Browsing by Author "Bennett, Nicholas James"
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- ItemUnlocking the M13 (f1 and fd) virion : investigation into the role of the pIII C-domain of F specific filamentous bacteriophage in infection : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand.(Massey University, 2009) Bennett, Nicholas JamesFf filamentous bacteriophage infect male (F+) strains of Escherichia coli and are assembled at the cell membranes, by a secretion-like, non-lethal process. The pIII protein, located at one end of the virion-filament, is required at both the beginning and the end of the phage life cycle. During infection, the N-terminal domains of pIII, N2 and N1, bind to the primary and secondary host receptors, F pilus and TolA protein, respectively. At the end of the life cycle, the pIII C-domain mediates the termination and release of virions. Thus, both entry and release involve structural transitions of the virus coupled to membrane transactions of the virion proteins. "Unlocking” of the highly stable virion presumably results in membrane integration during entry, whereas a reverse event, “locking” of the virion, occurs upon detachment from the membrane at termination step of assembly/secretion. Recently, it was shown that the pIII C-domain plays an active role at the step of entry. This finding implicates the C-domain of pIII in “unlocking” of the virion, presumably resulting in the exposure of the membrane anchor at the very C-terminus of pIII (Bennett & Rakonjac, 2006). To further this work, this thesis has mapped the portion of the pIII C-domain required for infection, by constructing a set of nested deletions of the C-domain fused to the receptor binding domains N1 and N2, and then determined the infectivity of phage carrying the mutant proteins. This mapped the portion of the C-domain required for phage infection is different to that required for termination of assembly. The different requirement for entry and release suggests that the two processes are carried out by distinct mechanisms and/or depend on different sets of accessory proteins. In addition, a system was designed for the efficient production and purification of very short virions, the length of which is 1/20 that of the wild-type f1. These short virions, called microphage, are the first step towards the structural analyses of the phage termini cap structures, of which one contains pIII in the “locked” conformation.
- ItemUnlocking the M13 (F1) virion : investigation into the role of pIII C domain of F specific filamentous bacteriophage in infection : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2005) Bennett, Nicholas JamesFilamentous phage adsorb to the host cells by binding of the N2 domain of pIII to the tip of the F pilus. Binding of N1 domain of pIII to the secondary receptor (Tol A), triggers the opening of the virion by a poorly understood mechanism. Filamentous phage assembly is a secretion-like process. The assembly is terminated and virion released from the membranes by C domain of pIII. Because the infection is a reversal of assembly, it can be hypothesized that the C domain of pIII plays an active role in the infection. To test this hypothesis, we have set up a system in which virions carried a mixture of two types of mutant pIII molecules: i) functional N1N2 domains fused to a short C domain that can be incorporated but cannot terminate assembly and release the phage from the membrane: ii) C domain only, which can terminate phage assembly, but lacks the receptor-binding domains N1N2. The infectivity of the particles was as low as 0.21% that of the positive control setup in which virions carried a mixture of wild-type pIII and C domain. Therefore, a functional C domain covalently linked to the receptor domain N1N2 is required for infection. These findings suggest that simple binding of N1 domain of pIII to the periplasmic receptor TolA is not sufficient for infection. Rather, this interaction may, via functional C domain of pIII, trigger a conformational change required for the downstream events which result in the virion uncoating and DNA entry. To add further weight to this model, a "microphage" producing system was designed to produce short phage particles suitable for Cryo-EM structural analysis.