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Electron micrographs of bacteriophage T4. The well-recognized T4 morphology was nature's prototype of the NASA lunar excursion module. (A) Extended tail fibers recognize the bacterial envelope, and its prolate icosahedral head contains the 168,903-bp dsDNA genome. Reprinted with permission of M. Wurtz, Biozentrum, Basel, Switzerland. (B) The DNA genome is delivered into the host through the internal tail tube, which is visible protruding from the end of the contracted tail sheath. Courtesy of W. Rüger.

Electron micrographs of bacteriophage T4. The well-recognized T4 morphology was nature's prototype of the NASA lunar excursion module. (A) Extended tail fibers recognize the bacterial envelope, and its prolate icosahedral head contains the 168,903-bp dsDNA genome. Reprinted with permission of M. Wurtz, Biozentrum, Basel, Switzerland. (B) The DNA genome is delivered into the host through the internal tail tube, which is visible protruding from the end of the contracted tail sheath. Courtesy of W. Rüger.

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Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, inte...

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... phages ( Fig. 1) have been major model systems in the development of modern genetics and molecular biology since the 1940s; many investigators have taken advantage of their useful degree of complexity and the ability to derive detailed genetic and physiological information with relatively simple experiments. Bacteriophages T2 and T4 were instru- ...
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... phages build some of the most complex virus parti- cles known ( Fig. 1 and 8). They devote more than 40% of their genetic information to the synthesis and assembly of the pro- late icosahedral heads, tails with contractile sheaths, and six tail fibers that contribute to their very high efficiency of infec- tion. ...
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... of the genes for the structural proteins are tran- scribed in the clockwise direction on the standard genomic map. The genes responsible for each substructure are largely clustered, with these clusters distributed over more than half of the genome (Table 1; Fig. 3). There are some interesting ex- ceptions to the clustering. ...
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... packaging mechanism cuts the DNA when the head is filled, and it appears that EndoVII trims branches of DNA even after packaging has been initiated (523). The head full of DNA is about 3% longer than the genome size, accounting for the circular permutation of T4 genomes, with terminal redundancy of each genome; this cir- cular permutation is the basis for the circular T4 genetic map (1048, 1049, 1073). Shorter or longer phage heads are occa- sionally formed, due to assembly errors that are increased by specific mutations in some head genes (256) or by incorpora- tion of arginine analogues (194). ...
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... One of the most interesting and potentially instructive ex- amples of an orthologous protein is thymidylate synthase (td or TS). A number of stretches of amino acids are highly con- served between Bacteria, Eucarya and T4, facilitating precise alignment and analysis; these are indicated in red on the crystal structure of the T4 enzyme shown in Fig. 10 (also see pdb 1TIS). The two stretches indicated in yellow are totally differ- ent between the T4 enzyme and all other thymidylate syn- thases; these regions are largely hydrophobic in T4 but hydro- philic in other members of the family. They lie on the surface of the enzyme, where presumably they are involved in the interaction between thymidylate ...
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... in other members of the family. They lie on the surface of the enzyme, where presumably they are involved in the interaction between thymidylate synthase and other enzymes of the nucleotide synthesis complex described above. When these two segments are excluded and the core regions are used for alignment, the phylogenetic relationship shown in Fig. 11 is obtained. The tree suggests that the T4 enzyme branched off somewhere before the split between Eucarya and Bacteria. The apparently ancient branch point is not just due to faster evo- lution of viral proteins than of proteins of their hosts, since, for example, herpesviruses appear to branch off much later in the Eucarya lineage, ...
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... TS also has one sequence near the N terminus that is otherwise unique to archaeal thymidylate synthases, which are sufficiently different from those of bacteria and eukaryotes that they are more difficult to align unequivocally. Figure 11 also shows the distant relationship between thymidylate synthases and T4 HMase (gp42; also see pdb 1B5D). This is not too surprising since both enzymes catalyze the transfer of methyl (or hydroxy- methyl) to the same position on a pyrimidine monophosphate (dUMP and dCMP, respectively). ...
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... the regions in which the T4 enzyme is different from all others colored in yellow. The latter regions are largely hydrophilic for most thymidylate synthases but are hydrophobic for the T4 enzyme (which may facilitate its incorporation into the nucleotide-synthesizing complex). These regions were not included for the predicted evolutionary tree in Fig. 11. Structural coordinates are from reference 274 and were used to create this figure. Also see reference 143. VOL. 67,2003kinase activities are both required in host strains with the restriction system carried by the cryptic DNA element, prr (see above) ...

