Studies On The Cooperativity Of Chemotactic Regulatory Elements And The Effect Of The Second Messenger Molecule C-di-GMP On Chemotaxis Of Escherichia Coli

Our world is overflowing with the diversity of life.During the long evolution of about four billion years,the process of genetic mutation combined with selective environmental pressures has resulted in highly optimized evolutionary solutions to biological problems at the molecular level.Any form of life can respond to changes in the external environment to ensure its own survival and reproduction.The signal processing embodied in life is realized by various intracellular signal transduction networks.An important aspect of understanding the nature of life is to explore how these signal transduction networks function.Meanwhile,the study of bacteria is of great importance for areas that have a large impact on society,namely those of bionanotechnology and synthetic biology,food and drug industrial application,fuel production for commercial use and biomedicine,and so on.In this dissertation,we selected the model organism Escherichia coli as the research object.A comprehensive study of its chemotaxis signal transduction network will be performed.This system has been shown to be an excellent paradigm model for the study of general signaling transduction networks in far more complex organisms.Here,we revealed the cooperativity of the regulatory elements of the chemotaxis network at the single-cell level,and further expanded the understanding of the effect of the second messenger c-di-GMP on chemotaxis.In this thesis,investigation of the bacterial chemotaxis signal transduction network with quantitative experiments and computational simulations provided new insights into the design principles and influencing factors of transduction networks,and enhanced our comprehension of the nature of life.For the chemotaxis network of E.coli,previous studies were usually carried out in vitro based on biochemical experiments and usually at the population level.The population-averaged experimental results cannot accurately describe its functional properties and mechanisms.Here,by constructing a fluorescence resonance energy transfer(FRET)measurement system,we succeeded in detecting chemotactic signal responses in real-time at the single-cell level in vivo.Subsequently,systematic studies were carried out on the key regulatory elements of chemotaxis(the phosphatase CheZ and response regulatory protein CheY-P),revealing high cooperativity in the interaction between CheZ and CheY-P.This cooperativity suppressed the cell-to-cell variation of CheY-P concentration so that it fell within the operational range of the flagellar motor.Thus,we elucidated new mechanisms for the robustness and adaptability of the bacterial chemotaxis signaling network.To deepen our understanding of the molecular mechanisms underlying this cooperative effect,we established three origins of this high cooperativity by performing experiments with different mutants of CheZ.Besides the cooperativity due to the intrinsic allostery of the CheZ homodimer,one is from the localization of CheZ to the receptor clusters via interaction with the short form of the kinase CheAs located at the receptor cluster,and the other may be that CheY-P promotes the oligomerization of CheZ in the crowded intracellular environment,thereby exhibiting cooperative effect.This greatly deepened our understanding of the cooperativity of the chemotaxis network and provided new ideas and methods for studying other microbial chemotaxis networks and signal transduction networks in complex organisms.After explaining the characteristics of the chemotaxis network,we further conducted studies on the effect of the second messenger c-di-GMP on chemotaxis.The ubiquitous bacterial second messenger molecule c-di-GMP affects the operation of the flagellar motor at the output end of the chemotaxis network through the effector protein YcgR.However,systematic research on its effect on E.coli chemotaxis is still lacking.At the downstream output level of the chemotaxis network,by performing dynamic fluorescence measurements at the single-cell level,we revealed for the first time the dynamic exchange properties of c-di-GMP::YcgR at flagellar motors.Meanwhile,we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR.At the upstream input level of the chemotaxis network,we found that c-di-GMP had no significant effect on the dose-response and stimulusadaptive properties of the chemotaxis network.Thus,we systematically elucidated the effect of c-di-GMP on the upstream and downstream of E.coli chemotaxis network.In order to study the joint effects on bacterial chemotaxis,namely the reduction of the swimming speed and the reduction of the tumbling frequency of E.coli caused by the elevated c-di-GMP levels,we performed a stochastic simulation of the chemotactic motion,which was based on a coarse-grained description of the intracellular chemotaxis signaling pathway and a computational model coupling chemotaxis signaling and swimming.Lastly,we also used the microfluidic assay to elucidate the effect of c-di-GMP on chemotactic motility.In summary,we demonstrated the mechanism by which E.coli can maintain normal chemotactic performance in the solution environment with elevated c-di-GMP levels.From the revealing of the cooperativity in the chemotaxis network to the elucidating of the influence of external factor(the second messenger molecule c-diGMP)on it,systematic studies of the chemotaxis network were carried out from inside to outside.This provided strong support for exploring other microbial chemotaxis signal transduction networks and opened up new ways to deepen the understanding of signal transduction networks in other complex living systems.Our studies of the chemotaxis signal transduction network in E.coli promoted our comprehension of the nature of life,allowing us to see the beauty of science behind life and to really appreciate life.