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 proton-dependent 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 sodium-ion 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 PomAB in a stator deletion background resurrected rotation speed in a stepwise manner, whereas production of MotAB in a PomAB-driven motor decreased rotation speed stepwise. These results strongly indicate that MotAB is incorporated into the force-generating PomAB-occupied stator ring, slowing down motor rotation. MotAB production in a stator deletion background did not restore rotation. However, swimming assays revealed that MotAB is sufficient to drive flagellar rotation in a subpopulation of cells, strongly suggesting that both stators are able to function together in a single motor. To clearly characterise the role of MotAB and PomAB in the hybrid motor of S. oneidensis MR-1 further biophysical studies are required. The genome wide bioinformatic analysis of all sequenced bacterial genomes revealed that dual or multiple stator complexes along with a single flagellar system are surprisingly widespread among bacterial species. Moreover, stator complex homology comparison in S. oneidensis MR-1 indicated that MotAB has recently been acquired by lateral gene transfer as a consequence of adaptation to a fresh-water environment. Thus, the flagellar motor might still be in a process of optimisation. Collectively, I hypothesize that S. oneidensis tunes its flagellar motor by exchanging stator complexes and that stator swapping represents a common mechanism applicable to other bacteria to adapt to changing environments

Mechanism of bidirectional thermotaxis in Escherichia coli.

In bacteria various tactic responses are mediated by the same cellular pathway, but sensing of physical stimuli remains poorly understood. Here, we combine an in-vivo analysis of the pathway activity with a microfluidic taxis assay and mathematical modeling to investigate the thermotactic response of Escherichia coli. We show that in the absence of chemical attractants E. coli exhibits a steady thermophilic response, the magnitude of which decreases at higher temperatures. Adaptation of wild-type cells to high levels of chemoattractants sensed by only one of the major chemoreceptors leads to inversion of the thermotactic response at intermediate temperatures and bidirectional cell accumulation in a thermal gradient. A mathematical model can explain this behavior based on the saturation-dependent kinetics of adaptive receptor methylation. Lastly, we find that the preferred accumulation temperature corresponds to optimal growth in the presence of the chemoattractant serine, pointing to a physiological relevance of the observed thermotactic behavior

Enterale Absorption von Paracetamol, Talinolol und Amoxicillin nach oraler Gabe mit 240 ml Wasser und 240 ml Saccharose-Lösung

Erfassung der gastrointestinalen Volumenverteilung und Volumenabsorption von 240 ml Arzneistoffsuspension (Paracetamol, Amoxicillin, Talinolol) unter kalorischen und akalorischen Bedingungen mittels MRT-Bildgebung mit gleichzeitiger Untersuchung der Arzneistoffabsorption.Detection of gastrointestinal volume distribution and volume absorption of 240 ml drug suspension containing acetaminophen, amoxicillin, talinolol under caloric and non-caloric conditions using MRI imaging with simultaneous investigation of drug absorption

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 proton-dependent 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 sodium-ion 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 PomAB in a stator deletion background resurrected rotation speed in a stepwise manner, whereas production of MotAB in a PomAB-driven motor decreased rotation speed stepwise. These results strongly indicate that MotAB is incorporated into the force-generating PomAB-occupied stator ring, slowing down motor rotation. MotAB production in a stator deletion background did not restore rotation. However, swimming assays revealed that MotAB is sufficient to drive flagellar rotation in a subpopulation of cells, strongly suggesting that both stators are able to function together in a single motor. To clearly characterise the role of MotAB and PomAB in the hybrid motor of S. oneidensis MR-1 further biophysical studies are required. The genome wide bioinformatic analysis of all sequenced bacterial genomes revealed that dual or multiple stator complexes along with a single flagellar system are surprisingly widespread among bacterial species. Moreover, stator complex homology comparison in S. oneidensis MR-1 indicated that MotAB has recently been acquired by lateral gene transfer as a consequence of adaptation to a fresh-water environment. Thus, the flagellar motor might still be in a process of optimisation. Collectively, I hypothesize that S. oneidensis tunes its flagellar motor by exchanging stator complexes and that stator swapping represents a common mechanism applicable to other bacteria to adapt to changing environments

MotX and MotY Are Required for Flagellar Rotation in Shewanella oneidensis MR-1▿ †

The single polar flagellum of Shewanella oneidensis MR-1 is powered by two different stator complexes, the sodium-dependent PomAB and the proton-driven MotAB. In addition, Shewanella harbors two genes with homology to motX and motY of Vibrio species. In Vibrio, the products of these genes are crucial for sodium-dependent flagellar rotation. Resequencing of S. oneidensis MR-1 motY revealed that the gene does not harbor an authentic frameshift as was originally reported. Mutational analysis demonstrated that both MotX and MotY are critical for flagellar rotation of S. oneidensis MR-1 for both sodium- and proton-dependent stator systems but do not affect assembly of the flagellar filament. Fluorescence tagging of MotX and MotY to mCherry revealed that both proteins localize to the flagellated cell pole depending on the presence of the basal flagellar structure. Functional localization of MotX requires MotY, whereas MotY localizes independently of MotX. In contrast to the case in Vibrio, neither protein is crucial for the recruitment of the PomAB or MotAB stator complexes to the flagellated cell pole, nor do they play a major role in the stator selection process. Thus, MotX and MotY are not exclusive features of sodium-dependent flagellar systems. Furthermore, MotX and MotY in Shewanella, and possibly also in other genera, must have functions beyond the recruitment of the stator complexes

Mutations targeting the plug-domain of the Shewanella oneidensis proton-driven stator allow swimming at increased viscosity and under anaerobic conditions

Shewanella oneidensis MR-1 possesses two different stator units to drive flagellar rotation, the Na+-dependent PomAB stator and the H+-driven MotAB stator, the latter possibly acquired by lateral gene transfer. Although either stator can independently drive swimming through liquid, MotAB-driven motors cannot support efficient motility in structured environments or swimming under anaerobic conditions. Using ΔpomAB cells we isolated spontaneous mutants able to move through soft agar. We show that a mutation that alters the structure of the plug domain in MotB affects motor functions and allows cells to swim through media of increased viscosity and under anaerobic conditions. The number and exchange rates of the mutant stator around the rotor were not significantly different from wild-type stators, suggesting that the number of stators engaged is not the cause of increased swimming efficiency. The swimming speeds of planktonic mutant MotAB-driven cells was reduced, and overexpression of some of these stators caused reduced growth rates, implying that mutant stators not engaged with the rotor allow some proton leakage. The results suggest that the mutations in the MotB plug domain alter the proton interactions with the stator ion channel in a way that both increases torque output and allows swimming at decreased pmf values. This article is protected by copyright. All rights reserved