Most bacteria swim through fluids by rotating helical flagella, driven at their base by a macromolecular rotary motor. The motor drives the flagellum against the viscous resistance of the fluid, and in doing so it propels the cell forward like a miniature submarine. Under conditions of high mechanical load, the motor runs at a low speed and produces a large torque. In contrast, under conditions of low load, the motor runs at a high speed and produces only a small torque.
What is less well-understood is how the motor adapts to changes in the mechanical environment. Bacteria interact with surfaces, swim in fluids with different viscosities, interact with mucus lining our guts, and so on. What effect do changes in mechanical load have on the motor?
To answer this question, we set out to measure the motor’s response to varying mechanical loads over a wide range of operating conditions. To make progress, we had to solve a challenging technical hurdle. How does one quickly and controllably change the load on a motor 50 nm in size, and measure its output at the same time? How does one do this so that the load change is reversible and has a large dynamic range?
We came up with an interesting solution to this problem. We tethered bacterial cells on a surface by single flagella. With the flagellum immobilized, and the motor still running, the cell body rotates, and the motor operates under a very high load. We then applied a rapidly-rotating electric field. This external field induces a rotating dipole on the cell. Under the right conditions, the induced dipole lags the external field by a small amount and their interaction causes an external torque on the cell. We used the external torque to drive the tethered cell forward, and in so doing, reduced the load taken up by the motor. Turning the external field off increased the load again. Thus, the cell body acted both as a speedometer for the motor and as a handle with which to control the load on the motor.
We found that when we decreased the motor load, the motor’s speed went down in steps, due to a loss of the torque-generating stator units that drive it. Increasing motor load by turning the external field off reversed this process. Thus, the motor re-builds itself in response to changing mechanical demands. When load goes down, it releases excess torque-generating units. When load goes up, it recruits additional units. It’s as if your car’s engine removed or added components while you are driving down the freeway at 70 mph. This process allows the motor to always match the output with demand, a remarkable feat for a molecular machine that is just tens of nanometers in size.
To understand this process from a theoretical perspective, we teamed up with Prof. Rob Phillips from Caltech, who is an expert in using physical principles to model and understand biological phenomena. Together, we developed a statistical physics-based model that not only captured the experimental observations, but also made novel quantitatively testable predictions. We are now developing new experiments to test those predictions.
In all forms of life, important biological processes are carried out by molecular machines that are often made from smaller units that self-assemble inside the cell. Despite a long tradition of research into the function of such molecular machines, little is known about how their assembly itself might be regulated to accommodate varying environmental conditions. This work advances our understanding of how interactions between protein subunits can be tuned by external stimuli to functionally control the formation of macromolecular assemblies.
by Navish Wadhwa and Howard Berg
