Purified tails from bacteriophage T5
Ontology highlight
ABSTRACT:
SUBMITTER: Romain Linares
PROVIDER: EMPIAR-11319 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Structural basis of bacteriophage T5 infection trigger and <i>E. coli</i> cell wall perforation.
Linares Romain R Arnaud Charles-Adrien CA Effantin Grégory G Darnault Claudine C Epalle Nathan Hugo NH Boeri Erba Elisabetta E Schoehn Guy G Breyton Cécile C
Science advances 20230324 12
Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail (<i>Siphoviridae</i>) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-ele ...[more]
Similar Datasets
Project description: Not available
| EMPIAR-11327 | biostudies-other
Project description:In a recent study, we identified and characterized the long-elusive replicative single-stranded DNA-binding protein of bacteriophage T5, which we showed is related to the eukaryotic transcription coactivator PC4. Here, we provide an extended discussion of these data, report several additional observations and consider implications for the recombination-dependent replication mechanism of the T5 genus, which is still poorly understood.
| S-EPMC3897522 | biostudies-literature
Project description:Coliphage T5 injects its DNA in 2 steps: the first step transfer (FST) region 7.9% is injected and its genes are expressed and only then does the remainder (second step transfer, SST) of its DNA enter the cell. In the FST region, only 2 essential genes (A1 and A2) have been identified and a third (dmp) non-essential gene codes for a deoxyribonucleotide 5' monophosphatase. Thirteen additional putative ORFs are present in the FST region. Numerous properties have been attributed to FST region, including SST, host DNA degradation, inhibition of host RNA and protein synthesis, restriction insensitivity and protection of T5 DNA. These effects do not occur following infection with an A1 mutant. The A2 gene seems only to be involved in SST transfer. This is puzzling since there are more seemingly unrelated effects than there are essential genes to accomplish them and it is possible that some important genes were not identified. This review attempts to analyze these problems that were first identified in the 1970-80 s. In particular, an attempt is made to determine which potential ORFs are conserved in evolution (and thus likely to be important); by comparing T5 to 10 newly isolated and completely sequenced T5-like phages. A similar approach is used to identify conserved repeats, inverted repeats and palindromes that occur in all T5-like phages in the region containing the injection stop signal (iss) and the terminase substrate. Finally, an attempt is made to re-analyze the mechanism whereby T5 protects itself from the enzymes that degrade host DNA, from the RecBCD nuclease and from restriction enzymes. For all of these FST effects new hypotheses and possible new genetic and biochemical approaches are envisaged.
| S-EPMC4743489 | biostudies-literature
Project description:Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.
| S-EPMC5180179 | biostudies-literature
Project description:Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ?500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3'-5' direction is more robust and can be distinguished from the 5'-3' activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation.
| S-EPMC3632103 | biostudies-literature
Project description:The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the ?70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP ? subunit and moderately inhibits RNAP interaction with the discriminator region of ?70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.
| S-EPMC7472727 | biostudies-literature
Project description:The large (90-nm) icosahedral capsid of bacteriophage T5 is composed of 775 copies of the major capsid protein (mcp) together with portal, protease, and decoration proteins. Its assembly is a regulated process that involves several intermediates, including a thick-walled round precursor prohead that expands as the viral DNA is packaged to yield a thin-walled and angular mature capsid. We investigated capsid maturation by comparing cryoelectron microscopy (cryo-EM) structures of the prohead, the empty expanded capsid both with and without decoration protein, and the virion capsid at a resolution of 3.8 Å for the latter. We detail the molecular structure of the mcp, its complex pattern of interactions, and their evolution during maturation. The bacteriophage T5 mcp is a variant of the canonical HK97-fold with a high level of plasticity that allows for the precise assembly of a giant macromolecule and the adaptability needed to interact with other proteins and the packaged DNA.
| S-EPMC6800373 | biostudies-literature
Project description:Phage genome editing is crucial to uncover the molecular mechanisms of virus infection and to engineer bacteriophages with enhanced antibacterial properties. Phage genetic engineering relies mostly on homologous recombination (HR) assisted by the targeted elimination of wild-type phages by CRISPR-Cas nucleases. These strategies are often less effective in virulent bacteriophages with large genomes. T5 is a virulent phage that infects Escherichia coli. We found that CRISPR-Cas9 system (type II-A) had ununiform efficacies against T5, which impairs a reliable use of CRISPR-Cas-assisted counterselection in the gene editing of T5. Here, we present alternative strategies for the construction of mutants in T5. Bacterial retroelements (retrons) proved to be efficient for T5 gene editing by introducing point mutations in the essential gene A1. We set up a protocol based on dilution-amplification-screening (DAS) of phage pools for mutant enrichment that was used to introduce a conditional mutation in another essential gene (A2), insert a new gene (lacZα), and construct a translational fusion of a late phage gene with a fluorescent protein coding gene (pb10-mCherry). The method should be applicable to other virulent phages that are naturally resistant to CRISPR/Cas nucleases.
| S-EPMC8108384 | biostudies-literature
Project description:The vast majority of phages, bacterial viruses, possess a tail ensuring host recognition, cell wall perforation and safe viral DNA transfer from the capsid to the host cytoplasm. Long flexible tails are formed from the tail tube protein (TTP) polymerised as hexameric rings around and stacked along the tape measure protein (TMP). Here, we report the crystal structure of T5 TTP pb6 at 2.2?Å resolution. Pb6 is unusual in forming a trimeric ring, although structure analysis reveals homology with all classical TTPs and related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin). Structures of T5 tail tubes before and after interaction with the host receptor were determined by cryo-electron microscopy at 6?Å resolution. Comparison of these two structures reveals that host-binding information is not propagated to the capsid through conformational changes in the tail tube, suggesting a role of the TMP in this information transduction process.
| S-EPMC5717097 | biostudies-literature
Project description:Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.
| S-EPMC5292684 | biostudies-literature