Browsing by Author "Gold VAM"

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    A Single-Plasmid Inducible-Replication System for High-Yield Production of Short Ff (f1, M13 or fd)-Phage-Derived Nanorods
    (John Wiley and Sons Ltd., 2025-04-01) León-Quezada RI; Miró MG; Khanum S; Sutherland-Smith AJ; Gold VAM; Rakonjac J
    Ff (f1, M13 or fd) filamentous phages have been used for myriad applications including phage display, assembly of nanostructures and as carriers of agents used for diagnostic and therapeutic purposes. Recently, short Ff phage-derived functionalised nanorods have emerged as a superior alternative to full-length filamentous phages for applications from lateral flow assays to cell- and tissue-targeting. Their advantages, such as shorter length and the lack of antibiotic resistance genes, make them particularly promising for expanding the current scope of Ff bionanotechnology and biomedical applications. Limitations to the widespread use of Ff-derived nanorods include a requirement for two plasmids and the relatively low production efficiency. This is due to the presence of only the positive Ff origin of replication, allowing replication of only the positive strand. Here we describe a single-plasmid negative origin-containing inducible-replication system for nanorod production. These improvements simplify and increase nanorod production by two orders of magnitude compared with the constitutive positive origin-only production system. The high concentration of nanorods allows formation of higher-order structures, such as stacks and rafts, as imaged by transmission electron microscopy. In summary, our system will facilitate production and expand the applications of Ff-derived biological nanorods.
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    Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly
    (2022-11-04) Conners R; León-Quezada RI; McLaren M; Bennett NJ; Daum B; Rakonjac J; Gold VAM
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    CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage
    (Springer Nature Limited, 2021-11-02) Conners R; McLaren M; Łapińska U; Sanders K; Stone MRL; Blaskovich MAT; Pagliara S; Daum B; Rakonjac J; Gold VAM
    The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
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    CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
    (2/11/2021) Conners R; McLaren M; Łapińska U; Sanders K; Stone MRL; Blaskovich MAT; Pagliara S; Daum B; Rakonjac J; Gold VAM
    The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.