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Substrate targeting and recognition in the flagellar type III secretion pathway 10.17863/CAM.50254 Bryant, Owain Fraser, Gillian Margaret The bacterial flagellum is a complex molecular machine that enables bacteria to move as well as having additional roles in biofilm formation, adhesion and host cell invasion. The sequential assembly of the flagellar rod, hook and filament requires the export of thousands of structural subunits across the bacterial cell membrane by a specialised flagellar type III secretion system (fT3SS) located at the base of each flagellum. These subunits are unfolded and exported into a narrow 20Å wide channel in the centre of each flagellum energised by the proton motive force (PMF) and facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. Unfolded subunits transit the length of the channel to the flagellum tip where they fold into the nascent structure. The order of subunit export is determined by the stage of flagellum assembly. Early subunits are exported to assemble the rod and hook substructures after which a substrate specificity switch in the fT3SS allows late subunit export. The first aim of this study was to determine how the export signals that reside within early flagellar subunits contribute to export. In this thesis it was found that early flagellar subunits dock at the cytoplasmic domain of FlhB to correctly position an export signal at the subunit extreme N-terminus. The distance between the extreme N-terminal signal and the FlhB gate recognition motif (GRM) was found to be critical for subunit export. Evidence is presented to show that the extreme N-terminal signal of early flagellar subunits converts the FliPQR components of the export gate from a closed to open conformation, allowing subunits to enter the central channel within the flagellum. Further evidence was presented to show that the FlhA component of the flagellar export machinery is involved in export gate opening. Increasing the proton-motive force (PMF) improved subunit export by a strain encoding a FlhA variant defective in export gate opening, indicating that the PMF energises opening of the export gate. The data are compatible with the view that early subunits dock at the cytoplasmic domain of FlhB to correctly position the extreme N-terminal export signal to trigger PMF-driven opening of the export gate. The late filament structural subunits are delivered from their site of synthesis in the cytoplasm to the flagellar export machinery at the membrane by their cognate chaperones. Using motility and export assays, and in vitro and in vivo affinity chromatography pull down assays, data are provided that build on what is currently known about the sequence of binding events between chaperoned subunits and the flagellar export machinery. Specifically, evidence is presented to show that the FlgN chaperone binds the FliJ component of the ATPase complex after FlgN has docked and been released from the cytoplasmic domain of FlhA. The final aim of this study was to determine whether the flagellar export chaperones regulate activation of the PMF-driven fT3SS export machinery. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. This thesis showed that the flagellar export chaperones regulate FliJ activation of FlhA. The data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available. University of Cambridge 2020-04-25 Thesis Doctoral Doctor of Philosophy (PhD) en Department of Pathology https://www.repository.cam.ac.uk/handle/1810/303175 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/26072efe-0a6f-4f18-8e36-e056b1945a9c/download c4ff7ba36d67e4c345e325b0611b273d https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/92bf680b-d349-491c-be42-6d2dc68c7002/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ bacterial flagella biogenesis Type III secretion system proton motive force protein export ATPase