Citations

... For longer sequences, circularization methods typically involve the use of RNA ligases and ribonucleases. Commonly used ligases include T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1), and T4 RNA ligase 2 (T4 Rnl 2), all of which are ATP-dependent enzymes that catalyze the connection of RNA 5'-phosphate ends and 3'-OH ends through three nucleotide transfer steps to generate circRNAs [44,45]. ...
Article
Circular RNAs (circRNAs) have emerged as a large class of stable and conserved RNAs that are derived primarily from back-splicing of pre-mRNAs and expressed in a cell- and tissue-specific fashion. Recent studies have indicated that a subset of circRNAs may undergo translation through cap-independent pathways mediated by internal ribosome entry sites (IRESs), m6A modifications, or IRES-like short elements. Considering the stability and low immunogenicity of circRNAs, in vitro transcribed circRNAs hold great promise in biomedical applications. In this review, we briefly discuss the noncoding and coding functions of circRNAs in cells, as well as the methods for the in vitro synthesis of circRNAs and current advances in the applications of circRNAs in biomedicine.
... We next assessed the activity of mSCD-B5 on double-stranded DNA (dsDNA). To do this, mSCD-B5 deamination reactions were performed directly on, or following heat denaturation of, dsDNA from bacteriophages containing 5mC (Xp12), 5hmC (T4gt, a mutant of T4 deficient in DNA αand β-glucosyltransferases and as a result contains 5hmC in its genome (Miller et al. 2003)), or unmodified C (Lambda). Using LC-MS, we observed minimal deaminase activity on intact dsDNA from all bacteriophages, while heat-denatured (ssDNA) samples showed substantial deamination, with 35% of 5mC and 27% of 5hmC converted (Supplementary Figure 2E). ...
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Mining phages for new enzymatic activities continues to be important for the development of new tools for biotechnology. In this study, we used MetaGPA—a method linking genotype to phenotype in metagenomic data—to identify deoxycytidine deaminases, a protein family highly associated with cytosine modifications in metaviromes. Unexpectedly, a subset of these deaminases exhibited a preference for 5-methylcytosine (5mC) over cytosine (C) in both mononucleotide and single-stranded DNA substrates. In a methylome sequencing workflow, preferential deamination of 5mC by these enzymes enabled direct conversion of methylated cytosine while completely eliminating any background deamination of unmodified cytosine. This direct conversion allows for precise identification of methylated sites at single-base resolution with unmatched sensitivity enabling broad applications for the simultaneous sequencing of genome and methylome.
... The functions of the proteins encoded by pangenomic genes were verified using the genome annotation of Escherichia phage T4 (NC_000866.4) [28]. ...
... Another example is the branch containing the genomes of Enterobacteria phages phage Enterobacteria phage RB51, Enterobacteria phage RB68, Escherichia phage wV7, Escherichia phage vB EcoMSa45lw, Escherichia phage PE37, Escherichia phage vB_EcoM_G10400, Escherichia phage vB_EcoM_G4507, Enterobacteria phage RB33, Escherichia phage RB32, Enterobacteria phage RB27, Escherichia phage RB14, Enterobacteria phage RB10, Enterobacteria phage RB3, Enterobacteria phage RB5, Enterobacteria phage RB6, Enterobacteria phage RB7, Enterobacteria phage RB9. As one can see, this branch contains a group of RB phages [28]. Although the RB-phage group is highly diverse in composition and contains representatives of, at least, two genera [28], many of these phages, which were isolated from a lake near the Cold Spring Harbor village, are closely related. ...
... As one can see, this branch contains a group of RB phages [28]. Although the RB-phage group is highly diverse in composition and contains representatives of, at least, two genera [28], many of these phages, which were isolated from a lake near the Cold Spring Harbor village, are closely related. The branches of this tree are almost identical to the branches on the phylogenomic tree constructed using the alignment-free method [31] (Fig. 4) and on the trees obtained by maximum likelihood after alignment of genes or amino acid sequences for the major capsid proteins of the phages ( Fig. 5 and 6, respectively). ...
