Goldstone, Robert J. (2012) Investigating the relationship between quorum sensing, motility, and the type 3 secretion system of Yersinia pseudotuberculosis. PhD thesis, University of Nottingham.
Over the course of the last two decades, research into the role of quorum sensing (QS) in regulating diverse bacterial behaviours has exploded, and around twelve years ago, a QS network was identified in the enteropathogenic bacterium Yersinia pseudotuberculosis, which was shown to control motility and cellular clumping. This thesis seeks to expand this regulatory relationship and explore the causes and consequences of the link between QS and motility, which affects pleiotropic processes including the type 3 secretion system (T3SS) and biofilm formation. Indeed, the clumping phenotype first explored by Atkinson et al. (1999), is linked to QS-dependent regulation of the T3SS, since the deletion of several QS genes results in liquid culture biofilm (LCB) formation. This is concomitant with T3S protein secretion into culture supernatant, which occurs under normally non-inducing conditions, while deleting the T3SS structural component yscJ prevents secretion and LCB formation. De-repression of the T3SS and the development of LCBs also occurs following mutation of the flagella regulators flhDC and fliA, revealing that QS and the flagella system co-regulate LCBs. However, interestingly it was found that LCB formation and secretion also occurs following mutation of the flagella structural gene flhA. The ΔflhA mutant represents a flagella-minus strain, in which the underlying regulatory circuit mediated by FlhDC and FliA is intact, suggesting that an element of the flagella structure that depends on FlhA activity acts as a check-point governing expression of the T3SS.
Both QS and the flagella system positively regulate biofilm formation by Y. pseudotuberculosis on the surface of the nematode worm, Caenorhabditis elegans. Surprisingly, the up-regulated T3SS was found to be responsible for mediating down-regulation of biofilm formation by Y. pseudotuberculosis QS mutants, since subsequent deletion of yscJ could restore biofilms to wild-type levels. This suggested that a component of the injectisome was capable of influencing cellular processes in addition to its role in secretion. In light of the link regulatory link between flagella and T3S, this raised the possibility that the injectisome could play a role in the reciprocal regulation of motility. Since the genetic regulatory network underpinning expression of the T3SS is intact in the ΔyscJ mutant, like the ΔflhA mutant for flagella, the ΔyscJ mutant can reveal the role of the injectisome structure in modulating gene expression. By phenotypic observation, it was determined that the ΔyscJ mutant displayed aberrant flagella mediated motility, swimming vigorously under conditions in which the wild-type did not, and, similar to the over-production of Yop proteins in the ΔflhA mutant, the ΔyscJ mutant over-produces flagellin. This suggests that a component of the T3SS injectisome acts as a checkpoint to regulate motility, which appears to be at the level of transcription, since the ΔyscJ mutant displays up-regulation of the flagella regulators flhDC and fliA. Indeed, the relationship between T3S and motility appears to require a direct influence on QS, since subsequent mutation of ypsI and ytbI abolishes ΔyscJ-dependent hyper-motility, the ΔyscJ mutant displays altered expression of the QS system genes. Furthermore, for the emerging transcriptional relationship between these systems, the flagella and QS mutants which are up-regulated for the production of Yop proteins also over-express the virulence regulator virF, completing the transcriptional regulatory circuit which appears to be crucial for the regulation of lifestyle choices by Y. pseudotuberculosis.
|Item Type:||Thesis (PhD)|
|Faculties/Schools:||UK Campuses > Faculty of Medicine and Health Sciences > School of Molecular Medical Sciences|
|Deposited By:||Mr Robert J Goldstone|
|Deposited On:||15 Oct 2012 12:59|
|Last Modified:||15 Oct 2012 12:59|
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