maturation of...
An essential step in the morphogenesis of tailed bacteriophages is the joining of heads and tails to form infectious virions. Our understanding of the maturation of complete virus particles remains incomplete. Through an unknown mechanism, phage T4 gene product 4 (gp4) plays an essential role in the head-tail joining step of T4-like phages. Alignment of T4 gp4 homologs identified a type II restriction endonuclease motif. Purified gp4 from both T4 and a marine T4-like bacteriophage, YC, have non-specific nuclease activity in vitro. Mutation of a single conserved amino acid residue in the endonuclease fold of T4 and YC gp4 abrogates nuclease activity. When expressed in trans, the wild type T4 gp4, but neither the mutated T4 protein nor the YC homolog, rescues a T4 gene 4 amber mutant phage. Thus the nuclease activity appears essential for morphogenesis, potentially by cleaving packaged DNA to enable the joining of heads to tails.
Topics: Bacteriophage T4; Capsid; Capsid Proteins; Codon, Nonsense; Endonucleases; Mass Spectrometry; Microscopy, Electron, Transmission; Morphogenesis; Virion; Virus Assembly
PubMed: 32056848
DOI: 10.1016/j.virol.2020.01.008 -
Viruses Jul 2018The mechanisms by which bacteriophage T4 converts the metabolism of its host to one dedicated to progeny phage production was the subject of decades of intense research... (Review)
Review
The mechanisms by which bacteriophage T4 converts the metabolism of its host to one dedicated to progeny phage production was the subject of decades of intense research in many labs from the 1950s through the 1980s. Presently, a wide range of phages are starting to be used therapeutically and in many other applications, and also the range of phage sequence data available is skyrocketing. It is thus important to re-explore the extensive available data about the intricacies of the T4 infection process as summarized here, expand it to looking much more broadly at other genera of phages, and explore phage infections using newly-available modern techniques and a range of appropriate environmental conditions.
Topics: Bacteriophage T4; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genome, Viral
PubMed: 30037085
DOI: 10.3390/v10070387 -
Scientific Reports Oct 2020A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical...
A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.
Topics: Bacteria; Bacteriophage T4; CRISPR-Cas Systems; Gene Editing; Genetic Engineering; Viral Plaque Assay
PubMed: 33106580
DOI: 10.1038/s41598-020-75426-6 -
Proceedings of the National Academy of... May 2017Viruses are incapable of autonomous energy production. Although many experimental studies make it clear that viruses are parasitic entities that hijack the molecular...
Viruses are incapable of autonomous energy production. Although many experimental studies make it clear that viruses are parasitic entities that hijack the molecular resources of the host, a detailed estimate for the energetic cost of viral synthesis is largely lacking. To quantify the energetic cost of viruses to their hosts, we enumerated the costs associated with two very distinct but representative DNA and RNA viruses, namely, T4 and influenza. We found that, for these viruses, translation of viral proteins is the most energetically expensive process. Interestingly, the costs of building a T4 phage and a single influenza virus are nearly the same. Due to influenza's higher burst size, however, the overall cost of a T4 phage infection is only 2-3% of the cost of an influenza infection. The costs of these infections relative to their host's estimated energy budget during the infection reveal that a T4 infection consumes about a third of its host's energy budget, whereas an influenza infection consumes only ≈ 1%. Building on our estimates for T4, we show how the energetic costs of double-stranded DNA phages scale with the capsid size, revealing that the dominant cost of building a virus can switch from translation to genome replication above a critical size. Last, using our predictions for the energetic cost of viruses, we provide estimates for the strengths of selection and genetic drift acting on newly incorporated genetic elements in viral genomes, under conditions of energy limitation.
Topics: Animals; Bacteriophage T4; Energy Metabolism; Host-Pathogen Interactions; Humans; Alphainfluenzavirus; Kinetics; Models, Biological; Viruses
PubMed: 28512219
DOI: 10.1073/pnas.1701670114