Bacterial Flagellar Motors as Microfluidic Actuators

2004 ◽  
Author(s):  
Ryan Pooran ◽  
Mohamed Al-Fandi ◽  
Steve Tung ◽  
Jin-Woo Kim ◽  
Nalini Kotagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Adhesion Force ◽  
Flagellar Motor ◽  
Hydrodynamic Load ◽  
Microfluidic Systems ◽  
Hydrodynamic Loading ◽  
Flagellar Motors ◽  
Flagellar Filament ◽  
Biochemical Analyses ◽  
Multiple Cells

Microfluidic actuators are extremely important components of microfluidic systems used in biological, chemical and biochemical analyses. We are in the process of developing hybrid microsystem devices that utilize flagellar motors in tethered Escherichia coli (E.coli) cells as actuators. In this study we describe the robustness of the flagellar motor actuators. To examine the robustness of the actuators we observe the behavior of the cells when exposed to a hydrodynamic load. With this method we are able to determine the torque generated by the flagellar motor and the adhesion force of the flagellar filament to the substrate. Using hydrodynamic loading we are also able to synchronize the rotation of multiple cells. Finally, we present data which represents the lifetime of the flagellar motor actuators.

scholarly journals Biphasic chemotaxis ofEscherichia colito the microbiota metabolite indole

2020 ◽  
Vol 117 (11) ◽  
pp. 6114-6120 ◽  
Author(s):  
Jingyun Yang ◽  
Ravi Chawla ◽  
Kathy Y. Rhee ◽  
Rachit Gupta ◽  
Michael D. Manson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Bacterial Chemotaxis ◽  
Dynamic Responses ◽  
Time Dependent ◽  
Flagellar Motor ◽  
Wild Type ◽  
Gi Tract ◽  
Flagellar Motors

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposedEscherichia colito a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response—wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.

scholarly journals Applying torque to the Escherichia coli flagellar motor using magnetic tweezers

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Maarten M. van Oene ◽  
Laura E. Dickinson ◽  
Bronwen Cross ◽  
Francesco Pedaci ◽  
Jan Lipfert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
External Load ◽  
Magnetic Bead ◽  
Magnetic Tweezers ◽  
Flagellar Motor ◽  
Motor Speed ◽  
Motor Torque ◽  
Rotary Engine ◽  
Flagellar Motors ◽  
Before And After

Abstract The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor’s response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor’s performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level.

scholarly journals Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H+-Driven and Na+-Driven Motors in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1466-1472 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Junghoon Yang ◽  
Hajime Fukuoka ◽  
Michio Homma ◽  
David F. Blair
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Electrostatic Interactions ◽  
General Feature ◽  
Flagellar Motor ◽  
Motor Patterns ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Flagellar Rotation

ABSTRACT In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

Application of a PDMS Microsieve for the Patterning of Flagellar Motors in a Microfluidic System

2005 ◽  
Author(s):  
Ryan Pooran ◽  
Steve Tung ◽  
Jin Woo Kim
Keyword(s):  
Experimental Data ◽  
Escherichia Coli ◽  
Microfluidic System ◽  
Substrate Material ◽  
Flagellar Motor ◽  
Escherichia Coli Cells ◽  
Flagellar Motors

We are in the process of developing hybrid micro devices that utilize the flagellar motors of Escherichia coli cells as actuators. For the micro devices it is important that the flagellar motors be tethered at specific locations on a given substrate in order to maximize its performance as actuators. In this paper we present results that demonstrate an effective method for patterning the flagellar motor actuators onto a substrate material. We also present the first experimental data which discusses the performance of the actuator in a microfluidic pump.

scholarly journals The Structure, Composition, and Role of Periplasmic Stator Scaffolds in Polar Bacterial Flagellar Motors

