Stable sub-complexes observedin situsuggest a modular assembly pathway of the bacterial flagellar motor

AbstractThe self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. InSalmonellaandEscherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intactLegionella pneumophila,Pseudomonas aeruginosaandShewanella oneidensiscells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub-complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub-complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed.

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

eLife ◽

10.7554/elife.43487 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 17

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.

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Loss of the Bacterial Flagellar Motor Switch Complex upon Cell Lysis

mBio ◽

10.1128/mbio.00298-21 ◽

2021 ◽

Author(s):

Mohammed Kaplan ◽

Elitza I. Tocheva ◽

Ariane Briegel ◽

Megan J. Dobro ◽

Yi-Wei Chang ◽

...

Keyword(s):

Self Assembly ◽

Bacterial Species ◽

Cell Lysis ◽

Flagellar Motor ◽

Bacterial Flagellar Motor

The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor.

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Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor

Nature Structural & Molecular Biology ◽

10.1038/nsmb.3172 ◽

2016 ◽

Vol 23 (3) ◽

pp. 197-203 ◽

Cited By ~ 37

Author(s):

Matthew A B Baker ◽

Robert M G Hynson ◽

Lorraine A Ganuelas ◽

Nasim Shah Mohammadi ◽

Chu Wai Liew ◽

...

Keyword(s):

Self Assembly ◽

The Self ◽

Flagellar Motor ◽

Domain Swap ◽

Bacterial Flagellar Motor

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Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor

10.1101/2021.10.27.466211 ◽

2021 ◽

Author(s):

Tatsuro Nishikino ◽

Yugo Sagara ◽

Hiroyuki Terashima ◽

Michio Homma ◽

Seiji Kojima

Keyword(s):

Three Dimensional ◽

Dimensional Structure ◽

Growth Defect ◽

Flagellar Motor ◽

Inner Membrane ◽

Assembly Mechanism ◽

Conduction Pathway ◽

Ion Conducting ◽

The Stability

Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolicinterface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

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Torque-dependent remodeling of the bacterial flagellar motor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904577116 ◽

2019 ◽

pp. 201904577 ◽

Cited By ~ 6

Author(s):

Navish Wadhwa ◽

Rob Phillips ◽

Howard C. Berg

Keyword(s):

Free Energy ◽

Self Assembly ◽

Scientific Literature ◽

Protein Complexes ◽

Flagellar Motor ◽

Motor Speed ◽

Binding Rate ◽

Motor Load ◽

Bacterial Flagellar Motor

Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.

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The structural complexity of the Gammaproteobacteria flagellar motor is related to the type of its torque-generating stators

10.1101/369397 ◽

2018 ◽

Cited By ~ 3

Author(s):

Mohammed Kaplan ◽

Debnath Ghosal ◽

Poorna Subramanian ◽

Catherine M. Oikonomou ◽

Andreas Kjær ◽

...

Keyword(s):

Legionella Pneumophila ◽

Pathogenic Bacteria ◽

Cell Envelope ◽

Shewanella Oneidensis ◽

Structural Complexity ◽

High Viscosity ◽

Flagellar Motor ◽

Flagellar Motors ◽

Wide Range ◽

A Cell

AbstractThe bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque-generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine thein situmacromolecular structures of the flagellar motors of three Gammaproteobacteria species:Legionella pneumophila,Pseudomonas aeruginosa, andShewanella oneidensisMR-1, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the stator system of the motor and its structural complexity. Motors with a single H+-driven stator system have only the core P- and L-rings in their periplasm; those with dual H+-driven stator systems have an extra component elaborating their P-ring; and motors with Na+- (or dual Na+-H+)- driven stator systems have additional rings surrounding both their P- and L-rings. Our results suggest an evolution of structural complexity that may have enabled pathogenic bacteria likeL. pneumophilaandP. aeruginosato colonize higher-viscosity environments in animal hosts.

