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Viruses Sep 2020The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity- and . Gene encodes the well-known T4 lysozyme (commonly called T4L) that...
The bacteriophage T4 genome contains two genes that code for proteins with lysozyme activity- and . Gene encodes the well-known T4 lysozyme (commonly called T4L) that functions to break the peptidoglycan layer late in the infection cycle, which is required for liberating newly assembled phage progeny. Gene product (gp5) is the tail-associated lysozyme, a component of the phage particle. It forms a spike at the tip of the tail tube and functions to pierce the outer membrane of the host cell after the phage has attached to the cell surface. Gp5 contains a T4L-like lysozyme domain that locally digests the peptidoglycan layer upon infection. The T4 Spackle protein (encoded by gene ) has been thought to play a role in the inhibition of gp5 lysozyme activity and, as a consequence, in making cells infected by bacteriophage T4 resistant to later infection by T4 and closely related phages. Here we show that (1) gp61.3 is secreted into the periplasm where its N-terminal periplasm-targeting peptide is cleaved off; (2) gp61.3 forms a 1:1 complex with the lysozyme domain of gp5 (gp5Lys); (3) gp61.3 selectively inhibits the activity of gp5, but not that of T4L; (4) overexpression of gp5 causes cell lysis. We also report a crystal structure of the gp61.3-gp5Lys complex that demonstrates that unlike other known lysozyme inhibitors, gp61.3 does not interact with the active site cleft. Instead, it forms a "wall" that blocks access of an extended polysaccharide substrate to the cleft and, possibly, locks the enzyme in an "open-jaw"-like conformation making catalysis impossible.
Topics: Bacteriophage T4; Crystallography, X-Ray; Escherichia coli; Genome, Viral; Muramidase; Protein Conformation; Viral Proteins
PubMed: 32987925
DOI: 10.3390/v12101070 -
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 -
Virology Journal Dec 2010The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can... (Review)
Review
The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.
Topics: Bacteriophage T4; Bacteriophage T7; Crystallography, X-Ray; DNA Replication; Macromolecular Substances; Models, Biological; Models, Molecular; Protein Structure, Quaternary; Protein Structure, Tertiary; Scattering, Small Angle; Viral Proteins
PubMed: 21129204
DOI: 10.1186/1743-422X-7-359 -
Journal of Bacteriology Jul 2011Like most phages with double-stranded DNA, phage T4 exits the infected host cell by a lytic process requiring, at a minimum, an endolysin and a holin. Unlike most...
Like most phages with double-stranded DNA, phage T4 exits the infected host cell by a lytic process requiring, at a minimum, an endolysin and a holin. Unlike most phages, T4 can sense superinfection (which signals the depletion of uninfected host cells) and responds by delaying lysis and achieving an order-of-magnitude increase in burst size using a mechanism called lysis inhibition (LIN). T4 r mutants, which are unable to conduct LIN, produce distinctly large, sharp-edged plaques. The discovery of r mutants was key to the foundations of molecular biology, in particular to discovering and characterizing genetic recombination in T4, to redefining the nature of the gene, and to exploring the mutation process at the nucleotide level of resolution. A number of r genes have been described in the past 7 decades with various degrees of clarity. Here we describe an extensive and perhaps saturating search for T4 r genes and relate the corresponding mutational spectra to the often imperfectly known physiologies of the proteins encoded by these genes. Focusing on r genes whose mutant phenotypes are largely independent of the host cell, the genes are rI (which seems to sense superinfection and signal the holin to delay lysis), rIII (of poorly defined function), rIV (same as sp and also of poorly defined function), and rV (same as t, the holin gene). We did not identify any mutations that might correspond to a putative rVI gene, and we did not focus on the famous rII genes because they appear to affect lysis only indirectly.
Topics: Amino Acid Sequence; Bacteriophage T4; Base Sequence; Escherichia coli; Lysogeny; Molecular Sequence Data; Mutation; Viral Proteins
PubMed: 21571993
DOI: 10.1128/JB.00138-11 -
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 -
Virology Journal Dec 2010Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image... (Review)
Review
Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.
Topics: Bacteriophage T4; Crystallography, X-Ray; Imaging, Three-Dimensional; Macromolecular Substances; Microscopy, Electron; Models, Biological; Models, Molecular; Myoviridae; Viral Tail Proteins
PubMed: 21129200
DOI: 10.1186/1743-422X-7-355 -
PloS One 2017Increasing isolation of the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia has caused alarm worldwide due to the limited treatment options...
