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Assembly of the Bacterial Flagellum : How Salmonella Exports Flagellar Proteins and Controls Hook Length

Assembly of the Bacterial Flagellum : How Salmonella Exports Flagellar Proteins and Controls Hook Length


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ERHARDT, Marc, 2011. Assembly of the Bacterial Flagellum : How Salmonella Exports Flagellar Proteins and Controls Hook Length [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Erhardt2011Assem-12838, title={Assembly of the Bacterial Flagellum : How Salmonella Exports Flagellar Proteins and Controls Hook Length}, year={2011}, author={Erhardt, Marc}, address={Konstanz}, school={Universität Konstanz} }

eng Erhardt, Marc Erhardt, Marc 2011-05-02T07:35:25Z Assembly of the Bacterial Flagellum : How Salmonella Exports Flagellar Proteins and Controls Hook Length terms-of-use 2013-04-06T22:25:04Z Bacteria propel themselves through liquid environments using rotation of a propeller like organelle, the flagellum. Flagella are energized by the membrane ion gradient and enable bacteria to swim towards nutrients and away from harmful substances. This unique nanomachine shares structural and functional similarities to the needle-like injectisome complex that pathogenic bacteria employ to inject virulence factors into eukaryotic host cells. Bacterial flagella and injectisomes contain a specialized protein export system, termed ’type III secretion’, that functions to deliver structural subunits and effector proteins to the outside of the cytoplasmic membrane. Type III secretion systems are made of multiple proteins, however, the function of individual subunits and the molecular mechanism of protein translocation is poorly understood.<br />The first part of this thesis reports that the flagellar type III secretion system functions as a proton-driven protein exporter and demonstrates that many components of the apparatus have a facilitating role and are dispensable for the actual protein translocation process. Treatment with a protonophore that disrupts the membrane proton gradient of the cell prevented export of flagellar substrates. In a mutant strain deleted for the flagellar-specific ATPase FliI, we observed weak swarming motility and rare formation of flagella. Hydrolysis of ATP had been considered to provide energy for protein translocation via the flagellar type III export apparatus but these findings demonstrate that flagellar secretion in Salmonella enterica requires the proton motive force while ATP hydrolysis is not essential.<br />For efficient export function of the flagellar type III secretion system, six integral membrane proteins (FliOPQR FlhAB), three soluble proteins (FliHIJ) and the rotor- switch complex (FliGMN) are needed. We sought mutants that allowed for export of a model substrate into the periplasm in the absence of the rotor-switch complex, the C-ring. We isolated mutants in known and unknown flagellar regulatory loci that resulted in at least two-fold increased expression of the flagellar master operon, flhDC. The increased flhDC expression coincided with elevated levels of hook-basal-body formation. These results indicate that the C-ring functions primarily as the rotor of the flagellum and provides a secondary, facilitating role during type III secretion as a affinity cup-like structure that enhances the specificity and efficiency of the export process.<br />We next measured export of a flagellar-specific model substrate in a battery of export-apparatus mutants. Export of a hook-β-lactamase fusion protein into the periplasm confers quantifiable ampicillin resistance. We found that the soluble components of the flagellar type III secretion system, the cytoplasmic C-ring and the membrane protein FliO are dispensable for export. Overexpression of a single membrane protein, FliP, resulted in significant export of the reporter substrate, indicating that FliP forms the central channel of the secretion apparatus. Finally, we present the first molecular-level hypothesis for the organization and mechanism of the flagellar type III secretion system.<br />For efficient transmission of rotational energy from the flagellar basal-body to the rigid, extracellular filament, a flexible coupling structure is needed. The length of this flexible joint, the hook, is tightly controlled in Salmonella enterica by an intrinsic control mechanism. A molecular ruler, FliK, measures the length of the hook and transmits this information back to the FlhB component of the secretion apparatus at the base of the flagellum. Here, an interaction between the carboxy-terminus of FliK and FlhB induces a specificity switch in the flagellar type III secretion apparatus from secretion of rod- hook-type substrates to secretion of late substrates, including the filament subunits. Several models for the mechanism of length control have been proposed, including a ’static ruler’ and a ’measuring cup’, while failing to explain all published data on hook length control.<br />The second part of this thesis reports that FliK acts as an infrequent molecular ruler that is intermittently secreted during hook polymerization. We refuted the previous ’cup model’ for flagellar hook length control by demonstrating normal hook length control in the absence of the rotor-switch complex that was thought to act as a ’measuring cup’. FliK deletion variants that were previously reported to control hook length without secretion were in fact secreted. By uncoupling hook polymerization from FliK expression, we demonstrated that secreted FliK immediately triggers the specificity switch if the ruler is secreted in elongated hooks greater than the physiological length. The probability of a productive interaction of FliK with FlhB, which results in the specificity switch, is an increasing function of hook length. The experimental hook length data displayed excellent agreement with a mathematical model of the Infrequent Ruler hypothesis. Finally, the velocity of FliK secretion correlated inversely with hook length, which provides a possible molecular mechanism for hook length control by FliK. 2011

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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