Study On The Second Messenger Molecule And The Internal Load Of Flagellar Motor In Escherichia Coli

In the long course of history,human beings have never ceased to explore the nature of the law of life.All organisms including not only multicellular animals and plants,but also small single-celled bacteria and fungi can respond to external stimuli.The development of biology has demonstrated that many events changing human understanding of life are inextricably linked to microorganism.In this paper,we took Escherichia coli(E.coli)as the research object,and applied the concepts and methods of physics to studying the relationship between its structure and function.How can the external stimuli be perceived?Why should the internal signal be amplified?What is the connection between bacterial individuals and groups?Through the quantitative research on the target object,it is further extended to other organisms to clarify the laws of energy,matter and information at a certain time and space.In the first chapter,mainly the research progress and the related background knowledge of E.coli were introduced.First,the morphology and structure,metabolism and proliferation of E.coli were intuitively described.Second,combining structure and function of E.coli receptor protein and signal protein in vivo,we introduced the chemotactic signal transduction network and bacterial movement behavior.Third,the flagellar motor serves as the power force driving E.coli to move in the liquid.We introduced the base part,the external connection part of the motor,and the spiral flagella.Furthermore,we explained the switch,the output torque of the motor,the dynamic exchange of the stator protein in response to the external environment.Finally,this paper offered a brief description of the application principle of proteorhodopsin protein in E.coli and some common techniques in the current study.In the second chapter of this paper,we introduced the culture of bacteria and the method of strain transformation:preparation steps of culture solution and motility medium buffer,storage conditions of the strains,polymerase chain reaction(PCR)technology,target gene deletion,construction and transformation of plasmids,as well as fluorescent protein labeling.In the third chapter of this paper,the second messenger molecule(c-di-GMP)adjusts motility and promotes surface aggregation of bacteria were introduced.In E.coli,the protein YcgR containing the PilZ domain acts as an effector protein of the second messenger molecule,and the c-di-GMP::YcgR can inhibit the rotation of the flagellar motor.In the past two decades,the problem of the specific position of the protein c-di-GMP::YcgR interacts with the flagellar motor has been a heated debate in academic circles.To study this problem,we built a two-layer optical path microscope.Through the simultaneous analysis of the bright field and fluorescent images of the rotating bacteria,we found that the site of interaction with the c-di-GMP::YcgR protein is mainly located at the C-ring of the motor,not at the stator.In addition,since the torque-speed curve is one of the important properties of the motor.We also measured the effect of the second messenger molecule on the torque-speed curve with the motor rotating counterclockwise(CCW).We further studied the effect of the second messenger molecule on motor switch and bacterial movement behavior.In the fourth chapter of this paper,we measured the internal load of the E.coli flagellar motor by analyzing the fluctuation of the motor speed.In the experimental part,we recorded "motor resurrection" data under different loads(1.0?m;0.75?m;0.5?m),and then performed wild-type motor experiments.In the theoretical derivation part,we described the relationship between the external load(small bead)and the internal load(motor rotor)of the rotating motor through the Langevin equation.It is deduced that the internal frictional drag coefficient is independent of the external load and the number of stators,and corresponds to the same position of the "knee" in the power spectrum analysis of motor speed under different loads and different numbers of stators.The results of the motor resurrection experiments also verify the theoretical derivation.In the supplementary experiment,we ruled out the effect of long flagella and sampling frequency.Our results supply an important parameter for future model of the flagellar motor.In the fifth chapter of this paper,we explored other aspects around the flagellar motor.First,we characterized the cell body PMF(proton motive force)by motor rotation,and then studied the response of bacteria to the pH of the solution.Second,we measured the response latency of single-motor and multi-motor bacteria.In the last chapter of the paper,we summarized the work of this paper and made a research prospect.The two main works are to study the kinetic mechanism of bacterial flagellar motor.In the first work,we studied the effect of a small molecule(the second messenger molecule)on the properties of the motor and its mechanism,and discovered that c-di-GMP affects both the rotation speed and directional bias of the motor,thereby promoting the accumulation of bacteria on a solid surface.The effects of c-di-GMP we characterized here are consistent with the key role that c-di-GMP plays in the transition between motile and sedentary forms of bacterial life.In the second work,we measured the rotational drag coefficient of the rotor by fluctuation analysis.Our measurement supplies an important parameter for future updated modeling of the flagellar motor.