Flagellar motor tuning - The hybrid motor in Shewanella oneidensis MR-1

Bacteria are exposed to constantly changing environments. An efficient way to navigate towards favourable conditions is flagella-mediated motility. Flagellar rotation is achieved by the bacterial flagellar motor, composed of the rotor and stator complexes which surround the rotor in a ring-like structure. As an exception among the Shewanella species, the fresh-water organism S. oneidensis MR-1 harbours two different stator complexes, the sodium-ion dependent PomAB and the protondependent MotAB, differentially supporting rotation of a single polar flagellum. Both PomAB and MotAB are simultaneously present and required for full speed under low sodium-ion conditions. Although tightly anchored to the cell wall, stators are constantly exchanged even during ongoing rotation. Moreover, sodium-ion and proton-dependent stators can function with the same rotor. This raises the question of how PomAB and MotAB contribute to rotation of a single flagellum and whether PomAB and MotAB coexist in the stator ring of S. oneidensis MR-1, forming a hybrid motor. Here, I report a novel model for the dynamic adaptation of the rotor-stator configuration in response to the environmental sodium ion level in S. oneidensis MR-1. Transcriptional fusions to lucB revealed that both pomAB and motAB are concurrently transcribed. By using fluorescence microscopy, functional fusions of mCherry to the B-subunits revealed that in sharp contrast to MotB, a fraction of PomB is polarly positioned independently of the sodium-ion concentration. At low sodium-ion concentration, PomB and MotB appear to coexist in the flagellar motor. However, in the absence of PomAB, MotB is recruited to the flagellated pole independently of the sodiumion concentration. Interestingly, induced production of PomAB displaces polar MotB from the motor and confines it to the membrane. By quantifying single sfGfp molecules fused to PomB, I could show that the number of PomB in the stator ring is reduced from nine to five complexes when cells were shifted from a high to a low sodium-ion concentration. Thus, the incorporation efficiency of PomAB is directly modified in response to the sodium-ion concentration, whereas the association of MotAB into the stator ring rather depends on the presence of PomAB. Furthermore, two auxiliary proteins, MotX and MotY, were identified and shown to be essential for functionality of both PomAB and MotAB. Localisation studies revealed that, in contrast to Vibrio MotXY are not required for recruitment of the stator complexes to the flagellated pole. Taken together, my data support the model of dynamic stator swapping to tune the flagellar motor in response to environmental conditions, e.g. the availability of sodium ions. The concurrent presence of PomB and MotB at low sodium-ion concentration suggests the existence of a hybrid motor in S. oneidensis. Since it remains to be demonstrated whether MotAB stators are functionally incorporated in this hybrid motor, the second aim of this work was to biophysically analyse the contribution of MotAB and PomAB to motor rotation at the single cell level. To this end, a ‘bead assay’ and a ‘tethered cell assay’ were established. These set-ups required the delocalisation of the polar filament to a lateral position, the preparation of a highly specific antibody against the modified filament and, for the bead assay the attachment of polystyrene beads to the filament. While the bead assay was limited to short-term measurements, the tethered cell assay was optimised for long-term studies. The optimisation now permits a constant buffer exchange as well as the modulation of the stator complex level by an inducible promoter upstream of pomAB and motAB. Single cell analysis comparing the wild-type and the PomAB-driven motor revealed a significantly higher rotation speed for the wild-type motor at low sodium-ion concentration. Moreover, induced production of