Studies On The Dynamic Properties Of Flagellar Motor And Motility Mechanism In Pseudomonas Aeruginosa

Motility and chemotaxis are two core issues in bacterial biophysics-related research,which play an important role in biological processes such as the exploration of the environment and the spreading of bacterial infections.As an opportunistic pathogen,Pseudomonas aeruginosa can use its single polar flagellum and type Ⅳ pili to achieve swimming,swarming and twitching motility.Previous studies on P.aeruginosa-related infection confirmed that flagella-driven motility was closely related to the increase of incidence rate and mortality of acquired pneumonia and burn wound sepsis.Therefore,an in-depth understanding of the motility behavior of P.aeruginosa is of great significance to further clarify its pathogenic mechanism.The dynamic properties of flagellar motors and single-cell swimming mechanism are the two cores of this study:the former focuses on the intracellular working mechanism of this macromolecular machine,while the latter focuses on the dynamic behavior of extracellular flagellar filaments during swimming motility.Bacterial motility is rarely random,but is controlled by a complex intracellular signal transduction system.After gaining a comprehensive understanding of the motor behavior of P.aeruginosa,we used computer simulation technology to determine the kinetic factors for its efficient chemotaxis at the single-cell level,and experimentally observed the close connection between its flagellar motor and the chemotactic network.For the flagellar motor of P.aeruginosa,there has been a lack of powerful method to study it at the single-cell level.In this study,we developed a bead assay by introducing a cysteine mutation point into the flagellin FliC and using the biotinstreptavidin system to attach microspheres to shortened flagellar filaments.The realtime output properties of the motor can be characterized by observing the rotation of the beads.Using this bead assay,we found that the two sets of stator units contribute differently to the motor output.The MotAB stators generate higher total torque to support faster movement,whereas the MotCD stators provide excellent speed stability for the motor.By changing the external environment,we found that the wild-type strain can dynamically adjust the stator composition on the motor,which greatly enhances its environmental exploration ability.In addition,the combined use of long-term output signal acquisition and nonlinear filtering data processing methods makes it possible to accurately measure the number of stators on the motor and the speed generated per stator unit.P.aeruginosa use the electrochemical H+gradient to power the rotation of transmembrane motors,which drive the rotation of semi-rigid,helical flagellar filaments that allow cells to move.In order to more comprehensively characterize the motility behavior of single cells,we used the coupling between free sulfhydryl groups and maleimide to fluorescently label the flagellar filaments of living cells to observe their dynamic behavior,and proposed that it follows a swimming pattern covering"pull-wrap-push".By analyzing the near-surface swimming trajectories of the cells,we found that cells in push and pull states maintain the same flagellar filament chirality and conformation.In addition,we observed similar reversal events without flagellar polymorphic transformation in other peritrichous strains,which greatly deepened our understanding of the mechanism of flagellar filament rearrangement.Based on the systematic experimental study of the internal dynamic properties and external motility mechanisms of P.aeruginosa flagellar motors,we performed computer simulations of single-cell chemotactic behavior using a chemotactic simulation method based on the coarse-grained signaling pathway.The simulation results suggest that the synergistic assembly of the two stator components and the novel motility mechanism involving the wrap state are two key factors for the efficient chemotaxis of P.aeruginosa.Therefore,the close relationship between motor behavior and chemotactic phenotype is supported by reliable data.Then we carried out experiments using chromosomal fluorescent protein fusion technology,and found the consistency of spatial positioning between flagellar motor and chemotactic receptor cluster.Three factors affecting the distribution of the latter were found one after another,and they were the polar determinant flhF,flagellar motor-related protein and cell body curvature.By knocking out the core genes(fliG,fliF)of flagellar motor assembly,we discovered a motor integrity-dependent chemoreceptor cluster assembly mechanism in a bacterial system.From micron-scale individual swimming behavior to nano-scale macromolecular machine dynamics to intracellular molecular regulation mechanisms,the research scales range from large to small.The extension of the research dimension has deepened our understanding of the motile behavior of P.aeruginosa,and also provided a perfect experimental technique for further related research.The results in this thesis provide theoretical reserves for single-cell kinematics for further understanding of the mechanism of P.aeruginosa-related host-pathogen interaction and the actual physiological issues such as biofilm colonization and expansion.They also have reference significance for the study of motile behavior of other microorganisms.