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The Bacterial Flagellum : New Insights into Regulation, Bioassembly, and Type III Secretion

The Bacterial Flagellum : New Insights into Regulation, Bioassembly, and Type III Secretion

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SINGER, Hanna Magdalena, 2014. The Bacterial Flagellum : New Insights into Regulation, Bioassembly, and Type III Secretion [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Singer2014Bacte-29073, title={The Bacterial Flagellum : New Insights into Regulation, Bioassembly, and Type III Secretion}, year={2014}, author={Singer, Hanna Magdalena}, address={Konstanz}, school={Universität Konstanz} }

2014 2014-10-06T08:08:41Z eng The bacterial flagellum is a self-assembling, nanomachine-like structure used for locomotion of many bacteria, including Salmonella. The attached helical filament can reach lengths over 10 times the length of the cell body. Rotation of the structure allows the bacteria to move in their environment. Surprised by its structural complexity and mode of action, creationists have used the flagellum to challenge Darwin’s evolution theory and suggest it to be an example of intelligent design due to its "irreducible complexity".<br /><br /><br /><br />The focus of the first part of this work was to investigate the complex regulation of flagellar gene expression. The flagellar operon flhDC is a master regulator of flagella synthesis and resides at the top of a highly regulated flagellar gene expression hierarchy. In response to environmental stimuli, flhDC itself is regulated by a variety of positive and negative regulators and has previously been described to autoregulate itself. In Chapter 1, we presented the LuxR-homolog RflM (previously known as EcnR) as an FlhDC-dependent repressor of flhDC transcription. In a feedback loop, the FlhD<sub>4</sub>C<sub>2</sub> protein complex activates rflM and the repressor function of RflM on flhDC accounts for the formerly described autoinhibitory effect of FlhDC.<br /><br /><br /><br />Bacterial flagella and in particular the presence of flagellin play an important role during Salmonella invasion and subsequent infection of epithelial cells and macrophages. The biosynthesis of flagella is therefore spatiotemporally regulated depending on the stage of invasion. Factors needed for bacterial movement, invasion of host cells, and persistence are required at different time points during host infection and thus, regulatory cross-talk between the flagellar, the Salmonella pathogenicity island 1 (Spi1), and the type I fimbrial regulons is considered to be of great importance. In Chapter 2, we identified HilD, one of the key regulators of the Spi1 system as a positive regulator of the flagellar master operon, flhDC. Contradicting, the Spi1-associated regulator RtsB is known to negatively regulate flhDC. A reasonable general proposal is that flagellar biosynthesis is temporally controlled during the infection process and simultaneous activation and repression might allow for fast adaptation to external stimuli that arise within a given niche.<br /><br /><br /><br />Flagellar movement is mediated by rotation of helical filaments. The hook structure, which functions as a universal joint between the basal body and the filament, transmits the rotary motion from the base to the filament. In Salmonella, hook length is tightly controlled. During hook assembly, a molecular ruler, FliK, is secreted via the flagella-specific type III secretion system (T3SS). FliK thereby measures the length of the assembled hook and eventually transmits the information of hook completion back into the cell. FliK interaction with FlhB induces a conformational change within the secretion apparatus resulting in a switch in substrate specificity. The T3SS then switches from secretion of hook-type substrates to secretion of late (filament)-type substrates. In Chapter 3, we demonstrated that the molecular ruler FliK is intermittently secreted throughout hook-basal body assembly. FliK took temporal measurements of hook length and the switch in the secretion apparatus was induced as soon as the hook reached a minimal physiological length of >40 nm. Uncoupled FliK expression in elongated hooks resulted in an immediate switch in substrate specificity, which was in agreement with our mathematical model. Furthermore, the velocity of FliK secretion decreased with increasing hook length - a possible explanation how the probability of a productive interaction between FliK and FlhB is regulated.<br /><br /><br /><br />Little is known about the signals involved in type III secretion. In Chapter 4 of this thesis, we performed a preliminary study to characterize potential signal peptides and the role of the 5’-untranslated region (5’-UTR) in the T3S process. We uncoupled gene expression from potential mRNA effects of the untranslated region by expressing early- and late-type substrates from the arabinose promoter. Comparing the secretion capability, a clear difference in secretion efficiency was observed. Dissection of the protein sequences of different substrates indicated variable regions that were important for secretion. However, an overall tendency was pointing to a signal peptide within the unstructured N-terminus. We further analyzed the effect of the 5’-UTR in the type III secretion process. For early substrates like FlgB and FlgE, the native 5’-UTR lead to increased protein levels and significantly higher secretion efficiency compared to constructs using the 5’-UTR of araBAD. We conclude that secretion is facilitated by a combination of peptide and mRNA signals, which (depending on the secretory class) are recognized by the export apparatus or act together with substrate-specific chaperones.<br /><br /><br /><br />In the final part of this work, we used the flagellar secretion apparatus to establish a method for the purification of neuroactive peptides (Chapter 5). The flagellar secretion apparatus efficiently exports flagellar subunits at a rate up to 10,000 amino acid residues per second. A N-terminal fusion of a flagellar secretion substrate to a variety of peptides served as an export shuttle that targeted the fusion for type III-dependent secretion. Secretion from the bacterial cell into the growth medium circumvented the problem of inclusion body formation and allowed for easy purification from the culture supernatant. In a secretion-optimized strain, we could demonstrate secretion of peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria. As a proof of principle, we successfully applied this system to the purification of the μ-conotoxin SIIIA, an inhibitor of voltage-gated sodium channels NaV1.2. In electrophysiological tests on Xenopus oocytes expressing rat NaV1.2, activity of recombinant SIIIA was comparable to chemically synthesized SIIIA. terms-of-use The Bacterial Flagellum : New Insights into Regulation, Bioassembly, and Type III Secretion Singer, Hanna Magdalena 2014-10-06T08:08:41Z Singer, Hanna Magdalena

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