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
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Date
2015
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Massey University
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Abstract
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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Keywords
Gram-negative bacteria, Pathogenic bacteria, Bacterial cell walls, Secretins, Stress