Studies On The Mechanical Properties Of The Flagellar Motor Output In E.coli

Although bacteria is one of the oldest living organisms,the pathogenicity of bacteria is still one primary source of disease in our daily life from the view of modern medicine.The advent of antibiotics has solved most of the diseases caused by bacteria as modern medicine advanced.However,some superbacteria still exist the environment with antibiotics,leading to new problems about bacterial pathogenesis.Having been studied to some extent in past several decades,the specific pathogenic mechanism of bacteria,which is closely related to the motile behavior of bacteria,is still in urgent need to be solved.If these mechanisms could be well understood,new drugs or new treatment processes would be developed.Therefore,the most primitive question of how the mechanism of the movement of bacteria is operating was brought back to our view.In the last century,a series of important progresses on experimental design and theoretical modeling for studying this problem have been made by more and more biologists and physicists,and this study also belongs to the study of the behavior of bacteria,which will be divided into four chapters.Chapter one will introduce a series of studies on Escherichia coli in the last few decades,referring to the cell structure of Escherichia coli,individual swimming behavior,experimental and modeling studies of chemotaxis network,the flexibility of flagellar hook,the source of motor motion,flagellar motor stepping motion,MotB(motor component)exchange,the torque-speed curve of the motor,the dynamic nature of mechanical sensitivity,the research content of this paper and so on.In the second chapter,we mainly study the duty ratio of the motor,which has been debated both experimentally and theoretically in the past twenty years,and there has been no directly experimental evidence to solve this argument.We developed a technique combining optical tweezer,laser dark field and bright field imaging.We first used bead addition technique to measure the number of stators binding to motor at zero load,proving that the zero-load speed is independent of the stator number,thus determining that the duty ratio is close to 1.Therefore,some theoretical assumptions on the flagellar motor would be eliminated,the physical image of the motor becoming clearer and clearer.In summary,we developed a method to precisely measure the stator number for flagellar motor at zero load.This method could also solve the problem that the number of stators binding to motor is so few that this number would be unable to be determined previously.In terms of technological development,we realized the visualization of nano gold spheres at high temporal resolution and applied optical tweezers technology simultaneously,which provided an experimental possibility for future research on the interaction between optics and gold sphere.In the third chapter,we study the problem that the proton transport of the motor and its rotation is tightly or loosely coupled.Genally speaking,the efficiency of the flagellar motor of Escherichia coli at high load would reach the maximum,but number of protons consumed when the motor steps one revolution,is still not very precisely determined because the motor torque at high load is not measured accurately.But according to the trend of torque-speed curve,it could be assumed that the output torque of the motor is approximately equivalent to the torque when the motor is pulled and halted externally(stall torque).Therefore,the first part of this chapter is to on how to accurately measure the stall torque of the flagellar motor,including several early works on measuring the stall torque.Magnetic tweezer would be a suitable technique to measure the stall torque of flagellar motor,but the process of calibrating the torsional stiffness of magnetic tweezers is still unaccurate.In second part,we mainly talk about how to eliminate the uncertainties,such as by combining with optical tweezers or other techniques.In a word,we try to use magnetic tweezers to prove whether the motor's proton transport is tightly or loosely coupled with the rotation of motor experimentally.In the fourth chapter,based on our own work and the previous work in the past few years,we mainly summarize the above work and look forward to the future work.The first work was determining that the duty ratio of the Escherichia coli motor is close to 1,resolving the previous dispute and clarifying the physical images of the motor movement.The second work mainly introduced a possible way to accurately measure the stall torque of Escherichia coli,some of the problems we met now,and future improvements to solve these problems.Finally,considering the above work and previous works,we could use our newly developed technology combining optical tweezer,laser dark-field and bright-field imaging to more accurately study the properties of the flagellar motor,such as the torque-speed curve for motors with a specific number of stators and so on;in addition,we introduced magnetic tweezer for measurement of stall torque of Escherichia coli,and if succeeded,conversely,certain properties of magnetic tweezer could be investigated with the output torque of the flagellar motor,thus promoting the application of magnetic tweezer to other areas.