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    Extracytoplasmic stress responses induced by a model secretin : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2015) Spagnuolo, Julian
    Pathogenic bacteria export large proteins and protein complexes, including virulence factors, using dedicated transenvelope multiprotein machinery, collectively called secretion systems. Four of these protein export machines found in Gram-negative bacteria, type 2/3 secretion systems, filamentous phage assembly-secretion system and the type 4 pilus assembly system contain large homologous gated channels, called secretins, in the outer membrane. Secretins are radially symmetrical homomultimers (luminal diameter 6-8 nm) interrupted by an internal septum or gate. Expression of these channels imposes a fitness cost to bacteria. While stress induced by model secretin pIV has been previously investigated using microarrays, this thesis is the first RNAseq characterisation of secretin stress responses. Furthermore, this is the first comparison of stress imposed by a closed-gate secretin (wildtype pIV), vs. an isogenic leaky-gate variant, the latter serving as a model of an open-gate substrate-secreting channel. The high sensitivity to changes in gene expression and low background noise of the RNA-seq approach have greatly expanded the known secretin stress responses to include the SoxS, CpxR and RcsB/RcsAB regulons, in addition to the known involvement of the Psp response. A synthetic lethality analysis of candidate genes in these pathways suggested that the leaky-gate secretins, besides rendering the Psp response essential for survival, also stimulate the SoxS and RcsB/RcsAB regulons for protection of the cells. Knowledge of the secretin stress expanded by this work helped identify potential targets for development of much-needed antibiotics against toxinsecreting Gram-negative bacteria.
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    Analysis of gate residues in the type 2 secretin PulD : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2012) Whitaker, Rowan
    Secretins are gated outer-membrane channels with large internal pore sizes (6-10 nm). They are the outer membrane components of bacterial trans-envelope complexes that assemble/export filamentous bacteriophages as well as pili, complex protein toxins and virulence factors. 12-14 identical subunits form the radially symmetrical channels which share a common architecture - a 3-tiered barrel with middle septum. Secretins are essential components of Gram-negative Type 2/3 secretion systems, spanning the outer membrane and interacting with the inner membrane components of transport machinery. Since secretins have such large pore diameters a simple channel would allow noxious compounds through the normally impermeable outer membrane. The presence of a gate structure allows for the controlled opening and closing of secretin channels, in response to specific cues regulating protein export. Here I have determined gate-structural elements of the Klebsiella oxytoca Type 2 Secretin, PulD. Random mutagenesis coupled with selection for open or 'leaky'-gate phenotypes created a library of mutations which were mapped by DNA sequence analysis. Analysis of leaky mutants revealed 12 distinct missense point mutations in pulD. Additionally, two deletion mutants were isolated, spanning 5 and 9 amino acids, both conferring a leaky gate phenotype. Comparison of these pulD mutations with those previously identified in another secretin gene encoding the Escherichia coli filamentous phage f1 secretin pIV, reveals mutations in both are localised in two main clusters that correspond to regions within the secretin homology domain. Named GATE1 and GATE2, these clusters indicate functional gate regions in both secretins.