Chemie Chemistry + allied sciences Chemie Motility plays a key role for the superior survival strategy of many bacteria. Sophisticated, macromolecular machines, called flagella, serve as bacterial locomotion organelles. These flagella appear in distinct spatial arrangements along the bacterial cell, constituting the flagellation patterns, whose disruption is detrimental to motility. However, the number of flagellation patterns that have arisen in a plethora of bacterial species can be counted by the fingers of one hand. How these patterns are established in the first place, and how they are maintained during cell division, remains a yet unassessed task in the field. Two nucleotide-binding proteins, FlhF and FlhG, were identified to be crucial for the spatial regulation of flagella in most flagellated bacteria, which exhibit various flagellation patterns. This work presents a structural and biochemical characterization of the flagella regulating ATPase FlhG, which revealed its function as a molecular switch, having a dimeric, membrane-associated state and a mobile, monomeric state in the cytoplasm. This hallmark feature of MinD/ParA ATPases is conserved in FlhG of peritrichous B. subtilis as well as monotrichous S. putrefaciens. In both organisms FlhG interacts with the flagellar C-ring components FliM and FliN(Y) providing insight into its role as a flagellar C-ring assembly factor, coordinating the assembly of a FliM/FliN(Y) complex to FliG. Differences in the regulatory networks underlying different flagellation patterns were identified in species-specific interaction partners of FlhG, such as the flagellar master regulator FlrA in S. putrefaciens or the late divisome component GpsB in B. subtilis. These findings led to the hypothesis that the spatial arrangement of flagella is encoded in the structure of the interaction network of FlhF and FlhG. This hypothesis is supported by the occurrence of varying C-ring components in differently flagellated bacteria. This work also includes the implementation and successful application of 1H/2H exchange mass spectrometry in Marburg. Not only does this powerful tool allow the convenient investigation of protein dynamics, but also the rapid mapping of protein-protein and protein-ligand interfaces. Interface mapping, in particular, revealed the power of this method and was applied in various research projects. Shewanella Bacillus FlhG Fachbereich Chemie Schuhmacher, Jan Simon Schuhmacher Jan Simon C-ring Flagellum Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg Kirkpatrick CL + Viollier PH (2011) Poles Apart: Prokaryotic Polar Organelles and Their Spatial Regulation. 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Science 259(5102):1717-1723. 2015-12-16 https://doi.org/10.17192/z2015.0411 Shewanella Philipps-Universität Marburg 2015 English application/pdf doctoralThesis ths Dr. Bange Gert Bange, Gert (Dr.) Bacillus Untersuchung der Funktion und des Interaktionsnetzwerkes des Flagellaren Regulators FlhG opus:6466 monograph Geißel [Biologie] C-ring Investigating the Function and the Interaction Network of the Flagellar Regulator ATPase FlhG urn:nbn:de:hebis:04-z2015-04119 Mikroorganismus 2015-11-06 https://archiv.ub.uni-marburg.de/diss/z2015/0411/cover.png Enzym Motilität ist ein zentraler Aspekt der Überlebensstrategie von Bakterien. Viele Bakterien bewegen sich mit Hilfe komplexer makromolekularer Motoren, Geißeln oder Flagellen genannt. Diese sind in speziellen Mustern auf der Zelloberfläche verteilt und werden im Folgenden Flagellierungsmuster genannt. Trotz der unglaublichen Vielzahl unterschiedlicher Bakterien finden sich in der Natur nur eine Hand voll dieser flagellaren Muster. Darüber, wie diese Muster nach jeder Zellteilung reproduzierbar und präzise ausgebildet werden, liegen nur spärlich Informationen vor. Zwei Proteine, FlhF und FlhG, spielen in diesem Zusammenhang eine wichtige Rolle und führen zu unterschiedlichen Mustern in den jeweiligen Bakterien. Diese Arbeit beinhaltet eine biochemische und strukturelle Charakterisierung der ATPase FlhG, die die Funktion von FlhG als molekularen Schalter hervorhebt. FlhG kann einerseits als Dimer in Membran-assoziiertem Zustand vorliegen, andererseits als Monomer frei im Zytoplasma diffundieren. Homologe Proteine in den unterschiedlich flagellierten Bakterien B. subtilis und S. putrefaciens weisen dieselben charakteristischen Merkmale auf und deuten auf ein einheitliches Funktionsprinzip der ATPase hin. FlhG interagiert in beiden Organismen mit den Proteinen FliM und FliN(Y) des flagellaren C-rings und offenbart dabei seinen Beitrag zum Aufbau des flagellaren C-rings indem es die Interaktion des FliM/FliN(Y)-Komplexes mit FliG ermöglicht. Weitere Untersuchungen des Interaktionsnetzwerkes von FlhG in beiden Organismen zeigten sowohl speziesübergreifende (FliM/FliN(Y)) als auch speziesspezifische Interaktionspartner, darunter das Zellteilungsprotein GpsB in B. subtilis und der Hauptregulator der Flagellenbiosynthese FlrA in S. putrefaciens. Daraus lässt sich ableiten, dass die unter-schiedlichen Flagellierungsmuster nicht direkt durch FlhG bestimmt werden, je-doch in der Struktur des Interaktionsnetzwerkes von FlhG und FlhF kodiert sind. Darüber hinaus deutet die Variabilität des C-ring Proteins FliN(Y) in unterschiedlichen Bakterien, das mit der Ausbildung unterschiedlicher Muster korreliert, in dieselbe Richtung. Außerdem wurde im Rahmen dieser Arbeit in enger Zusammenarbeit mit der Massenspektrometrie Abteilung der Chemischen Fakultät der Universität Marburg eine Technik zur Untersuchung von Proteinen mit Hilfe von Wasserstoff/Deuterium Austausch etabliert. Die erfolgreiche Anwendung wird in dieser Arbeit am Beispiel von drei unterschiedlichen Projekten beschrieben und zeigt die Vorteile dieser Methode zur Bestimmung von Portein-Ligand und Protein-Protein Interaktionsoberflächen. FlhG