Increasing isolation of the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia has caused alarm worldwide due to the limited treatment options available. A potential treatment option for fighting this bacterium is 'phage therapy', the clinical application of bacteriophages to selectively kill bacteria. Bacteriophage DLP6 (vB_SmoM-DLP6) was isolated from a soil sample using clinical isolate S. maltophilia strain D1571 as host. Host range analysis of phage DLP6 against 27 clinical S. maltophilia isolates shows successful infection and lysis in 13 of the 27 isolates tested. Transmission electron microscopy of DLP6 indicates that it is a member of the Myoviridae family. Complete genome sequencing and analysis of DLP6 reveals its richly recombined evolutionary history, featuring a core of both T4-like and cyanophage genes, which suggests that it is a member of the T4-superfamily. Unlike other T4-superfamily phages however, DLP6 features a transposase and ends with 229 bp direct terminal repeats. The isolation of this bacteriophage is an exciting discovery due to the divergent nature of DLP6 in relation to the T4-superfamily of phages.
Topics: Bacteriophage T4; Microscopy, Electron, Transmission; Phylogeny; Promoter Regions, Genetic; Stenotrophomonas maltophilia; Terminator Regions, Genetic
PubMed: 28291834
DOI: 10.1371/journal.pone.0173341 -
Scientific Reports Mar 2019Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and...
Enzyme immobilization is widely applied in biocatalysis to improve stability and facilitate recovery and reuse of enzymes. However, high cost of supporting materials and laborious immobilization procedures has limited its industrial application and commercialization. In this study, we report a novel self-assembly immobilization system using bacteriophage T4 capsid as a nanocarrier. The system utilizes the binding sites of the small outer capsid protein, Soc, on the T4 capsid. Enzymes as Soc fusions constructed with regular molecular cloning technology expressed at the appropriate time during phage assembly and self-assembled onto the capsids. The proof of principle experiment was carried out by immobilizing β-galactosidase, and the system was successfully applied to the immobilization of an important glycomics enzyme, Peptide-N-Glycosidase F. Production of Peptide-N-Glycosidase F and simultaneous immobilization was finished within seven hours. Characterizations of the immobilized Peptide-N-Glycosidase F indicated high retention of activity and well reserved deglycosylation capacity. The immobilized Peptide-N-Glycosidase F was easily recycled by centrifugation and exhibited good stability that sustained five repeated uses. This novel system uses the self-amplified T4 capsid as the nanoparticle-type of supporting material, and operates with a self-assembly procedure, making it a simple and low-cost enzyme immobilization technology with promising application potentials.
Topics: Bacteriophage T4; Binding Sites; Capsid; Enzymes, Immobilized; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Virus Assembly
PubMed: 30890747
DOI: 10.1038/s41598-019-41378-9 -
Postepy Higieny I Medycyny... May 2010Bacteriophage T4 of the Myoviridae family is ubiquitous in the environment and living organisms. In microbiology it has become a universal research model for the... (Review)
Review
Bacteriophage T4 of the Myoviridae family is ubiquitous in the environment and living organisms. In microbiology it has become a universal research model for the mechanisms of many biological processes, including bacteriophage infection. T4 phage is a tailed phage, the most frequent bacteriophage group. It is made up of a head, a contractile tail, and dsDNA. Its tail is a complex structure composed of a collar and its whiskers, a tail tube, a base plate, short fibers, and tail fibers. All these elements cooperate in effective infection. The main host of bacteriophage T4 is Escherichia coli. Adsorption on the bacterial surface is crucial for infection. It depends on specific receptors: lipopolysaccharides and OmpC protein. The high bacteriophage specificity requires specific structures (compositions) of both bacterial and bacteriophage (gp12, gp37) elements. The introduction of phage DNA into the bacterium engages a group of proteins, for example those essential for effective tail contraction and membrane fusion and those with enzymatic activity. In the infected bacterial cell, T4 starts to control cell metabolism with phage replication and expression factors. The final stage of infection is assemblage and lysis. Here the role of bacterial and bacteriophage elements in the above processes is presented and their cooperation with regard to currently identified molecular regions of activity.
Topics: Bacteriophage T4; Capsid Proteins; Escherichia coli; Humans
PubMed: 20498502
DOI: No ID Found -
ACS Synthetic Biology Oct 2017Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of...
Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.
Topics: Bacteriophage T4; CRISPR-Cas Systems; Genetic Engineering; Plasmids; Point Mutation
PubMed: 28657724
DOI: 10.1021/acssynbio.7b00179