Studies On The Dynamics Of Escherichia Coli Flagellar Motor

Bacteria have a crucial influence on the global environment and human life.As the simplest biological system,Escherichia coli is a type of single-cell organism which normally inhabit human gut,and often used as a modern organism for labora-tory research.The motility of E.coli is one of the important conditions for its survival,chemotaxis and pathogenicity.It is closely related to the flagellar motor,a precision molecular rotary motor.Its molecular structure and motion mechanism are both dif-ferent from macro motors.The study of bacterial motility and motor mechanism has potential application in medicine and nano-engineering,such as targeted drugs deliv-ery in the human body,and the design of ultra-sensitive biosensors and micro-robots.In this dissertation,the dynamic of E.coli flagellar motor was studied.We ap-plied the methods of labeling latex beads to motor and using light-powered proton pump(proteorhodopsin protein)to control cellular proton motive force(PMF),and combined with statistical modeling to study the kinetics of stator turnover,the rela-tionship between the number of stators and CW bias(the probablility of motor turning clockwise),and switching interval distributions.These studies let us further under-stand the flagellar motor.In the first chapter,we started by introducing the background knowledge of E.coli,including the cell morphology and cellular structure,the motility and chemo-taxis system.The focus is on the research progress of E.coli flagellar motor.Then we briefly introduced the theory and application of proteohodopsin(PR)protein.We ended by analyzing the significance of the studies of E.coli and the problems to be solved.In the second chapter,the experimental methods were introduced,including the conditions of cultivating bacteria,the recipies for related reagents,and the plasmid transformation techniques and recombinant DNA techniques used in the transfor-mation of strains.The E.coli flagellar motor is dynamic,and stators will randomly bind or unbind to motor.Recent experiments have shown that the stator's stability was related to the external load.The higher the load,the stronger stator was bound to the motor.At high load in the steady state,the rotation speed would exhibit stepwise fluctuations as sta-tors coming on and off the motor.We used a high-resolution camera to record the time series of individual motor speeds.We analyzed the dwell-time distribution of stators and found the distribution is multi-exponential shape,which was contrary to that pre-dicted by the two-state model of the kinetics of stator turnover.There must exist a third state,which we called the hidden state.The lifetime of the hidden state is very short,and the number of stators in the hidden state is at most one.The same method can be used to study other fast dynamics in biology.E.coli rotary flagellar motor has two rotational states:clockwise(CW)and coun-ter clokwise(CCW)rotation.Measurements of the CW or CCW dwell time distribu-tions provide the basis for studying the motor-switching dynamics.We systematically analyzed the interval time distributions under different loads,different proton motive forces,and different stator numbers.With the increase of the torque,the interval dis-tribution changed from exponential to non-exponential shape.We introduced the non-equilibrium effect into the conformation spread model of motor switching,suc-cessfully explained this observation and predicted that as external load increases,the motor dose-response curve will shift to the left.Only about 0.2%of the energy input in the motor is used for motor switching,but the sensitivity of the motor to the level of signal protein CheY-P is greatly increased.The switching of flagellar motor is not only regulated by the chemotaxis network system,but also affected by changes in load,temperature and proton-motive-force.We used the light-powered proton pump(proteorhodopsin)to control PMF level by using different intensities of green light,and carried out the motor resurrection ex-periments.Based on this technology,we studied the quantitative relationship between CW bias and the number of stators under different levels of CheY-P.When the CheY-P concentration is low,the CW bias increases from zero with the number of stators;when the CheY-P concentration increases,the CW bias decreases from 1 as the number of stators increases.This shows that motors with different number of sta-tors had different sensitivities to CheY-P level.The dose-response curves suggested that as the number of stators increases,the hill coefficient decreased first and then in-creases.This may be due to the non-equilibrium effect of torque,but the specific mechanism remains to be further explored.We found that the observed and true rotational directions of bacterial flagellar motor maybe be reversed in the bead assay,which affected the experimental results on motor CW bias distribution.We suspected that because the flagellum hook was soft and bended when labeled with the latex bead,but it is not yet known how this phe-nomemon happened.We systematically studied how to reduce or eliminate the error.To reduce the error probability,we should choose the cell which was totally stuck to the glass,and with the bead attacted to a motor in the middle of the cell body.To eliminate the error,for strains with functional chemotaxis network,we can use the knowledge that attractants will reduce CW bias to distinguish the rotational direction of the motor;If the chemotaxis network was unfuntional,we can test the rotation di-rection by flowing motility buffer with acid PH;when flowing motility buffer with PH equal to 5.0,the cell should tend to rotate clockwise.In the last chapter,we summarized our work and presented our outlook on future works on the bacterial flagellar motor.