Article
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In this paper, we investigate the possibility of using the multiplex virtual PCR method to develop approaches to barcoding the genomes of T4-type bacteriophages. A comparison of the multiplex virtual PCR method and phylogenomic analysis based on alignment-free approach was performed. A dataset of Tequatrovirus genomes previously selected for pangenomic analysis was used for this study. The genomes used were preliminarily checked for their adequate annotation and, accordingly, sequencing accuracy. A study using the multiplex virtual PCR method was carried out for 67 genomes, barcodes were generated, and similarity parameters were determined. Based on the obtained data, a phylogenetic tree was constructed using the neighbor joining method. For the same set of Straboviridae bacteriophage genomes, a phylogenomic tree was constructed based on pairwise distance matriх between oligonucleotide 10-mer sets. Comparison of these two trees showed differences in branching between the control distantly related genomes from the genera Mosigvirus and Dhakavirus, and the branch of the genomes of bacteriophages of the genus Tequatrovirus. The authors of the work suggested that these differences may reflect the features of the multiplex virtual PCR method, which is more applicable for assessing the similarity of closely related genomes than within genomes with an identity degree of less than 75 %. It can be concluded that the multiplex virtual PCR method allows one to distinguish taxonomic groups of large bacteriophages, which include T4-type bacteriophages, at the genus level. Thus, using this method, within the genus of such bacteriophages one can more accurately compare the genomes of viruses with a length of about 170 kb. From a practical point of view, multiplex virtual PCR is quite applicable for including barcodes compiled on its basis in the bacteriophage passport for use in the practice of phage therapy.
... The functions of the proteins encoded by pangenomic genes were verified using the genome annotation of Escherichia phage T4 (NC_000866.4) [28]. ...
... Another example is the branch containing the genomes of Enterobacteria phages phage Enterobacteria phage RB51, Enterobacteria phage RB68, Escherichia phage wV7, Escherichia phage vB EcoMSa45lw, Escherichia phage PE37, Escherichia phage vB_EcoM_G10400, Escherichia phage vB_EcoM_G4507, Enterobacteria phage RB33, Escherichia phage RB32, Enterobacteria phage RB27, Escherichia phage RB14, Enterobacteria phage RB10, Enterobacteria phage RB3, Enterobacteria phage RB5, Enterobacteria phage RB6, Enterobacteria phage RB7, Enterobacteria phage RB9. As one can see, this branch contains a group of RB phages [28]. Although the RB-phage group is highly diverse in composition and contains representatives of, at least, two genera [28], many of these phages, which were isolated from a lake near the Cold Spring Harbor village, are closely related. ...
... As one can see, this branch contains a group of RB phages [28]. Although the RB-phage group is highly diverse in composition and contains representatives of, at least, two genera [28], many of these phages, which were isolated from a lake near the Cold Spring Harbor village, are closely related. The branches of this tree are almost identical to the branches on the phylogenomic tree constructed using the alignment-free method [31] (Fig. 4) and on the trees obtained by maximum likelihood after alignment of genes or amino acid sequences for the major capsid proteins of the phages ( Fig. 5 and 6, respectively). ...
Article
Full-text available
In this paper, we investigate the possibility of using the multiplex virtual PCR method to develop approaches to barcoding the genomes of T4-type bacteriophages. A comparison of the multiplex virtual PCR method and phylogenomic analysis based on alignment-free approach was performed. A dataset of Tequatrovirus genomes previously selected for pangenomic analysis was used for this study. The genomes used were preliminarily checked for their adequate annotation and, accordingly, sequencing accuracy. A study using the multiplex virtual PCR method was carried out for 67 genomes, barcodes were generated, and similarity parameters were determined. Based on the obtained data, a phylogenetic tree was constructed using the neighbor joining method. For the same set of Straboviridae bacteriophage genomes, a phylogenomic tree was constructed based on pairwise distance matriх between oligonucleotide 10-mer sets. Comparison of these two trees showed differences in branching between the control distantly related genomes from the genera Mosigvirus and Dhakavirus, and the branch of the genomes of bacteriophages of the genus Tequatrovirus. The authors of the work suggested that these differences may reflect the features of the multiplex virtual PCR method, which is more applicable for assessing the similarity of closely related genomes than within genomes with an identity degree of less than 75 %. It can be concluded that the multiplex virtual PCR method allows one to distinguish taxonomic groups of large bacteriophages, which include T4-type bacteriophages, at the genus level. Thus, using this method, within the genus of such bacteriophages one can more accurately compare the genomes of viruses with a length of about 170 kb. From a practical point of view, multiplex virtual PCR is quite applicable for including barcodes compiled on its basis in the bacteriophage passport for use in the practice of phage therapy.
