Study Of Noises In The Chemotaxis Network And Flagellar Motor Of Escherichia Coli

Bacteria are ubiquitous around the world.Bacterial motility directly affects their invasion of host and pathogenicity.Studying their motile behavior and the associated physical mechanisms can promote our understanding of the dynamic mechanisms of biological systems.We selected Escherichia coli,a widely used model bacterium,as the experimental object to study the mechanism related to chemotaxis in its motility.E.coli sense the external environment through the intracellular chemotaxis network,and control the rotation direction of the flagellar motor to chase for better living conditions,while the rotation of the flagellar motor is the power source to enable E.coli to swim.Noise(or fluctuation)in bacterial motility is the physical object we mainly studied in this thesis.Noise exists in various types of biological systems.It can be utilized by creatures to achieve physiological function,and it can also provide information to understand the dynamic mechanisms of physiological activities.The current research results of the noise phenomenon related to bacterial motility are still controversial.Quantitative study of the noise phenomenon in the chemotaxis network and flagellar motor of E.coli is not only helpful to understand bacterial motile mechanisms,but also of great significance to understand the noise of other biological systems.The rotation direction of the flagellar motor is mainly modulated by the local level of the intracellular chemotaxis signal molecule CheY-P.The fluctuation of the CheY-P concentration(or noise)will lead to the correlation between the rotation of two different motors on the same bacterium.However,several previous experiments showed that the CheY-P concentration had a slow fluctuation with a long characteristic time,while in recent years,two-motor rotation correlation experiments only found a fast fluctuation in the CheY-P concentration with a short characteristic time,and thus,there was a direct contradiction.To resolve this conflict,in chapter 3,we designed a new video-analysis algorithm that could automatically analyze the raw video with a low signal-to-noise ratio from the camera,so that we could choose a smaller bead in our assay to eliminate the influence of the hydrodynamic coupling between the motors.We found that both types of fluctuations with two well separated timescales exist simultaneously.Then,we performed theoretical and data analysis,and extracted the characteristic time and amplitude of the two types of fluctuations.We further found that the amplitude of the slow fluctuation was highly correlated with the motor switching property,which explained the contradiction between previous works.This work is our exploration of fluctuations in the chemotaxis signal molecule.The output end of the chemotaxis network is the flagellar motor.If the thermal noise is the main source of fluctuation in the motor rotation,we can extract the motor torque from this fluctuation through fluctuation theorem.In chapter 4,we systematically measured the fluctuation in flagellar motor rotation using a bead assay,removed the contribution of other noise sources by using appropriate mathematical analysis method,subsequently calculated the amplitude and characteristic time of this fluctuation.Surprisingly,we discovered that the amplitude of the fluctuation is far beyond that of the thermal noise.This suggested that there exists extra fluctuation in the flagellar motor torque,which is not constant as previously thought.We found that the timescale of torque fluctuation was extremely short,consistent with the timescale of flagellar motor stepping.This suggested that the torque fluctuation we discovered here was probably due to the stepping of the motor.The fluctuation in motor torque we characterized here,provided new information for the dynamic models of the flagellar motor.In order to overcome the problem that the fluctuation theorem cannot be used to measure the flagellar motor torque independently due to the fluctuation of the motor torque,we developed a new method to measure the maximum flagellar motor torque(stall torque).The stall torque of the flagellar motor is a key characteristic of motor function.From direct measurements of the stall torque,we can determine how many protons each torque-generating unit(stator)of the motor passes per revolution(the driving force of motor rotation is proton flow),and then test whether motor rotation and proton flux are tightly or loosely coupled.Previous direct measurements of the stall torque exhibited low precision.In chapter 5,we performed resurrection experiments for flagellar motors,combined magnetic and optical tweezers to stall the motor and get its deflection angle,then we used noise analysis to precisely calibrate the magnetic tweezers,finally,we performed direct measurements of the stall torque with higher precision.The calculated number of protons flowing through the motor per revolution is consistent with that predicted by the tight coupling model of proton flux and motor rotation,thereby supporting this model.In summary,noise plays an important role in the mechanism of bacterial motility.The fast fluctuation in the concentration of the chemotaxis signal molecule CheY-P can coordinate the rotation of multiple flagellar motors on the same bacteria,while the slow fluctuation can enhance bacterial chemotaxis.We utilized the bead assay,developed a new automatic video analysis algorithm,and combined them with mathematical physics tools designed for noise analysis,to quantitatively and systematically study the dynamic properties related to E.coli motility.In the chemotaxis network,we discovered two well separated timescales in the fluctuation of the concentration of the signal molecule CheY-P;in the flagellar motor,we discovered unanticipated fluctuation in the motor torque;we developed a new method combining magnetic tweezers and optical tweezers to measure the resurrection procedure of the motor and obtain the key physical quantity-the maximum torque of the motor.These studies enhanced our understanding of the dynamic mechanism of E.coli motility,the noise phenomena in E.coli,and the role of noise in biological systems.At the same time,the new experimental techniques and data analysis methods developed in this paper are also enlightening for the study of noises in other biological systems,and also have reference significance for the study of other biological molecular motors.