The role of the nucleotide-binding proteins FlhF and FlhG during flagellar biosynthesis

Flagella are bacterial organelles of locomotion and present one the smallest motors in the living organisms. Their architecture can be divided into a cytoplasmic C-ring, the membrane-embedded basal body and the extracellular hook and filament structures. While flagellar structure and constituents ar...

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Bibliographic Details
Main Author: Knauer, Carina
Contributors: Bange, Gert (Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2016
Chemie
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Summary:Flagella are bacterial organelles of locomotion and present one the smallest motors in the living organisms. Their architecture can be divided into a cytoplasmic C-ring, the membrane-embedded basal body and the extracellular hook and filament structures. While flagellar structure and constituents are conserved among the bacterial species, number and localization of flagella at the bacterial cell surface are not. Instead, they appear in species-specific patterns that are characterized by defined number and places of the flagella. For example, Shewanella putrefaciens exhibits one flagellum at one cell pole (monotrichious), while the food-borne pathogen Campylobacter jejuni features one flagellum at both cell poles (amphitrichous). In contrast, the Gram-positive bacterium Bacillus subtilis shows approximately 25 flagella that are regularly spaced at the lateral sides and are absent from the cell poles (peritrichous). Importantly, these patterns are reproduced during each cycle of cell division and have been used as an early criterion for the taxonomic classification of bacteria. An essential question for understanding bacterial cell physiology is how these flagellation patterns are maintained? During the past decade, the two nucleotide-binding proteins FlhF and FlhG have been identified as key players for the spatial and numerical regulation of flagella. Most notably, both proteins are highly conserved but manage different types of flagellation patterns. The major aim of this work was to understand the function of FlhF and FlhG in regulating flagellation patterns. I could show that FlhF and FlhG form a regulatory unit in the monotrichious Shewanella putrefaciens and the amphitrichous Campylobacter jejuni. Similar to the situation in the peritrichous B. subtilis, the N-terminal fraction of FlhG stimulates the GTPase activity of the homodimeric GTPase FlhF via a conserved ‘DQAxxLR’ motif (x = any amino acid). These findings suggest that the regulation of FlhF by FlhG is highly conserved among differently flagellated bacteria and does probably not account for the diversity FlhF/FlhG-dependent flagellation patterns. This notion is also supported by in-depth biochemical and structural analysis of the FlhG enzymes from Shewanella putrefaciens and Campylobacter jejuni. To better understand how the FlhF/FlhG unit can regulate different flagellation patterns, I next set out to identify interaction partners of FlhF and FlhG in the monotrichious Shewanella putrefaciens and the peritrichous B. subtilis. In Shewanella putrefaciens, I could show the FlhG interacts with the C-ring protein complex of FliM/FliN via the conserved ‘EIDAL’ motif of FliM. This is in contrast to the situation in B. subtilis where FlhG also interacts with the FliM/FliY complex, however, via a motif within the N-terminus of FliY. This finding presents the first differences between FlhF/FlhG-dependent regulation of a monotrichious and peritrichous flagellation pattern. My search for interaction partners of FlhF showed that the protein interacts with ribosomes, the SRP-RNA and the FliM/FliN (FliY complex). In monotrichious Shewanella putrefaciens, the three-domain protein FlhF interacts via its N-terminal and natively unfolded B-domain with the ribosome, the SRP-RNA and the FliM/FliN. Definition of the binding sites showed that they localize within the first 40 amino acids of the protein and seem to partially overlap. However, further studies need to clarify the molecular details. Similarly, the B-domain of FlhF from the peritrichous B. subtilis also interacts with the C-ring protein complex FliM/FliY via the FliY protein. While many questions remain open, I would like to suggest a working hypothesis that combines and reflects the current knowledge about FlhF/FlhG with the data obtained in this work.
Physical Description:109 Pages
DOI:https://doi.org/10.17192/z2016.0856