... Phage T4 specifically infects some particular strains of Escherichia coli. This specificity is partly due to the interaction between the tail fiber proteins of phage T4 and the outer membrane receptors of E. coli [36]. Phage λ specifically infects certain strains of E. coli by binding to the maltose-binding protein receptor on their outer membrane. ...
Article
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Bacteriophage (phage) AP1 has been reported to effectively lyse Acidovorax oryzae, the causative agent of bacterial brown stripe in rice. However, phage AP1 exhibits strain-specific lysis patterns. In order to enhance the potential of phages for biological control of rice bacterial brown stripe, this study investigated the possible mechanism of strain-specific infection by characterizing phage AP1 and its susceptible (RS-2) and resistant (RS-1) strains. Based on the current classification standards and available database information, phage AP1 was classified into the class Caudoviricetes, and it is a kind of podophage. Comparative analysis of the susceptible and resistant strains showed no significant differences in growth kinetics, motility, biofilm formation, or effector Hcp production. Interestingly, the resistant strain demonstrated enhanced virulence compared to the susceptible strain. Prokaryotic expression studies indicated that six putative structural proteins of phage AP1 exhibited varying degrees of binding affinity (1.90–9.15%) to lipopolysaccharide (LPS). However, pull-down assays and bacterial two-hybrid analyses revealed that only gp66 can interact with four host proteins, which were identified as glycosyltransferase, RcnB, ClpB, and ImpB through immunoprecipitation and mass spectrometry analyses. The role of LPS in the specific infection mechanism of phage AP1 was further elucidated through the construction of knockout mutant strains and complementary strains targeting a unique gene cluster (wbzB, wbzC, wbzE, and wbzF) involved in LPS precursor biosynthesis. These findings provide novel insights into the mechanisms of phage-host specificity, which are crucial for the effective application of phage AP1 in controlling rice bacterial brown stripe.
... Bacterial genomes encode at least a minimum essential set composed of two topoisomerases, a DNA Gyrase and a Topo I, and up to five topoisomerases ( Figure 1A). In addition, genes encoding for topoisomerases are also found on plasmids [3,4] and bacteriophages [5] suggesting their evolution by horizontal gene transfer and adaptation to the regulation of supercoiling homeostasis. ...
Article
Topoisomerases are the main enzymes capable of resolving the topological constraints imposed by DNA transactions such as transcription or replication. All bacteria possess topoisomerases of different types. Although bacteria with circular replicons should encounter similar DNA topology issues, the distribution of topoisomerases varies from one bacterium to another, suggesting polymorphic functioning. Recently, several proteins restricting, enhancing or modifying the activity of topoisomerases were discovered, opening the way to a new area of understanding DNA topology management during the bacterial cell cycle. In this review, we discuss the distribution of topoisomerases across the bacterial phylum and current knowledge on the interplay among the different topoisomerases to maintain topological homeostasis.
... The genome of BM7 contains three internal virion proteins, six baseplate wedge subunits, and tail sheath proteins, which are present in all previously iso- Ananna et al. Virus Research xxx (xxxx) 199491 lated long contractile-tailed phages, such as T4 or K. pneumoniae, infecting P545, vB_KpnMFBKp34, vB_KpnM_FBKp24, and ZCKP1 etc. (Bonilla et al., 2021;Li et al., 2020;Miller et al., 2003;Taha et al., 2018). The initiation of phage infection occurs through host cell binding by tail fiber protein, leading to conformational changes in the baseplate from a hexagonal dome-shaped to a planar star-shaped structure, causing the tail sheath to contract, creating a path by driving the tail tube in the host cell wall (Yap et al., 2016). ...
