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University of Cambridge

1. Bergen, Paul Michael. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB.

Degree: 2017, University of Cambridge

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

Subjects/Keywords: type III secretion systems; T3SS; Salmonella; Flagella; Bacterial Motility; FlhB; fT3SS; Ordered protein export; SctU

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Bergen, P. M. (2017). Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. (Thesis). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/267915

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Bergen, Paul Michael. “Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. ” 2017. Thesis, University of Cambridge. Accessed September 23, 2019. https://www.repository.cam.ac.uk/handle/1810/267915.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Bergen, Paul Michael. “Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. ” 2017. Web. 23 Sep 2019.

Vancouver:

Bergen PM. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. [Internet] [Thesis]. University of Cambridge; 2017. [cited 2019 Sep 23]. Available from: https://www.repository.cam.ac.uk/handle/1810/267915.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Bergen PM. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. [Thesis]. University of Cambridge; 2017. Available from: https://www.repository.cam.ac.uk/handle/1810/267915

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation


University of Cambridge

2. Bergen, Paul Michael. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB.

Degree: PhD, 2017, University of Cambridge

Flagella, the helical propellers that extend from the bacterial cell surface, illustrate how complex nanomachines assemble outside the cell. The sequential construction of the flagellar rod, hook, and filament requires export of thousands of structural subunits across the cell membrane and this is achieved by a specialised flagellar Type III Secretion System (fT3SS) located at the base of each flagellum. The fT3SS imposes a crude ordering of subunits, with filament subunits only exported once the rod and hook are complete. This “export specificity switch” is controlled by the FlhB component of the fT3SS export gate in response to a signal from the exported molecular ruler FliK, which monitors the length of the growing hook. This study seeks to clarify how rod and hook subunits interact with FlhB, and how FlhB switches export specificity. Rod and hook subunits possess a conserved gate recognition motif (GRM; Fxxxφ, with φ being any hydrophobic residue) that is proposed to bind a surface-exposed hydrophobic patch on the FlhB cytosolic domain. Mutation of the GRM phenylalanine and the final hydrophobic residue resulted in impaired subunit export and decreased cell motility. Isothermal titration calorimetry was performed to assess whether subunit export order is imposed at FlhB. These experiments showed that rod and hook subunits bind to FlhB with micromolar dissociation constants (5-45 μM), suggesting transient interactions. There was no clear correlation between subunit affinity for FlhB and the order of subunit assembly in the nascent flagellum. Solution-state nuclear magnetic resonance (NMR) spectroscopy supported prior data showing that rod and hook subunits interact with FlhB’s surface-exposed hydrophobic patch. NMR also indicated that residues away from the patch undergo a conformational change on subunit binding. FlhB autocleaves rapidly in its cytosolic domain, and the resulting polypeptides (FlhBCN and FlhBCC) are held together by non-covalent interactions between b-strands that encompass the autocleavage site. The autocleavage event is a prerequisite for the export specificity switch, but its function is unclear. Analysis of the cellular localization of FlhBCN and FlhBCC revealed that FlhBCC dissociated from the membrane export machinery, but only in the presence of FliK. Biochemical and biophysical studies of FlhB variants that undergo export specificity switching in the absence of FliK showed that these FlhB “autonomous switchers” were less stable than wildtype FlhB and their FlhBCC domain could dissociate from the export machinery in the absence of FliK. The results suggest that the export specificity switch involves a FliK-dependent loss of FlhBCC from the export machinery, eliminating the binding site for rod and hook subunits.

Subjects/Keywords: 616.9; type III secretion systems; T3SS; Salmonella; Flagella; Bacterial Motility; FlhB; fT3SS; Ordered protein export; SctU

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Bergen, P. M. (2017). Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. (Doctoral Dissertation). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/267915 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725595

Chicago Manual of Style (16th Edition):

Bergen, Paul Michael. “Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB.” 2017. Doctoral Dissertation, University of Cambridge. Accessed September 23, 2019. https://www.repository.cam.ac.uk/handle/1810/267915 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725595.

MLA Handbook (7th Edition):

Bergen, Paul Michael. “Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB.” 2017. Web. 23 Sep 2019.

Vancouver:

Bergen PM. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. [Internet] [Doctoral dissertation]. University of Cambridge; 2017. [cited 2019 Sep 23]. Available from: https://www.repository.cam.ac.uk/handle/1810/267915 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725595.

Council of Science Editors:

Bergen PM. Characterisation of the structure and function of the Salmonella flagellar export gate protein, FlhB. [Doctoral Dissertation]. University of Cambridge; 2017. Available from: https://www.repository.cam.ac.uk/handle/1810/267915 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725595

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