Studies On The Motility Behavior And The Dynamic Properties Of Flagellar Motor Of Escherichia Coli

Life is a miracle of nature,a peculiar physical phenomenon that goes against the tide of entropy death of the universe.Among the most thoroughly studied living organisms,Escherichia coli stands out as an indispensable symbiotic bacterium in human body,a common pathogene in human society,the most widely used biochemical factory in the field of bioengineering,the carrier and platform of emerging medical technologies,and the most classic research model in the field of microbiology.However,to this day,E.coli remains enigmatic,attracting us to continue to explore.Therefore,further research on E.coli holds significant academic importance and extensive practical value.In this study.the motility behavior of E.coli and the dynamic properties of the flagellar motor were studied systematically.For the former,we focus on the dynamic behavior of the flagellar filaments in various environments,while for the latter,we focus on the dynamic exchange of the stators of the flagellar motor.On the basis of the experimental studies,we combined thermodynamics and fluid mechanics knowledge,and used simplified theoretical models,to explain and simulate various phenomena observed in our experiments.The motility behavior of E.coli in free liquid space follows the run-and-tumble pattern and can be described by a simple two-state equilibrium model.However,the flagellar rotary motor that drives this motility behavior exhibits non-equilibrium switching dynamics.To resolve this contradiction,we used a non-invasive detection method:we specifically labeled the flagellar filaments of E.coli with fluorescent dyes,and followed the polymorphic transformation of single flagellar filaments over a long period of time.By quantifying the probabilistic flow in the polymorphic phase space of the flagellar filaments,we determined the mesoscopic non-equilibrium nature of the swimming behavior of E.coli,and estimated the entropy production rate and energy dissipation rate of the system.We also proved that the concentration fluctuation of the chemotaxis signaling protein CheY-P does not significantly affect the non-equilibrium characteristics of the polymorphic system of flagellar filaments.Flagellar motors of E.coli provide the external energy input for the polymorphic system of flagellar filaments to maintain the non-equilibrium steady state.The stators,as the energy generating units of the flagellar motor,dynamically exchange between the flagella motor and the inner membrane of the cell,causing stepwise jumps in the rotation speed of the high-load motor.The traditional view describes the dynamic exchange of the stator as a simple bound-unbound two-state model,and predicts that the dwell time distribution of the stator follows a single exponential decay.However,most previous studies were based on fluorescent protein labeling techniques,which were limited by photobleaching.As a result,many important details were lost,making it impossible to reliably verify this prediction.Using bead tagging technology to track the rotation speed of individual motors over a long period of time with high resolution,we achieved non-invasive detection of the stator exchange.We found that the measured stator dwell time distribution follows a double exponential decay,rather than the predicted single exponential decay,indicating the presence of a third undiscovered hidden state in addition to the bound and unbound states.In order to quantitatively describe the stator exchange dynamics,a three-state model was established and the relevant dynamic parameters were calculated.We further explored the mechanism of the hidden state through experiments and analysis,revealing that,while maintaining the anchoring of the peptidoglycan layer,the stator will temporarily decouple from the FliG protein of the C ring,forming the hidden state.Based on the systematic study of the polymorphic transformation dynamics of single flagellar filament,we focused on the cooperative mechanism of multiple flagellar filaments to explore its impact on bacterial motility.In the natural environments,E.coli often encounter limited movement space,and escape from certain extremely narrow environments through reversal.Specifically,in quasi-one-dimensional restricted environments,existing research had been limited to monotrichous bacteria,which did not involve the cooperative regulation among multiple flagella.To fill this academic gap,we constructed a quasi-one-dimensional microfluidic device,and observed the cooperative dynamic behavior of multiple flagellar filaments during bacterial reversal by specific fluorescent labeling.Through fluid dynamics analysis,we elucidated the physical mechanism of the reversal of E.coli and the cooperative strategy between multiple flagella.In addition,we also found that increasing the channel width and continuous exposure to short-wavelength excitation light promote reversal.The hydrodynamic interaction between bacteria and solid-liquid interface not only participates in the physical mechanism of reversal,but also results in many interesting motile phenomena.Hydrodynamics simulations of peritrichous bacteria without tumble showed that when the cell body is fixed on the flat solid-liquid interface,the flagellar filaments form bipolar flagellar bundles along the long axis of the cell body.However,there has been no experimental verification of this simulation result so far.Through experiments on E.coli in a similar fluid dynamics environment,we verified the presence of bipolar flagellar bundles.During this process,we found that a few of the flagellar filaments swung abnormally in counterclockwise direction,and we speculated that the hook obtained a small amount of rotational stiffness.This caused the hook to rotate around the symmetry axis of the flagellar motor while also rotating around its own molecular axis.In addition,we also found that the flagellar bundles of some bacteria moving in circles near the solid-liquid interface showed a splitting phenomenon,and this phenomenon had no significant effect on the swimming speed of the bacteria.From the dynamic properties of flagellar motors,to the polymorphic transformation of flagellar filaments and the motility behavior of individual bacteria,several unexpected discoveries have been made at multiple scales and levels.These studies and discoveries have expanded our cognizance of the motility of E.coli and deepened our understanding of the physiological behaviors of bacteria,such as swimming and colonizing in the gut and other complex native environments.In addition,our research methods and ideas also have a certain reference value for the dynamics of other living systems.