Biophysical Characterization of Flagellar Motor Functions

Ford, Katie; Chawla, Ravi; Lele, Pushkar P.

doi:10.3791/55240

Cited by 7 publications

(16 citation statements)

References 26 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Overnight cultures were diluted in fresh TB (10 g l −1 tryptone; 5 g l −1 NaCl) at 30°C and grown to an OD 600 of 0.5–0.6. Cells were washed twice in motility buffer containing lactate (0.055 M NaCl, 10 mM EDTA, 10 mM potassium phosphate, pH 7.0) and sheared 70 times to shorten the filaments (Ford et al ., ). These strains carried cysteine groups in the flagellin or hook proteins ( hag T209C / flgET 123C ) in order to facilitate tethering to maleimide‐coated coverslips (MicroSurfaces, Inc.).…”

Section: Methodsmentioning

confidence: 97%

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Diethmaier

Chawla

Canzoneri

et al. 2017

Molecular Microbiology

Self Cite

View full text Add to dashboard Cite

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS-DegU two-component system. Here we report a role for flagella in the regulation of the K-state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU-P, which inhibits the expression of ComK, the master regulator for the K-state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hagA233V) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU-P levels through an unknown signaling mechanism. This flagellar-load based mechanism ensures that cells in the motile subpopulation have a 10-fold enhanced likelihood of entering the K-state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.

show abstract

Section: Methodsmentioning

confidence: 97%

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Diethmaier

Chawla

Canzoneri

et al. 2017

Molecular Microbiology

Self Cite

View full text Add to dashboard Cite

show abstract

“…The situation is reversed when the filaments rotate CW. Thus, it is possible to discriminate between 20 the two modes when a bacterium swims near a surface. We analyzed each cell that swam in circular trajectories near the surface and determined the mean speeds for the two directions.…”

Section: Cell Swims Faster In the Pusher Modementioning

confidence: 99%

“…In the canonical chemotaxis network, chemoreceptors sense extracellular ligands and modulate the activity of the chemotaxis kinase. The kinase 20 regulates CheY-P levels to promote switching in the direction of motor rotation (11). Flagellar switching reverses the swimming direction in H. pylori.…”

Section: Introductionmentioning

confidence: 99%

“…CW bias has been measured in the model species Escherichia coli by tracking the rotation of tethered cells, where a single flagellar filament is attached to a glass surface while the cell freely rotates (15). Alternatively, the bias is determined by sticking the cell to the surface and monitoring the rotation of a probe bead attached to a single filament (20,21). Such single motor assays have been employed successfully in E. coli because the filaments are spaced apart on the 5 cell body.…”

Section: Introductionmentioning

confidence: 99%

“…Our work provides a framework to study chemotaxis signaling and flagellar switch-behavior in H. pylori. 20 To determine the behavior of flagellar motors in H. pylori, we tracked cell motility in the bulk fluid with a phase contrast microscope. The positions of single cells were quantitatively determined from digital videos with the aid of particle tracking (see Materials and Methods).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Anisotropic random walks reveal chemotaxis signaling output in run-reversing bacteria

Antani

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

10The bias for a particular direction of rotation of the flagellar motor is a sensitive readout of chemotaxis signaling, which mediates bacterial migration towards favorable chemical environments. The rotational bias has not been characterized in Helicobacter pylori, which limits our understanding of the signaling dynamics. Here, we determined that H. pylori swim faster (slower) whenever their flagella rotate counterclockwise (clockwise) by analyzing their 15 hydrodynamic interactions with bounding surfaces. The anisotropy in swimming speeds helped quantify the fraction of the time that the cells swam slower to report the first measurements of the bias. A stochastic model of run-reversals indicated that the anisotropy promotes faster spread compared to isotropic swimmers. The approach further revealed that the diffuse spread of H. pylori is likely limited at the physiological temperature due to increased reversal frequencies. 20 Thus, anisotropic run-reversals make it feasible to study signal-response relations in the chemotaxis network in non-model bacterial species. Page 2 of 22 Impact Statement Anisotropy in run and reversal swimming speeds promotes the spread of H. pylori and reveals temperature-dependent behavior of the flagellar switch. cell was observed, B C , to yield: FG HC*3,C = J K LM J KThe error associated with the calculation of FG HC*3,C values decreases with increasing B C . But, 15 different cells were observed for different durations; hence the FG HC*3,C values were allocated weights that corresponded to their respective durations:

show abstract

Torque, but not FliL, regulates mechanosensitive flagellar motor-function

Chawla

Ford

Lele

2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

The stator-complex in the bacterial flagellar motor is responsible for surface-sensing. It remodels in response to perturbations in viscous loads, recruiting additional stator-units as the load increases. Here, we tested a hypothesis that the amount of torque generated by each stator-unit modulates its association with the rotor. To do this, we measured stator-binding to the rotor in mutants in which motors reportedly develop lower torque compared to wildtype motors. First, we employed a strain lacking fliL. Contrary to earlier reports, measurements indicated that the torque generated by motors in the fliL strain was similar to that in the wildtype, at high loads. In these motors, stator-binding was unchanged. Next, experiments with a paralyzed strain indicated that the stator-binding was measurably weaker when motors were unable to generate torque. An analytical model was developed that incorporated an exponential dependence of the unit’s dissociation rate on the force delivered to the rotor. The model provided accurate fits to measurements of stator-rotor binding over a wide range of loads. Based on these results, we propose that the binding of each stator-unit is enhanced by the force it develops. Furthermore, FliL does not play a significant role in motor function in E. coli.

show abstract

Biophysical Characterization of Flagellar Motor Functions

Cited by 7 publications

References 26 publications

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Viscous drag on the flagellum activates Bacillus subtilis entry into the K‐state

Anisotropic random walks reveal chemotaxis signaling output in run-reversing bacteria

Torque, but not FliL, regulates mechanosensitive flagellar motor-function

Contact Info

Product

Resources

About