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The Dynamic Ion Motive Force Powering the Bacterial Flagellar Motor

Frontiers in Microbiology ◽

10.3389/fmicb.2021.659464 ◽

2021 ◽

Vol 12 ◽

Author(s):

Anaïs Biquet-Bisquert ◽

Gilles Labesse ◽

Francesco Pedaci ◽

Ashley L. Nord

Keyword(s):

Mechanical Energy ◽

Dynamical Behavior ◽

Motive Force ◽

Cellular Respiration ◽

Flagellar Motor ◽

Motor Speed ◽

Inner Membrane ◽

Ion Flow ◽

Bacterial Flagellar Motor ◽

The Imf

The bacterial flagellar motor (BFM) is a rotary molecular motor embedded in the cell membrane of numerous bacteria. It turns a flagellum which acts as a propeller, enabling bacterial motility and chemotaxis. The BFM is rotated by stator units, inner membrane protein complexes that stochastically associate to and dissociate from individual motors at a rate which depends on the mechanical and electrochemical environment. Stator units consume the ion motive force (IMF), the electrochemical gradient across the inner membrane that results from cellular respiration, converting the electrochemical energy of translocated ions into mechanical energy, imparted to the rotor. Here, we review some of the main results that form the base of our current understanding of the relationship between the IMF and the functioning of the flagellar motor. We examine a series of studies that establish a linear proportionality between IMF and motor speed, and we discuss more recent evidence that the stator units sense the IMF, altering their rates of dynamic assembly. This, in turn, raises the question of to what degree the classical dependence of motor speed on IMF is due to stator dynamics vs. the rate of ion flow through the stators. Finally, while long assumed to be static and homogeneous, there is mounting evidence that the IMF is dynamic, and that its fluctuations control important phenomena such as cell-to-cell signaling and mechanotransduction. Within the growing toolbox of single cell bacterial electrophysiology, one of the best tools to probe IMF fluctuations may, ironically, be the motor that consumes it. Perfecting our incomplete understanding of how the BFM employs the energy of ion flow will help decipher the dynamical behavior of the bacterial IMF.

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Mechanosensitive remodeling of the bacterial flagellar motor is independent of direction of rotation

10.1101/2021.01.19.427295 ◽

2021 ◽

Author(s):

Navish Wadhwa ◽

Yuhai Tu ◽

Howard C. Berg

Keyword(s):

Self Assembly ◽

External Load ◽

Protein Complexes ◽

General Strategy ◽

Flagellar Motor ◽

Functional Regulation ◽

Bacterial Flagellar Motor ◽

Living Organisms ◽

Broad Interest ◽

Insight Into

Motility is critical for the survival and dispersal of bacteria, and it plays an important role during infection. How bacteria regulate motility is thus a question of broad interest. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load).SignificanceMacromolecular machines carry out most of the biological functions in living organisms. Despite their significance, we do not yet understand the rules that govern the self-assembly of large multi-protein complexes. The bacterial flagellar motor tunes the assembly of its torque-generating stator complex with changes in external load. Here, we report that clockwise and counterclockwise rotating motors have identical remodeling response to changes in the external load, suggesting a purely mechanical mechanism for this regulation. Autonomous control of self-assembly may be a general strategy for tuning the functional output of protein complexes. The flagellar motor is a prime example of a macromolecular machine in which the functional regulation of assembly can be rigorously studied.

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Cooperative Self-Assembly of Pyridine-2,6-Diimine-Linked Macrocycles into Mechanically Robust Nanotubes

10.26434/chemrxiv.8080043 ◽

2019 ◽

Author(s):

Michael J. Strauss ◽

Darya Asheghali ◽

Austin Evans ◽

Rebecca Li ◽

Anton Chavez ◽

...

Keyword(s):

Aspect Ratio ◽

High Aspect Ratio ◽

Self Assembly ◽

Gel Permeation Chromatography ◽

Synthetic Polymers ◽

Young’S Moduli ◽

Structural Rigidity ◽

Assembly Mechanism ◽

Low Dimensionality ◽

Harsh Conditions

Nanotubes assembled from macrocyclic precursors offer a unique combination of low dimensionality, structural rigidity, and distinct interior and exterior microenvironments. Usually the weak stacking energies of macrocycles limit the length or strength of the resultant nanotubes. Imine-linked macrocycles were recently found to assemble into high-aspect ratio (>103), lyotropic nanotubes in the presence of excess acid. Yet these harsh conditions are incompatible with many functional groups and processing methods, and lower acid loadings instead catalyze macrocycle degradation. Here we report pyridine-2,6-diimine-linked macrocycles that assemble into high-aspect ratio nanotubes in the presence of less than 1 equiv of CF3CO2H per macrocycle. Analysis by gel permeation chromatography and fluorescence spectroscopy revealed a cooperative self-assembly mechanism. Nanofibers obtained by touch-spinning the pyridinium-based nanotubes exhibit Young’s moduli of 1.48 GPa, which exceeds that of many synthetic polymers and biological filaments. These findings will enable the design of structurally diverse nanotubes from synthetically accessible macrocycles.

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