Study On The Interaction Mechanism Of Chemoreceptors And Chemotaxis Network Noise Of Escherichia Coli

The survival and reproduction of living organisms cannot be separated from the tendency to seek benefits and avoid harm.As one of the oldest,most widely distributed and simplest life forms on Earth,the regulatory network and internal mechanism of the chemotactic behavior of bacteria have been studied.On the one hand,these studies are conducive to our in-depth exploration of the conserved law of life evolution over hundreds of millions of years.On the other hand,they are helpful to our understanding of the physiological process of the chemotactic infection of bacteria as one of the pathogenic microorganisms,and provide some new research ideas for the prevention of disease caused by bacterial infection.In this paper,Escherichia coli was selected as the research object.The bacterium senses and explores the external environment through the chemotaxis signal transduction system,and regulates its movement pattern and migration behavior.As the bacterium moves toward favorable stimuli,an increase in ligand concentration bound to the chemoreceptor decreases receptor activity,thereby reducing the concentration of the diffusible protein CheY-P phosphorylated by the protein kinase CheA,and decreasing the probability of the bacterium undergoing a tumble.In addition,the chemotactic network has an adaptation mechanism.When the activity of the chemoreceptor changes due to ligand binding,the adaptive enzymes CheR and CheB will methylate and demethylate the chemoreceptor respectively,so that the activity of the chemoreceptor is gradually restored.Therefore,we are led to consider the following two questions:Firstly,when there is no change in external ligand concentration,the CW bias of bacterial flagellar motor is not fixed but fluctuates due to the noise in CheY-P concentration.Is this related to the activity of the receptor itself or the dynamic modification of the chemoreceptor by the adaptive enzyme CheR?Secondly,bacteria often have a variety of chemoreceptors,and these receptors play physiological functions in the form of clusters.What is the intrinsic mechanism of their interaction,and what effect will it have on the efficiency of bacterial chemotaxis sensing and adaptation?To address the above two problems,we comprehensively applied the techniques of gene editing,microfluidics,bead assay,and fluorescence resonance energy transfer(FRET).Based on previous studies,we explored the mechanism of noise generation in the chemotaxis network and the interaction of chemoreceptors in E.coli.The specific researches are as follows.In order to study the mechanism of noise generation in chemotaxis signal transduction network from the single-cell level,we compared the fluctuation noise of CheY-P and chemoreceptor activity in different bacteria under steady-state conditions by observing the CW bias of the flagellar motor.We also characterized the concentration of methyltransferase CheR in real-time by observing the recovery time of bacterial chemoreceptors to external saturated stimuli.By analyzing the correlation between the fluctuation of CheY-P concentration and chemoreceptor activity as well as CheR concentration,we found that the noise of CheY-P concentration was negatively correlated with the concentration of CheR and positively correlated with the activity of the chemoreceptors,indicating that the noise was caused by the concentration of CheR and the activity of the chemoreceptors.In order to explore the interaction effect between bacterial chemoreceptors,we labeled CheY and CheZ with fluorescent proteins and used FRET technique to observe the chemoreceptor adaptation time of Tar-only mutant and wild-type strain under different chemoreceptor activities.The experimental results were as follows.First,when the chemoreceptor was inactivated under saturated stimulation,the wild-type strain with multiple receptors showed a shorter recovery time.Second,the Tar-only mutant showed faster adaptation and higher sensitivity than the wild-type strain,and the difference became more obvious with the decrease of chemoreceptor activity.In addition,we also demonstrated that the different adaptation rates between the wild-type strain and the Tar-only strain was not caused by the difference in expression level of Tar chemoreceptor.Third,in absence of stimulation,the wild-type strain also showed a longer adaptation time than the mutant.By simulating the chemotaxis of bacteria with different adaptation rates in a stable stimulus concentration gradient,we confirmed that the interaction of different types of receptors can effectively promote the chemotaxis of E.coli under stable spatial gradient of attractant and ensure the minimum noise of cell distribution.In summary,we found that the expression of methyltransferase CheR can enhance the methylation level of bacterial chemoreceptors,promoting faster adaptation of bacteria to their environment and reducing noise in the chemotaxis signaling output.The chemoreceptor interaction effectively enhances the speed and sensitivity of bacterial chemotactic adaptation.Under stable stimulation concentration gradients,the cooperative action of different chemoreceptors effectively promotes E.coli chemotaxis,and ensures minimal cell distribution noise.In this study,we carefully quantified the changes of chemoreceptor adaptation time with chemoreceptor activity through precise concentration control of chemical stimulus concentration and long-term stable measurement of cell chemotactic responses,providing a unique interpretation of the chemoreceptor interaction mechanism and further deepening the understanding of the chemotaxis system.