Soman, V. | Search bioRxiv

bioRxiv2020

The Response of Bacterial Flagellar Motor to Stepwise Increase in NaCl Concentration

Many species of bacteria use flagella to navigate in its environment. The flagellum is a 7-10 m long helical filament with a rotary motor at its base embedded in the cell membrane and almost a dozen stator complexes. Proton motive force across the cell membrane powers the flagellar motors of E.coli and Salmonella. The motor stochastically switches between clockwise and counter-clockwise direction. A chemotaxis system causes the motor to change its direction, but the process is more complex as the switch is sensitive to load and proton motive force as well. NaCl is significant with regard to the flagellar motor as it affects the stator dynamics, proton motive force, and osmotaxis at higher concentration. Chemotaxis helps the bacteria for its growth and survival. E.colis natural habitat has high osmolarity and the organism uses use various mechanisms for osmoregulation. However, the role of flagellar motor to adapt to the changes in osmolarity, or osmotaxis, is not well studied. In this work, we dissipated the membrane potential of bacteria in pH 7 using step-wise increase in concentration of NaCl in motility buffer and studied the output of E.colis flagellar motor using tethered bead assay and swimming Salmonella enteritidis cells. We observed decrease in motor speed and switching rates with stepwise increase in NaCl concentration in the motility buffer. The mean speed of the motors decreased with NaCl concentration. The population of swimming cells tumbled more with increase in concentration of NaCl. At the single motor level, the motors biased to CCW rotation with decrease in membrane potential. In this study, we present our observations of the flagellar motor in high NaCl concentration, and explore how NaCl can be used to study various aspects of the bacterial flagellar motor. Statement of significanceSodium ion has been significant in the both the cellular energetics and the function of bacterial flagellar motor. Growing evidence show that the effect of sodium ions was not what hitherto thought it would be. It is involved in the sodium energetics, dissipate membrane potential, affect the flagellar stator dynamics of bacteria. Being an osmolyte, it influences the osmotaxis of bacteria. In this work, we studied the effect of NaCl on the response of the single bacterial flagellar motor of E.coli and swimming cells of Salmonella enteritidis. We observed that the effect of NaCl on the output of the flagellar motor was significant and it may affect the cells in various ways.

biophysics↗

bioRxiv2020

Effect of cell size and tethering geometry on rotation rate and torque of E.coli cells

The bacterial flagellum has a rotary motor embedded in its membrane surrounded by stator-units. Torque is generated by electro-steric interactions between rotor and stator-units. Chemotactic signals entail the motor to switch its direction of rotation. However, other factors such as protonmotive force and torque are involved in switching. In this work, we used peritrichously flagellated E.coli that stochastically tethers on surfaces in random geometries and studied how the cell size and position of the rotational axis affect the output of the motor. We developed a Cell Tethering Analysis Program (CTAP) to measure the length of the cells, the axis of rotation, rotational frequency of tethered cells. A D/L ratio (diameter traced by the cell body to the length of the cell body) was used to quantify the location of the rotational axis wherein, a D/L ratio of 1 and 1.9 means the axis of rotation is at the center of the cell body and near the tip of the cell body respectively. We performed experiments in controlled conditions and quantified the effect of cell size and tethering geometry on the output of the flagellar motor. The estimated torque of the tethered cells was 951 pN.nm and 1390 pN.nm for a D/L ratio of [~]1.0 and [~]1.9 respectively. As the torque increased, the motors rotated exclusively in CCW direction. We conclude that quantifying the cell size and tethering geometry is significant to characterize the output of the flagellar motors in a tethered cell assay.

biophysics↗