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaotian Zhou ◽  
Anna Roujeinikova
Keyword(s):  
Cytoplasmic Membrane ◽  
Flagellar Motor ◽  
New Paradigm ◽  
Binding Studies ◽  
Theoretical Concepts ◽  
Flagellar Motors ◽  
Flagellar Filament ◽  
Electron Cryotomography ◽  
And Function

In the bacterial flagellar motor, the cell-wall-anchored stator uses an electrochemical gradient across the cytoplasmic membrane to generate a turning force that is applied to the rotor connected to the flagellar filament. Existing theoretical concepts for the stator function are based on the assumption that it anchors around the rotor perimeter by binding to peptidoglycan (P). The existence of another anchoring region on the motor itself has been speculated upon, but is yet to be supported by binding studies. Due to the recent advances in electron cryotomography, evidence has emerged that polar flagellar motors contain substantial proteinaceous periplasmic structures next to the stator, without which the stator does not assemble and the motor does not function. These structures have a morphology of disks, as is the case with Vibrio spp., or a round cage, as is the case with Helicobacter pylori. It is now recognized that such additional periplasmic components are a common feature of polar flagellar motors, which sustain higher torque and greater swimming speeds compared to peritrichous bacteria such as Escherichia coli and Salmonella enterica. This review summarizes the data available on the structure, composition, and role of the periplasmic scaffold in polar bacterial flagellar motors and discusses the new paradigm for how such motors assemble and function.

scholarly journals A Chimeric N-Terminal Escherichia coli-C-Terminal Rhodobacter sphaeroides FliG Rotor Protein Supports Bidirectional E. coli Flagellar Rotation and Chemotaxis

2005 ◽  
Vol 187 (5) ◽  
pp. 1695-1701 ◽  
Author(s):  
Karen A. Morehouse ◽  
Ian G. Goodfellow ◽  
R. Elizabeth Sockett
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Sphaeroides ◽  
Protein Interactions ◽  
Flagellar Motor ◽  
E Coli ◽  
C Terminus ◽  
Flagellar Motors ◽  
Serovar Typhimurium ◽  
Essential Function ◽  
Flagellar Rotation

ABSTRACT Flagellate bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium typically express 5 to 12 flagellar filaments over their cell surface that rotate in clockwise (CW) and counterclockwise directions. These bacteria modulate their swimming direction towards favorable environments by biasing the direction of flagellar rotation in response to various stimuli. In contrast, Rhodobacter sphaeroides expresses a single subpolar flagellum that rotates only CW and responds tactically by a series of biased stops and starts. Rotor protein FliG transiently links the MotAB stators to the rotor, to power rotation and also has an essential function in flagellar export. In this study, we sought to determine whether the FliG protein confers directionality on flagellar motors by testing the functional properties of R. sphaeroides FliG and a chimeric FliG protein, EcRsFliG (N-terminal and central domains of E. coli FliG fused to an R. sphaeroides FliG C terminus), in an E. coli FliG null background. The EcRsFliG chimera supported flagellar synthesis and bidirectional rotation; bacteria swam and tumbled in a manner qualitatively similar to that of the wild type and showed chemotaxis to amino acids. Thus, the FliG C terminus alone does not confer the unidirectional stop-start character of the R. sphaeroides flagellar motor, and its conformation continues to support tactic, switch-protein interactions in a bidirectional motor, despite its evolutionary history in a bacterium with a unidirectional motor.

scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mohammed Kaplan ◽  
Debnath Ghosal ◽  
Poorna Subramanian ◽  
Catherine M Oikonomou ◽  
Andreas Kjaer ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Bioinformatics Analysis ◽  
Cell Envelope ◽  
Shewanella Oneidensis ◽  
Flagellar Motor ◽  
Flagellar Motors ◽  
A Cell ◽  
Animal Hosts

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here, we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor’s stator system and its structural elaboration. Motors with a single H+-driven stator have only the core periplasmic P- and L-rings; those with dual H+-driven stators have an elaborated P-ring; and motors with Na+ or Na+/H+-driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

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