Article
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Bacteriophages or bacteria infecting viruses are genetically diverse. Due to the emergence of antimicrobial-resistant bacteria, lytic bacteriophages are gaining enormous attention for treating superbug infections. Klebsiella pneumoniae is one of the eight most significant nosocomial pathogens and is addressed as a critical priority pathogen by WHO, requiring alternative treatment options. We reported two highly lytic bacteriophages, Klebsiella phage Kpn BM7 and the novel Klebsiella phage Kpn BU9, isolated from hospital wastewater and exhibiting lytic activity against different clinical isolates. Whole-genome analysis revealed that phages BM7 and BU9 belong to class Caudoviricetes. Phage BM7, with a genome length of 170,558 bp, is a member of the genus Marfavirus and the species Marfavirus F48. While phage BU9, with a genome length of 60,450 bp, remains unclassified. Neither phage harbors any lysogenic, toxin, or antimicrobial resistance genes. Both phages can steadily survive up to 40 °C and at pH 5–7. The optimal MOI was 0.1 for BM7 and 1 for BU9, with short latent periods of 10 and 25 min and burst sizes of 85 PFU/cell and 12 PFU/cell, respectively. This is the first carbapenem-resistant K. pneumoniae targeting lytic phages to be reported from Bangladesh. This study suggests that BM7 and BU9 are potential candidates for targeting carbapenem-resistant K. pneumoniae.
... Bacteriophages (or phages), Earth's most abundant biological entity, represent unparalleled genetic diversity. Yet, despite their ubiquity, much of this diversity remains functionally uncharacterized-even in the most well-studied phages [1][2][3][4][5] . While functional genomics in phages has lagged, functional genomics in microbes has made great progress. ...
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Understanding core and conditional gene essentiality is crucial for decoding genotype-phenotype relationships in organisms. We present PhageMaP, a high-throughput method to create genome-scale phage knockout libraries for systematically assessing gene essentiality in bacteriophages. Using PhageMaP, we generate gene essentiality maps across hundreds of genes in the model phage T7 and the non-model phage Bas63, on diverse hosts. These maps provide fundamental insights into genome organization, gene function, and host-specific conditional essentiality. By applying PhageMaP to a collection of anti-phage defense systems, we uncover phage genes that either inhibit or activate eight defenses and offer novel mechanistic hypotheses. Furthermore, we engineer synthetic phages with enhanced infectivity by modular transfer of a PhageMaP-discovered defense inhibitor from Bas63 to T7. PhageMaP is generalizable, as it leverages homologous recombination, a universal cellular process, for locus-specific barcoding. This versatile tool advances bacteriophage functional genomics and accelerates rational phage design for therapy.
... To explore the biological functions of phage-derived proteins, gene deletion has been the primary method historically employed, providing critical insights into gene essentiality for the phage infection process [6,7]. However, mutagenesis targeting only the catalytic residues rather than deleting the entire protein offers a possibility to elucidate the enzyme's role within a specific molecular context. ...
... The generation of catalytically inactive proteins preserves the other potential functions of the studied protein, such as involvement in protein-protein or protein-nucleic acid interactions. Particularly in phages with complex genomes, like phage T4, which has numerous overlapping genes and gene splicing arrangements [7], targeted mutagenesis is crucial to avoid unintended effects on other genes' expression. ...
... However, in this scenario, phage infections must be carried out using E. coli strains that harbor additional plasmids and thus do not represent the wild-type E. coli. Furthermore, α-/β-gt serve as auxiliary genes [7,17,22], as the encoded proteins are pivotal in protecting phage DNA through glycosylation from host defense systems and host nucleases, including Cas nucleases [15,23]. Thus, DNA modifications such as 5hmdC and 5ghmdC prevent the effective use of CRISPR-Cas for targeted phage mutagenesis and counterselection. ...
Article
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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 first involves the addition of condensation reagents that form a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl groups of the linear RNA. 163 However, this method inevitably produces by-products such as 2', 5'-phosphodiester linkages from the same condensation reaction. Next, circularization of linear RNAs can be achieved by enzymatic ligation with DNA/RNA ligases. ...
Article
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Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that perform diverse roles in various cellular processes and have been implicated in many pathological conditions. Owing to their intrinsic stability and low immunogenicity, circRNAs have garnered significant interest for their therapeutic potential in multiple diseases, with advancements in efficient in vitro production methods and optimized delivery systems. In this review, we provide a comprehensive overview of current knowledge on circRNA biogenesis and functions, and summarize recent advances in various technologies for circRNA research, including their profiling, validation, and biosynthesis. We also discuss key delivery strategies and therapeutic applications, highlighting the promising prospects and current challenges for the clinical development of circRNA-based therapeutics. Research to date has shown that circRNAs are not merely splicing errors and that circRNA-based therapeutic platforms may have superior application prospects from bench to bedside.