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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.
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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
... The E. coli RNAP, which caps host NAD-RNAs ab initio during transcription (22), is also responsible for the transcription of T4 phage genes during infection (32). Thus, we supposed that T4 phage transcripts are analogously NAD-capped by the host RNAP. ...
... Apart from that distinct feature of E. coli NAD-RNA promoters, they share the AT-rich -10 element and a T-rich -35 element with the E. coli consensus promoter identified here. The consensus T4 phage promoter also contains an AT-rich -10 element and displays a tendency towards +1A TSSs (Supplementary Figure S6B) in agreement with the previously studied T4 promoter motif (32). 60 % (90 in total) of the T4 phage promoters identified here initiate with adenosine. ...
... Further, we detected similar amount of progeny released by T4 NudE.1 E64,65Q and T4 WT and similar burst sizes of both phages ( Figure 7C, Supplementary Table S9). This agrees with previous studies that assessed a NudE.1 deletion T4 phage (37) and described the auxiliary role of nudE.1 gene during T4 phage infection (32). Figure S8B). ...
Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli . NAD also acts as a key molecule in phage-host interactions, where bacterial immune systems deplete NAD to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during phage infections of bacteria and the mechanisms of their synthesis and degradation are unknown in this context. The T4 phage that specifically infects E. coli presents an important model to study phage infections, but a systematic analysis of the presence and dynamics of NAD-RNAs during T4 phage infection is lacking. Here, we investigate the presence of NAD-RNAs during T4 phage infection in a dual manner. By applying time-resolved NAD captureSeq, we identify NAD-capped host and phage transcripts and their dynamic regulation during phage infection. We provide evidence that NAD-RNAs are – as reported earlier – generated by the host RNA polymerase by initiating transcription with NAD at canonical transcription start sites. In addition, we characterize NudE.1 – a T4 phage-encoded Nudix hydrolase – as the first phage-encoded NAD-RNA decapping enzyme. T4 phages carrying inactive NudE.1 display a delayed lysis phenotype. This study investigates for the first time the dual epitranscriptome of a phage and its host, thereby introducing epitranscriptomics as an important field of phage research.
... However, a substantial portion of the viral genetic information remains unknown, with 75% of the more than 2.79 million viral genes cataloged lacking identified functions and without any corresponding homologous genes found in public databases [6]. Even for the well-studied model phage T4, there is a significant gap in knowledge, as 45% of its genes are of unknown function [7]. Identifying non-essential genes for phage would aid in the analysis of phage genomes [5,8], and current functional investigation methods include homologous recombination, CRISPR-Cas, and complete genome reconstruction in non-host systems [9]. ...
Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR–Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.
... The terminase-Viruses 2024, 16,192 4 of 21 DNA complex is then able to dock onto another DNA-empty procapsid, and the process repeats. In this manner, 171 kb of linear DNA representing~103% of the T4 genome, or the headful amount, is packaged into each capsid [43]. The additional~3% more than the full genome map length that is packaged is referred to as a terminal redundancy. ...
In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring–DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.
... [84] Enzymatic strategies Three types of polynucleotide ligases are commonly used in the enzymatic ligation of synthetic oligonucleotides: T4 RNA ligase 1, T4 DNA ligase, and T4 RNA ligase 2. They are encoded in the genome of the bacteriophage T4 and can ligate nicks in single-and/or double-stranded RNA constructs. [85,86] They assist in the circularization process by promoting the ATP-consuming synthesis of a phosphodiester bond between the 3′-hydroxyl (acceptor) and 5′-phosphate (donor) end groups in RNA or DNA. [87] Ribozymatic strategies Ribozymatic strategies are the most commonly used methods for RNA circularization, particularly when the production of large circular RNAs is required. ...
Circular RNAs (circRNAs) are a class of single-stranded RNAs with covalently closed structures. Owing to their not having 3' or 5' ends, circRNAs are highly durable and insusceptible to exonuclease-mediated degradation. Moreover, some circRNAs with certain structures are translatable, making them novel vaccines. Vaccines are efficient tools for immunotherapy, such as for the prevention of infectious diseases and cancer treatment. The immune system is activated during immunotherapy to fight against abnormal allies or invaders. CircRNA vaccines represent a potential new avenue in the vaccine era. Recently, several circRNA vaccines have been synthesized and tested in vitro and in vivo. Our review briefly introduces the current understanding of the biology and function of translatable circRNAs, molecular biology, synthetic methods, delivery of circRNA, and current circRNA vaccines. We also discussed the challenges and future directions in the field by summarizing the developments in circRNA vaccines in the past few years.
... To this end, recombination was used to replace gp141 expression in SEA1 with NanoLuc ® expression, creating SEA1∆gp141.NL. SEA1's gp141 is the homolog of an essential baseplate wedge subunit in the well-studied Escherichia coli phage T4, gp53 [25,26]. In each T4 virion, six copies of gp53 join adjacent wedge complexes and are required to stabilize the hexagonal foundation of the phage baseplate [27][28][29]. ...
... SEA1 was found to have modest genomic similarity to the well-studied Escherichia coli phage T4, approximately 69% identity over 57% query coverage, by BLAST comparison [30]. Only roughly 20% of genes in T4 are described as essential [25,26]. Among these essential genes, the gene encoding gp53 in T4 proved to be an attractive candidate that had not been previously investigated for this purpose. ...
