AbstractsBiology & Animal Science

Assembly, stability and regulation of flagellar motor in Escherichia coli

by Hui Li




Institution: Universität Heidelberg
Department: The Faculty of Bio Sciences
Degree: PhD
Year: 2010
Record ID: 1105435
Full text PDF: http://archiv.ub.uni-heidelberg.de/volltextserver/11255/


Abstract

Bacterial flagellar motor is a highly ordered and complex supramolecular structure with two functions: it is both a rotary engine that powers the rotation of the flagella, coupling swimming to chemotactic signaling, and a type III protein export apparatus needed for assembly of extracellular flagella. Motor biogenesis represents a formidable example of self-assembly, involving a number of different proteins that form multiple oligomeric rings in the cytoplasm, periplasm and plasma membrane. Although late extracellular steps of the flagellar assembly process are comparatively well understood, much less is known about the early steps of the motor structure formation in the cytoplasm and in the plasma membrane. We used fluorescence imaging to dissect the order of the motor assembly in Escherichia coli cells and fluorescence resonance energy transfer (FRET) to map in vivo the underlying protein interactions. We observed that the self-association of the membrane export apparatus protein FlhA is independent of the other motor components, thus it is likely to be the first step in motor assembly. It is followed by the recruitment of the MS-ring component FliF and the ordered association of other motor structures with the association of each subsequent structure appearing to stabilize the growing assembly. We further applied fluorescence recovery after photobleaching (FRAP) to show that parts of the assembled motor undergo dynamic protein exchange. In addition, we found an assembly-dependent stabilization of motor components against proteolytic degradation, indicating removal of the unassembled motor subunits through a protein quality control mechanism. Furthermore, we additionally demonstrated the involvement of DnaK/HtpG machinery in assembly of flagellar motor and chemosensory complexes and showed regulatory interactions between cyclic di-GMP binding protein YcgR and the stator component of flagellar motor MotA. Our results provide a novel and thus far the most comprehensive view of the early steps of flagellar motor assembly, enabling a better structural understanding of the flagellar export apparatus and other type III secretion systems. We believe that the observed combination of hierarchy and cooperativity in the assembly process could be common to many large intracellular complexes. Der bakterielle Flagellenmotor ist eine hochgeordnete und komplexe supramolekulare Struktur, die zwei Funktionen ausübt: Zum einen als rotierender Motor, der die Drehung der Flagellen antreibt und somit die Schwimmbewegung an die chemotaktische Wahrnehmung koppelt; Zum anderen als Typ III Proteinexportapparat, der zur Bildung der extrazellulären Flagellen benötigt wird. Die Biogenese des Motors ist ein eindrucksvolles Beispiel molekularer Selbstorganisation, bei der eine Vielzahl verschiedener Proteine mehrere oligomere Ringe in Zytoplasma, Periplasma und Plasmamembran bilden. Während die späten extrazellulären Schritte der Flagellenbildung vergleichsweise gut verstanden sind, ist über die frühen Schritte, die an der Bildung der…