Competitive Substrate Binding Coordinates the Two Antagonistic Motors of the Bacterial Type IV Pilus

Abstract

The bacterial type IV pilus is an intricate nano-machine that coordinates two antagonistic molecular motors to extend and retract a semi-flexible polymer to the environment. Filament retraction is driven by the strongest molecular motor known and used for a variety of tasks such as uptake of DNA, surface sensing, and motility. However, how both motors are coordinated to dynamically extend and retract pili is unknown. To fill this void, we genetically engineered a fluorescent label for the pilus fiber. This enables us to characterize the dynamic cycles of extension and retraction of the pilus in an unprecedented way. Careful characterization of pilus dynamics reveals that pili are short and individual cells make a new pilus every 10 seconds. This is in stark contrast to the current dogma of the field that was shaped by static cyro electron images. To explain this discrepancy, we introduce a simplistic three-state model that describes the mutually exclusive binding/unbinding of the motor proteins to the pilus machine by a Poisson process. We combined live cell super-resolution microscopy and Monte-Carlo simulations to determine the rate constants of the model. We explain how these rates determine biological function such as pilus length and rate of pilus assembly. Intriguingly, we find that the major throttle of pilus production is the unbinding step of the retraction motor. Furthermore, we measured pilus activity with and without surface contact by combining fluorescence microscopy and line-scanning optical tweezers. Cells show similar levels of pilus activity, overturning the current model that surface contact drives pilus retraction. Together, our results shape a comprehensive picture of the physical processes that determine pilus activity and suggest that the pilus was optimized for multitasking of different tasks such as DNA uptake and motility.