... For example, the disruption of genes involved in host DNA degradation in T4 can result in significantly reduced burst sizes with only a handful of progeny produced per infected cell [38,39]. Importantly, a burst size of at least 10 has been suggested to be a requirement for reliable plaque formation [25]. In order to rule out the possibility of low levels of replication in the absence of gp141, a one-step growth curve was performed [34]. ...
Engineered bacteriophages (phages) can be effective diagnostic reporters for detecting a variety of bacterial pathogens. Although a promising biotechnology, the large-scale use of these reporters may result in the unintentional release of genetically modified viruses. In order to limit the potential environmental impact, the ability of these phages to propagate outside the laboratory was targeted. The phage SEA1 has been previously engineered to facilitate food safety as an accurate and sensitive reporter for Salmonella contamination. In this study, homologous recombination was used to replace the expression of an essential baseplate wedge subunit (gp141) in SEA1 with a luciferase, NanoLuc®. This reporter, referred to as SEA1Δgp141.NL, demonstrated a loss of plaque formation and a failure to increase in titer following infection of Salmonella. SEA1Δgp141.NL was thus incapable of producing infectious progeny in the absence of gp141. In contrast, production of high titer stocks was possible when gp141 was artificially supplied in trans during infection. As a reporter, SEA1Δgp141.NL facilitated rapid, sensitive, and robust detection of Salmonella despite an inability to replicate. These results suggest that replication-deficient reporter phages are an effective method to obtain improved containment without sacrificing significant performance or the ease of production associated with many phage-based diagnostic methods.
... Unless we develop methods to fill the knowledge gap between phage genetic diversity and gene function, we will be seriously constrained in understanding the mechanistic ecology of phages in diverse microbiomes and harness them as engineerable antimicrobials and microbial community editors [13,14]. Gaps in phage gene-function knowledge exist even for some of the most well-studied canonical phages [15,16]. Nevertheless, the application of classical phage genetic tools to a few canonical phages over the last few decades has paved the way for generating foundational knowledge of the phage life cycle [15,17,18]. ...
... Gaps in phage gene-function knowledge exist even for some of the most well-studied canonical phages [15,16]. Nevertheless, the application of classical phage genetic tools to a few canonical phages over the last few decades has paved the way for generating foundational knowledge of the phage life cycle [15,17,18]. A number of recent technological innovations have also addressed the growing knowledge gap between phage-gene-sequence and the encoded function [19][20][21]. ...
... Recent work demonstrated that dCas12a is capable of inhibiting infection by phage λ when targeting the essential gene cro, suggesting that application of dCas12a with arrayed crRNAs might facilitate genome-wide fitness measurements in phages [46]. The ability to effectively block transcription at target sites distant from promoters makes dCas12a potentially well suited for repressing transcription of phage genes within operons that show overlapping genetic architecture [15,17,18,47,48] and those that are highly regulated or vary in expression levels [49][50][51] in a noncompetitive plaque assay. ...
Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage–host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.
... We focused on bacteriophage T4, a virulent Tevenvirinae phage, with an approximatelyAU : Pleasenote 200-nm long myovirus morphology and a 168,903-bp genome, which infects Escherichia coli [33,34]. This phage was selected as it was previously demonstrated to be internalized by mammalian cells, accumulating intracellularly within macropinosomes over time [12,13]. ...
There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.
... Some phages rely on the host RNAP to transcribe their genes. In this case, phage-encoded proteins direct the host RNAP to appropriate promoters throughout the infection [1,2] and/or phage-encoded antitermination factors allow host RNAP to access specific groups of phage genes located downstream of terminators [3][4][5]. Prototypical examples of such strategies are bacteriophages T4 and λ, correspondingly. ...
A nucleus-like structure composed of phage-encoded proteins and containing replicating viral DNA is formed in Pseudomonas aeruginosa cells infected by jumbo bacteriophage phiKZ. The PhiKZ genes are transcribed independently from host RNA polymerase (RNAP) by two RNAPs encoded by the phage. The virion RNAP (vRNAP) transcribes early viral genes and must be injected into the cell with phage DNA. The non-virion RNAP (nvRNAP) is composed of early gene products and transcribes late viral genes. In this work, the dynamics of phage RNAPs localization during phage phiKZ infection were studied. We provide direct evidence of PhiKZ vRNAP injection in infected cells and show that it is excluded from the phage nucleus. The nvRNAP is synthesized shortly after the onset of infection and localizes in the nucleus. We propose that spatial separation of two phage RNAPs allows coordinated expression of phage genes belonging to different temporal classes.
... Surface-exposed S-LPS is important for E. coli colonisation of host digestive tracts [8,36,37]. The OAg forms a protective surface layer around bacterial cells helping to withstand gut environmental challenge, including from host antimicrobial peptides (AMPs) [38] and gut-residing bacteriophages, such as the R-LPS-attacking coliphage T4 [39,40]. Originally isolated from human stool samples, E. coli K-12 strains however were found to be sensitive to coliphage T4 and unable to colonise the human gut [8]. ...
Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E . coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E . coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E . coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.
... A previous report suggested overexpressing plasmid-borne Gp49 in B. subtilis would not obviously affect the growth during the optimal growth condition [9]. As bacteria stay mostly in the stationary phase in the natural environment and complex phages like T4 carry abundant genes during infection to cope with different environments [17], it was necessary to evaluate the effect of Gp49 during the stationary phage since the genome of SPO1 is comparable to that of T4. We then repeated the growth attenuation experiment with an extended observation time. ...
Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a “host-takeover module”, a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism—no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development.
