Dataset Information

Structural studies of the nucleus-like assembly of jumbo bacteriophage 201φ2-1.

ABSTRACT: The jumbo phages encode proteins that assemble to form a nucleus-like compartment in infected cells. Here we report the cryo-EM structure and biochemistry characterization of gp105, a protein that is encoded by the jumbo phage 201φ2-1 and is involved in the formation of the nucleus-like compartment in phage 201φ2-1 infected Pseudomonas chlororaphis. We found that, although most gp105 molecules are in the monomeric state in solution, a small portion of gp105 assemble to form large sheet-like assemblies and small cube-like particles. Reconstruction of the cube-like particles showed that the particle consists of six flat head-to-tail tetramers arranged into an octahedral cube. The four molecules at the contact interface of two head-to-tail tetramers are 2-fold symmetry-related and constitute a concave tetramer. Further reconstructions without applying symmetry showed that molecules in the particles around the distal ends of a 3-fold axis are highly dynamic and have the tendency to open up the assembly. Local classifications and refinements of the concave tetramers in the cube-like particle resulted in a map of the concave tetramer at a resolution of 4.09 Å. Structural analysis of the concave tetramer indicates that the N and C terminal fragments of gp105 are important for mediating the intermolecular interactions, which was further confirmed by mutagenesis studies. Biochemistry assays showed that, in solution, the cube-like particles of gp105 are liable to either disassemble to form the monomers or recruit more molecules to form the high molecular weight lattice-like assembly. We also found that monomeric gp105s can self-assemble to form large sheet-like assemblies in vitro, and the assembly of gp105 in vitro is a reversible dynamic process and temperature-dependent. Taken together, our results revealed the dynamic assembly of gp105, which helps to understand the development and function of the nucleus-like compartment assembled by phage-encoded proteins.

SUBMITTER: Liu Z

PROVIDER: S-EPMC10149743 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Structural studies of the nucleus-like assembly of jumbo bacteriophage 201φ2-1.

Liu Zhe Z Xiang Ye Y

Frontiers in microbiology 20230417

The jumbo phages encode proteins that assemble to form a nucleus-like compartment in infected cells. Here we report the cryo-EM structure and biochemistry characterization of gp105, a protein that is encoded by the jumbo phage 201φ2-1 and is involved in the formation of the nucleus-like compartment in phage 201φ2-1 infected <i>Pseudomonas chlororaphis</i>. We found that, although most gp105 molecules are in the monomeric state in solution, a small portion of gp105 assemble to form large sheet-li ...[more]

PMID: 37138628

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Project description:Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores. The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.

Project description:BackgroundBacteriophages are the most genetically diverse biological entities in nature. Our current understanding of phage biology primarily stems from studies on a limited number of model bacteriophages. Jumbo phages, characterized by their exceptionally large genomes, are less frequently isolated and studied. Some jumbo phages exhibit remarkable genetic diversity, unique infection mechanisms, and therapeutic potential.MethodsIn this study, we describe the isolation of Sharanji, a novel Escherichia coli jumbo phage, isolated from chicken feces. The phage genome was sequenced and analyzed extensively through gene annotation and phylogenetic analysis. The jumbo phage was phenotypically characterized through electron microscopy, host range analysis, and survival at different pH and temperatures, and one-step growth curve assay. Finally, Sharanji mediated infection of E. coli is studied through fluorescence microscopy, to analyze its mechanism of infection compared to well-studied nucleus-forming jumbo phages.ResultsWhole genome sequencing reveals that Sharanji has a genome size of 350,079 bp and is a phage encompassing 593 ORFs. Genomic analysis indicates that the phage belongs to the Asteriusvirus genus and is related to E. coli jumbo phages PBECO4 and 121Q. Phenotypic analysis of isolated phage Sharanji, indicates that the phage size is 245.3 nm, and it is a narrow-spectrum phage infecting E. coli K12 strains, but not other bacteria including avian pathogenic E. coli. Infection analysis using microscopy shows that Sharanji infection causes cell filamentation. Furthermore, intracellular phage nucleus-like structures were not observed in Sharanji-infected cells, in contrast to infection by ΦKZ-like jumbo phages.ConclusionsOur study reports the isolation and characterization of Sharanji, one of the large E. coli jumbo phages. Both genotypic and phenotypic analyses suggest that Sharanji serves as a unique model system for studying phage-bacteria interactions, particularly within the context of non-nucleus-forming jumbo phages. Further exploration of jumbo phages holds promise for uncovering new paradigms in the study of microbial viruses.

Dataset Information

Structural studies of the nucleus-like assembly of jumbo bacteriophage 201φ2-1.

Publications

Structural studies of the nucleus-like assembly of jumbo bacteriophage 201φ2-1.

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